Sunney Xie

Peking University

DNA exists as single molecules in individual cells. Consequectly gene expression is stochastic. The correlation among a pair of two mRNAs in a single cell provides important information regarding gene-to-gene interaction, and can only be extracted from single cell measurtements. We have developed a method for measuring single-cell transcriptome with improved detection efficiency and accuracy, allowing measurements of the covariance matrix for any pair of detected mRNAs. For a particular human cell type, we uncovered ~148 correlated transcription modules (CTMs) from the gene expression data of ~1000 individual cells under a steady state condition. We found that the CTMs are cell type dependent.

We have also developed transposase-based methods for single-cell whole gene amplification (WGA), which have superseded previous methods. With the improved genome coverage of a new WGA method, we have developed a high-resolution single-cell chromatin conformation capture method, which allowed for the first 3D genome map of a human diploid cell [Tan et al Science 2018]. The 3D genome structures are found to be cell type dependent.

The underlying mechanisms of transcription factors binding on the genome, changing the 3D genome structures, and regulating gene expression or cell differentiation, which are critically important to many fields of biology, can now be investigated with these new measurements.

Peter Jomo Walla

Institute for physical and theoretical Chemistry, University of Braunschweig, Gausstrasse 17, 38106 Braunschweig

Over millions of years, nature has achieved a remarkable efficiency in harvesting diffuse light photons and directing them onto an energy-converting device, the photosynthetic reaction center. Nature provides evidence that there is no fundamental limit for harvesting and funnelling nearly all scattered sun-photons onto smaller conversion centers. Recently, we demonstrated that this can also be achieved by an artificial material containing light-harvesting pools of randomly oriented molecules that funnel energy to individual, aligned light-redirecting molecules.[1]

In our contribution we will present how the principle concept behind this material allows to collect and convert nearly all sun-light energy with high efficiency and how it can be synthesised in a simple and affordable way.

Single molecule experiments are presented that confirm the desired 3D-dimensional orientational and structural arrangement as well as ultrafast measurements of the energy path ways and transition dipole reorientation that demonstrate the high efficiency of the photon collection and funnelling in this material. We will present unpublished data observed with new materials that aim at collecting the entire solar spectral range and converting it using state-of-the art high-efficiency energy converters.

Steffen Mühle¹, Man Zhou², Arindam Ghosh¹, Jörg Enderlein¹

¹Friedrich-Hund Platz 1, 37077 Göttingen, Germany
²Clarendon Laboratory, Parks Road, Oxford, OX1 3PU

The conformational flexibility and dynamics of unfolded peptide chains is of major interest in the context of protein folding and protein functioning. The rate with which amino acids at different positions along the peptide chain meet sets an upper speed limit for protein folding. By using single-molecule photo-induced energy transfer (PET) spectroscopy, we have systematically measured end-to-end and end-to-internal site contact formation rates for several intrinsically disordered protein fragments (11 to 41 amino acids), and have also determined their hydrodynamic radius using dual-focus fluorescence correlation spectroscopy (2fFCS). For interpreting the measured values, we have developed a Brownian dynamics model (based on bead-rod chain dynamics in a thermal bath including hydrodynamic interactions) which quantitatively reproduces all measured data surprisingly well while requiring only two fit parameters. The model provides a complete picture of the peptides' dynamics and allows us to translate the experimental rates and radii into molecular properties of the peptides: We find a persistence length of ~0.5 nm and a hydrodynamic radius of ~0.35 nm per amino acid.

Katrin I. Willig^1,2, Waja Wegner^1,2, Heinz Steffens^1,2

¹Center for Nanoscale Microscopy and Molecular Physiology of the Brain, , Göttingen, Germany
²Max Planck Institute of Experimental Medicine, Göttingen, Germany, E-mail: kwillig@em.mpg.de

From all super-resolution microscopy techniques currently available, STED microscopy stands out for its imaging capabilities in tissue: It is live-cell compatible, able to record 3D images from inside transparent tissue and has a fast imaging speed.

Here I will present applications of STED microscopy to image neuronal structures in the brain of living mice. We image the cerebral cortex through a glass window, in order to observe the dynamics of dendritic spines in the molecular layer. Recently we have pioneered in vivo superresolution of the postsynaptic scaffolding molecule PSD95, one of the key components in the organization of synapses, which is thought to control synaptic strength by anchoring postsynaptic receptors. We have shown for the first time the dynamic organization of PSD95 over several hours in the visual cortex of a living mouse [1]. These results show that STED nanoscopy is a highly suitable tool for research in neuroscience, which can play a substantial role in the study of learning and memory.

Gerti Beliu¹, Andreas Kurz¹, Alexander Kuhlemann¹, Lisa Behringer-Pliess¹, Mara Meub¹, Natalia Wolf², Jürgen Seibel², Zhen-Dan Shi³, Martin Schnermann⁴, Jonathan Grimm⁵, Luke Lavis⁵, Sören Doose¹, Markus Sauer¹

¹Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
²Institute of Organic Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
³Imaging Probe Development Center, National Heart, Lung, and Blood Institute, National, Institutes of Health, Rockville, Maryland, 20850, USA
⁴Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA
⁵Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, USA

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins^1,2. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells³.

Stefan Wieser², Loic Reymond^1,2, Johannes Ziegler², Christian Knapp², Gabriel Wang³, Thomas Huser³, Verena Ruprecht^1,4

¹Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 08003 Barcelona, Spain
²ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Castelldefels, Spain
³University Bielefeld, 33615 Bielefeld, Germany
⁴Universitat Pompeu Fabra (UPF), Barcelona, Spain

Visualization of cell dynamics with sub-diffraction molecular resolution demands for advanced fluorescence imaging modalities that combine high resolution, sensitivity and speed with minimal photobleaching and phototoxicity for long-term cell observation. Structured Illumination Microscopy (SIM) [1] has been established as a highly promising alternative superresolution live cell imaging approach which we combine with localization microscopy allowing to image dense structures such as actin in parallel with single myosin or plasma membrane constituents using high NA objectives (up to 1.7).

In a recent study Balzarotti et. al. used a doughnut shaped spot to enhance the localization precision of one single molecule emitter (MINFLUX) by a factor of 8 [2]. We present a Structured Illumination Microscopy based Point Localization Estimator (SIMPLE) that reveals a 2- to 6-fold increase in single molecule localization precision compared to conventional centroid estimation methods. SIMPLE advances MINFLUX by using precisely phase-shifted sinusoidal wave patterns as nanometric rulers for simultaneous particle localization based on photon count variation over a 20 µm field of view. We validate SIMPLE in silico and experimentally on a TIRF-SIM setup revealing 6.5 nm localization precision at 50 photon counts. 

Claus A.M. Seidel, Jan-Hendrik Budde, Suren Felekyan, Costanza Girardi, Christian Herrmann, Ralf Kühnemuth, Thomas Peulen, Nicolaas van der Voort

Chair for Molecular Physical Chemistry, Heinrich Heine University, Universitätsstraβe 1, 40225 Düsseldorf, Germany. cseidel@hhu.de

We combine super-resolution microscopy via stimulated emission depletion (STED) and Multi-parameter Fluorescence Image Spectroscopy (MFIS) [1,2] to reach molecular resolution with sub-nanometer precision for studying biomolecules and their complexes. While STED-MFIS captures the spatial and temporal information of the cellular context with a resolution down to 20 nm, the concurrent measurement of Förster resonance energy transfer (FRET) between an excited donor and acceptor provides a zoom with Ångström precision. Thus, super-resolution FRET microscopy exploits these synergies to reach molecular resolution. I will present the complete workflow from (I) super-resolution FRET microscopy over (II) FRET-specific data analysis to (III) integrative FRET-restrained structural models . MFIS uses multi-parameter fluorescence detection (MFD) [1] to capture the complete fluorescence information on the biomolecules by registering all eight characteristic fluorescence parameters in a single measurement. This allows us to study the formation of homo- and hetero-complexes by homo- and hetero-FRET in live cells simultaneously. We developed a refined analysis of MFIS-FRET data to achieve a significant noise reduction by species-selective averaging and to infer the structural properties, molecular stoichiometry and interaction affinities of molecular complexes in living cells by applying detailed models to resolve the corresponding FRET-parameters (i.e. distances) and species fractions. Using FRET restraints and computer simulations, we established an automated workflow to generate integrative structural models of biomolecular assemblies that can be deposited in the new protein data bank, PDB-dev [3, 4]. We studied Guanylate binding proteins (GBPs) that undergo a conformational transition for GTP-controlled oligomerization to exert their function as part of the innate immune system of mammalian cells - attacking intra-cellular parasites by disrupting their membranes. We identified GBP's intrinsic flexibility, a GTP-triggered association of the GTPase-domains and an assembly-dependent GTP-hydrolysis as functional design principles that control their reversible oligomerization in polar assemblies and the subsequent formation of condensates [5]. Further examples for FRET studies with molecular resolution are the structural characterization of GPRs in cells [6] and the recovery of multiscale dynamics in chromatin [7].

Shimon Weiss

Department of Chemistry & Biochemistry, University of California Los Angles, USA

We have been developing targetable voltage sensing inorganic nanoparticles (vsNPs) that are designed to self-insert into the cell membrane and non-invasively optically record, via the quantum confined Stark effect, action potential on the single-particle level, at multi-sites and in a large field-of-view. We synthesized a library of vsNPs with different compositions and geometries (type-I and type-II quantum dots and nanorods) and developed a high-throughput screen for optimization of their performance. We have explored several strategies for imparting these vsNPs with membrane-protein-like properties, including functionalization with libraries of peptides, lipids, and nanodiscs. We have developed screening assays for improving the efficiency of vsNPs’ membrane insertion and for their voltage sensitivity once embedded in the membrane. We have demonstrated membrane voltage sensing by membrane-inserted vsNPs in WT HEK cells using valinomycin and modulated concentration of potassium ions in a microfluidic chamber, and by patch-clamped in primary cultured cortical neurons. We will discuss limitations and trade-offs and suggest how to further improve these vsNPs. vsNPs hold promise for advancing electrophysiological investigations of the nervous system on the nanoscale. Membrane insertion of vsNPs also serve as an example for the emerging field of ‘biomimetic membrane proteins’.

Jelle Hendrix

Hasselt University, Dynamic Bioimaging Lab, Agoralaan C (BIOMED), B3590 Diepenbeek, Belgium

Lentiviruses such as the HIV poses a global health problem, and retroviruses such as the Moloney murine leukemia virus are interesting for gene therapy. In both, the integrase enzyme plays a pivotal role. We employ single-molecule Förster resonance energy transfer to provide detailed insights in the quaternary structure (stoichiometry and structure) of the enzyme complex. Interestingly, we do this in the context of infectious viral particles, allowing to study the fate of individual virions throughout replication. Although in the past we used acceptor photobleaching intensity based FRET quantifications, curently we employ multicolor pulsed interleaved excitation. We characterized different fluorescent protein pairs for their performance in smFRET, and work towards a realtime analysis of viral enzyme structure in 4D. I will provide an overview of the methods that we use, and discuss the various insights we could obtain in lenti/retroviral replication mechanisms.

Johann Bohlen¹, Izabela Kaminska², Sara Rocchetti¹, Florian Selbach¹, Renukka Yaadav¹, Guillermo Acuna³, Philip Tinnefeld¹

¹Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, 80539 München, Germany.
²Institute of Physical Chemistry of the Polish Academy of Sciences, 01-224 Warsaw, Poland.
³Department of Physics, Université de Fribourg, Ch. du Musée 3, CH-1700 Fribourg, Switzerland.

After its first isolation in 2004, graphene reached major interest in the fields of optics, electronics and mechanics due to its unique properties.¹ The optical interaction between a monolayer of graphene and a fluorescent emitter is well described with the distance dependence of d^-4.² Another important value to explain the energy transfer between the graphene monolayer and a fluorescent dye is d₀ which is the distance where 50% energy transfer efficiency occurs. This value can vary from 8 to 20 nm depending on the height placement and the emitter.^3,4 For a controlled height positioning of the emitter, DNA origami technique⁵ is the method of choice. With the self-assembled DNA origami structures, fluorescent dyes can be attached with the accuracy of 0.34 nm up to a distance of 120 nm away from the graphene monolayer. In this study, we show the influence of different fluorescently labelled DNA origami structures in the presence of a graphene monolayer by measuring the fluorescence lifetime, intensity and calculating the resulting energy transfer efficiency.⁶ The presented strategy broadens the palette of possibilities for fabrication of reliable graphene-based sensors and biological assays.

Jan-Hendrik Budde¹, Suren Felekyan², Costanza Girardi³, Ralf Kühnemuth⁴, Nicolaas van der Voort⁵, Claus A. M. Seidel⁶

Molecular Physical Chemistry, HHU Düsseldorf

Stimulated Emission Depletion (STED) microscopy [1] and Multiparameter Fluorescence Image Spectroscopy (MFIS) [2,3] were combined to selectively measure and characterize biomolecular systems on surfaces and in living cells with molecular resolution. While MFIS allows for detailed spectroscopic analysis and provides Ångström resolution via Förster Resonance Energy Transfer (FRET), STED microscopy overcomes the diffraction limit and localizes molecules with a resolution down to 30 nm. Thus, macromolecules can be localized with nanometer accuracy (STED), while monitoring their structure with Ångström resolution (FRET). The fruitful combination of both techniques is demonstrated in a benchmark study using double dye labeled DNA duplexes as spectroscopic ruler. Systematic distance variation within a FRET pair up to the practical resolution limits of STED allows us to map the localization of macromolecules with high precision and to resolve their inter- and intramolecular structural and dynamic features. Simultaneously different procedures  to determine the resolution are tested. Finally, the applicability in cellular imaging is demonstrated , allowing to unravel processes in living cells down to the single-molecule level .

C. Eggeling^1,2,3, E. Sezgin³, P. Kellner¹, I. Urbancic³, F. Schneider³, F. Reina^1,2

¹Friedrich‐Schiller‐University Jena, Institute of Applied Optics and Biophysics, 07743 Jena, Germany
²Leibniz Institute of Photonic Technology e.V., Department Biophysical Imaging, 07745 Jena, Germany
³University of Oxford, MRC Human Immunology Unit, OX3 9DS Oxford, United Kingdom e-mail: christian.eggeling@uni-jena.de

Molecular interactions are key in cellular signaling. They are usually ruled by the organization and mobility of the involved molecules. We present different fluorescence spectroscopic tools that are able to determine such organization mobility and potentially extract interaction dynamics. Specifically, the direct and non-invasive observation of the interactions in the living cell is often impeded by principle limitations of conventional far-field optical microscopes, for example with respect to limited spatio-temporal resolution. We depict how novel details of molecular membrane dynamics can be obtained by using advanced microscopy approaches such as the combination of super-resolution STED microscopy with fluorescence correlation spectroscopy (STED-FCS) or spectral detection. We highlight how STED-FCS and spectral STED microscopy can reveal novel aspects of membrane bioactivity such as of the existence and function of potential lipid rafts.

Mariano Gonzalez Pisfil^1,2, Marcelle König¹, Rhys Dowler¹, Benedikt Krämer¹, Sumeet Rohilla¹, Felix Koberling¹, Rainer Erdmann¹

¹PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany, info@picoquant.com
²Humboldt-Universität zu Berlin, Biology/Molecular Biophysics, Invalidenstr.42, 10115 Berlin, Germany

Fluorescence Correlation Spectroscopy (FCS) is an established tool for understanding the dynamics of complex cellular processes. By applying the approach scanning FCS (sFCS), significant improvements can be obtained when studying slowly diffusing species, as is often the case in cell membranes. In sFCS, the excitation volume is scanned rapidly through the sample allowing for the simultaneous measurement of the diffusion of species at multiple locations within the sample. This significantly increases the statistical accuracy. In addition, shorter residence times of the fluorophores lead to lower photon doses experienced by each detected molecule, reducing the risk of photobleaching. This is especially important for sensitive fluorophores or when performing combined Stimulated Emission Depletion (STED) and FCS measurements. An added advantage of the scanning process is the ability to determine the observation volume without prior calibration.

Here, we show results obtained with a confocal time-resolved fluorescence microscope (MicroTime 200 STED equipped with a FLIMbee galvo scanner, PicoQuant). We performed sFCS measurements on supported lipid bilayers (SLBs) which are commonly used as a simple model system for biological membranes. As the thickness of a bilayer is just a few nanometers, the diffusion properties determined with FCS or sFCS usually correspond to an average over both leaflets. We utilize the fluorescence lifetime information in order to achieve an axial nanometric localization based on Metal Induced Energy Transfer (MIET) [1]. The measured lifetime values enable us to separate the different molecular diffusion properties within the upper and the lower leaflet of the SLB measured on graphene.

Lukas Lau¹, Bálint Rehó², György Vámosi², Katalin Tóth¹

¹Division Biophysics of Macromolecules, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
²Department of Biophysics and Cell Biology, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary

When investigating intracellular dynamics on molecular level, Fluorescence (Cross-) Correlation Spectroscopy (F(C)CS) is a wide-spread method of choice. FCS typically retrieves information about concentrations, diffusion characteristics and species, but can even provide information about structural aspects of the molecular micro-environment. FCCS additionally indicates co-mobility i.e. binding behavior of molecules. However, on confocal setups spatial information can only be assessed at cost of time resolution.

Whereas Single Plane Illumination Microscopy (SPIM) is typically used in imaging applications, it also has significant potential for single molecule dynamics. SPIM-F(C)CS circumvents the spatio-temporal confocal microscopy F(C)CS limitations, as it allows for an entire plane to be acquired at the same time. It also reduces bleaching, as only a thin slice of the sample is illuminated.

We extended this approach further with an alternated excitation scheme providing additional insight. We show that simultaneous measurements of FRET and F(C)CS are viable. They give an overview not only of the diffusional and binding behavior inside live cells, but at the same time molecular proximity can be investigated. This extends the tool-set of microscopy methods measuring the dynamic behavior of fluorescent probes in live cells. The capabilities of this method are shown on the RAR-RXR nuclear receptor system.

Pamina M. Winkler¹, Raju Regmi^1,2, Valentin Flauraud³, Hervé Rigneault², Jürgen Brugger³, Jérôme Wenger², María F. García-Parajo^1,4

¹ICFO-Institut de Ciences Fotoniques, The Barcelona Institute of Science and Technology, 08860 Barcelona, Spain
²Aix-Marseille Université, CNRS, Institut Fresnel, Centrale Marseille, France
³Microsystems Laboratory, Ecole Polytechnique Fédérale de Lausanne, Switzerland
⁴ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain

Resolving the various interactions of lipids and proteins in the eukaryotic plasma membrane with high spatiotemporal resolution is of upmost interest [1]. Here, we present planar plasmonic antenna arrays with different nanogap sizes (10-45 nm) combined with fluorescence correlation spectroscopy (FCS) to resolve dynamic nanoscopic heterogeneities in mimetic and living plasma membranes. Our innovative approach confines the excitation light within the fully accessible planarized hotspot region of the nanoantennas yielding giant fluorescence enhancement factors of up to 10⁴-10⁵ times together with nanoscale detection volumes in the 20 zeptoliter range [2]. We exploit these planar nanoantenna arrays to investigate the dynamics of individual fluorescently labelled lipids in membrane regions as small as 10 nm in size with microsecond time resolution.
The existence of nanoscale assemblies of sterol and sphingolipids on mimetic as well as on living cell membranes has been questioned due to their highly transient and nanoscopic character. Our results on model lipid membranes reveal the coexistence of transient nanoscopic domains in both microscopically phase-separated regions with characteristic sizes < 10nm and lifetimes between 30 μs to 150 μs [3]. Currently, we are increasing the complexity of our mimetic system by incorporating hyaluronic acid (HA) to our multi-component lipid membranes. HA is an abundant glycoprotein of the extracellular matrix and recent work points to the glycocalyx as an important local organizer of the biological membrane [4]. Our current experiments combining atomic force microscopy and plasmonic nanoantennas aim at deciphering how HA contributes to phase segregation and nanoscale dynamic partitioning of mimetic biological membranes.

J. Wrachtrup

Institute for Quantum Science and Technology, IQST and Center for Applied Quantum Technologies, University of Stuttgart, Germany, wrachtrup@physik.uni-stuttgart.de

Novel quantum technologies have lead to the development of quantum sensors with potential application in life science. By combining e.g. optical microscopy with nuclear magnetic resonance it becomes possible to measure and image cellular structures, label-free, with chemical specificity and nanoscale spatial resolution. In the talk I will discuss various quantum sensors based on spin defects in materials like diamond. With such a system we measure a wealth of quantities including electric and magnetic fields, temperature, and force. We measure those quantities under ambient conditions and with unprecedented accuracy [1-4]. I will present a variety of applications including imaging of cellular structures.

Ling Xin^1,2, Mo Lu³, Steffen Both⁴, Markus Pfeiffer³, Maximilian J. Urban^1,2, Chao Zhou^1,2, Hao Yan⁵, Thomas Weiss⁴, Na Liu^1,2, Klas Lindfors³

¹Max Planck Institute for Intelligent Systems, Heisenbergstrasse 3, 70569 Stuttgart, Germany
²Kirchhoff Institute for Physics, Heidelberg University, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany
³Department of Chemistry, University of Cologne, Luxemburger Straße 116, 50939 Köln, Germany
⁴4th Physics Institute and Stuttgart Research Center of Photonic Engineering, University of Stuttgart, 70569 Stuttgart, Germany
⁵Department of Chemistry & Biochemistry, Biodesign Institute, Arizona State University, Tempe, AZ 85287-5601, USA

Plasmonic nanoantennas allow for enhancing the spontaneous emission, altering the emission polarization, and shaping the radiation pattern of quantum emitters. A critical challenge for the experimental realizations is positioning a single emitter into the hotspot of a plasmonic antenna with nanoscale accuracy. We demonstrate a dynamic light-matter interaction nanosystem enabled by the DNA origami technique. A single fluorophore molecule can autonomously and unidirectionally walk into the hotspot of a plasmonic nanoantenna along a designated origami track. Successive fluorescence intensity increase and lifetime reduction are in situ monitored using single-molecule fluorescence spectroscopy, while the fluorophore walker gradually approaches and eventually enters the plasmonic hotspot. Our scheme offers a dynamic platform, which can be used to develop functional materials, investigate intriguing light-matter interaction phenomena as well as to serve as prototype system for examining theoretical models.

Stefan W. Hell

Max Planck Institute for Biophysical Chemistry, Göttingen, Max Planck Institute for Medical Research, Heidelberg, hell@nanoscopy.de

Throughout the 20th century it was widely accepted that a light microscope relying on conventional optical lenses cannot tell apart details that are much finer than about half the wavelength of light, or 200-400 nanometers, due to diffraction. However, in the 1990s, the viability to overcome the diffraction barrier was realized and microscopy concepts defined that can resolve fluorescent features down to molecular dimensions. In this short talk, I will discuss the simple yet powerful principles that allow neutralizing the limiting role of diffraction [1,2]. In a nutshell, feature molecules residing closer than the diffraction barrier are transferred to different (quantum) states, usually a bright fluorescent state and a dark state, so that they become discernible for a brief period of detection. Thus, the resolution-limiting role of diffraction is overcome, and the interior of transparent samples, such as living cells and tissues, can be imaged at the nanoscale.

Hari Shroff

Section on High Resolution Optical Imaging (HROI), National Institute of Biomedical Imaging and Bioengineering, Bethesda MD 20814, USA

We describe theoretical and practical advances in algorithm and software design, resulting in ten to several thousand-fold faster deconvolution and multiview fusion than previous methods. First, we adapt methods from medical imaging, showing that an unmatched back projector accelerates Richardson-Lucy deconvolution by at least 10-fold, in most cases requiring only a single iteration. Second, we show that improvements in 3D image-based registration with GPU processing result in speedups of 10-100-fold over CPU processing. Third, we show that deep learning can provide further accelerations, particularly for deconvolution with a spatially varying point spread function. We illustrate the power of our methods from the subcellular to millimeter spatial scale, on diverse samples including single cells, nematode and zebrafish embryos, and cleared mouse tissue. Finally, we show that our methods facilitate the use of new microscopes that improve spatial resolution, including dual-view cleared tissue light-sheet microscopy and reflective lattice light-sheet microscopy. 

Arindam Ghosh, Akshita Sharma, Alexey I. Chizhik, Sebastian Isbaner, Daja Ruhlandt, Roman Tsukanov, Ingo Gregor, Narain Karedla, Jörg Enderlein

III. Institute of Physics – Biophysics, Georg August University, Friedrich-Hund-Platz 1,37077 Göttingen, Germany.

Super-resolution microscopy methods which are based on single-molecule localization (SMLM) such as PALM [1] , STORM [2] , fPALM [3] , dSTORM [4]  or PAINT [5]  have found manifold applications from fundamental physics to life sciences. These methods achieve lateral localization accuracies of a few nanometers, but encounter big challenges when it comes to the localization along the optical axis (third dimension). Recently, Metal-Induced Energy Transfer or MIET [6 , 7] was introduced as a technique for axial localization of fluorescent emitters with nanometer accuracy [8 , 9]. It exploits the energy transfer from an excited fluorophore to surface plasmons in a thin metal film. Here, we show that using graphene as the “metal” layer , one can increase the localization accuracy of MIET by nearly an order of magnitude We demonstrate this potential of graphene-based MIET (gMIET) by axially localizing single emitters, and by estimating supported lipid bilayer (SLB) thickness values with Ångström accuracy. We also present preliminary results concerning the structure and dynamics of mitochondrial membranes.

Michel Orrit

MoNOS, Leiden Institute of Physics, Huygens-Kamerlingh Onnes Lab. 2300 RA Leiden, Netherlands, orrit@physics.leidenuniv.nl

Several optical methods give access to single molecules. Single gold nanoparticles strongly interact with light through their plasmon resonance, and are chemically and photochemically stable. Anti-Stokes photoluminescence of single gold nanorods depends on the absolute temperature of the particles and can be used as absolute thermometer [1].

Photothermal contrast can be pushed to single-molecule sensitivity for organic conjugated polymers [2], or applied to the study of nanosecond dynamics of steam nanobubbles [3].

Non-absorbing protein molecules can be detected individually by their polarizability only [4]. Similar experiments can be performed on diffusing objects. Plasmonic shifts from individual diffusing molecules are brief but frequent and can be analyzed in a statistical manner similar to FCS.

Plasmonic fluorescence enhancement applies to dyes with low quantum yields. We have shown that the redox activity of single molecules can be monitored in real time, exhibiting the redox cycles of individual dye molecules [5] or of single protein molecules.

Ronald Walsworth

Harvard-Smithsonian

The nitrogen–vacancy (NV) quantum defect in diamond is a leading modality for magnetic, electrical, and temperature sensing at short length scales (nanometers to millimeters) under ambient conditions. This technology has wide-ranging application across the physical and life sciences — from NMR spectroscopy at the scale of individual proteins and cells to improved biomedical diagnostics to magnetic imaging of neuronal networks.  I will provide an overview of quantum diamond sensors and their diverse applications.

Tim Schröder¹, Gordon Hedley², Florian Steiner¹, Felix Hofmann³, Max B. Scheible⁴, Philip Tinnefeld¹, John Lupton³, Jan Vogelsang^1,3

¹Department Chemie and Center for NanoScience, Ludwig-Maximilians-Universitaet Muenchen, Butenandtstraße 5-13 Haus E, 81377 Muenchen, Germany
²School of Chemistry, University of Glasgow, University Avenue, Glasgow, G128QQ, United Kingdom
³Institut für Experimentelle und Angewandte Physik, Universität Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany
⁴GATTAquant GmbH, Am Schlosshof 8, 91355 Hilppoltstein, Germany

High chromophore densities are present in conjugated polymers, light harvesting complexes and are also mandatory for small but bright fluorescence point light sources. DNA nanotechnology allows us to place dyes with nanometer accuracy at defined positions to investigate dye-dye interactions on the single molecule or aggregate level.[1] Small distances below 1.0 nm between two dyes lead to quenching of fluorescence. With increasing distance, dynamics in the DNA origami structure are observed. Once quenching is suppressed at a distance of 2.4 nm, energy transfer processes such as singlet-singlet annihilation or singlet-triplet annihilation have to be considered. Fortunately, these annihilation processes lead to single photon emission, i.e. photon antibunching and are time dependent. For these reasons, the information of the average annihilation rates must be hidden in the photon stream. Here we introduce time-gated analysis of the photon statistics with sub-ns resolution to unravel annihilation processes in well-defined multi-chromophoric DNA-origami structures. We can distinguish between the number of physical emitters and the number of possible excitations during their excited state lifetime. Our study fills a gap of examining the interactions of dyes relevant for superresolution microscopy with dense labeling for single-molecule biophysics and the design of homogenous DNA origami nanobeads.

Ben Schuler

Department of Biochemistry and Department of Physics, University of Zurich, Switzerland

The functions of proteins have traditionally been linked to their well-defined three-dimensional, folded structures. It is becoming increasingly clear, however, that many proteins perform essential functions without being folded. Single-molecule spectroscopy is a versatile approach for investigating the structure and dynamics of such unfolded or ‘intrinsically disordered’ proteins (IDPs). The combination of single-molecule Förster resonance energy transfer (FRET) with nanosecond correlation spectroscopy, microfluidic mixing, and other advanced methods can be used to probe intra- and intermolecular distance distributions, reconfiguration dynamics, and interactions on a wide range of timescales, and even in heterogeneous environments, including live cells. I will illustrate the use of single-molecule spectroscopy for these highly dynamic and structurally heterogeneous systems.

Jörg Enderlein

III. Institute of Physics – Biophysics, Georg August University, Friedrich-Hund-Platz 1, D-37077 Göttingen, Germany, Email: jenderl@gwdg.de

Recent years have seen a tremendous development in high- and super-resolution techniques of fluorescence microscopy, such as STED, PALM or STORM. However, for nearly all of them, the axial resolution is typically a factor 3-5 worse than the lateral resolution, similar to most diffraction-limited optical microscopy techniques. I will present our recent work on Metal Induced Energy Transfer or MIET imaging [1] which achieves nanometer resolution along the optical axis. It uses the effect that by placing a fluorescent molecule close to a metal, its fluorescence properties change dramatically. In particular, one observes a strongly modified lifetime of its excited state (Purcell effect). This coupling between an excited emitter and a metal film is strongly dependent on the emitter’s distance from the metal. We have used this effect for mapping the basal membrane of live cells with an axial accuracy of ~3 nm [2,3,5]. The method is easy to implement and does not require any change to a conventional fluorescence lifetime microscope; it can be applied to any biological system of interest, and is compatible with most other super-resolution microscopy techniques that enhance the lateral resolution of imaging. Moreover, it is even applicable to localizing individual molecules, thus offering the prospect of three-dimensional single-molecule localization microscopy with nanometer isotropic resolution for structural biology [4,6]. I will also present recent application of MIET for leaflet-resolved imaging and spectroscopy of lipid bilayers, using graphene monolayers as the quenching substrates, which allows for achieving even sub-nanometer axial resolution. We will exemplify this on (i) distance measurements across lipid bilayers, for (ii) measuring structure and dynamics of lipid bilayers in leaflet-dependent manner (by combining MIET with Fluorescence Lifetime Correlation Spectroscopy = FLCS [7]), and for (iii) measuring fast conformational dynamic in single-stranded DNA molecules.

Acknowledgments

JR acknowledges financial support by the Deutsche Forschungsgemeinschaft (DFG) under Germany's Excellence Strategy - EXC 2067/1 - 390729940.

Christian Gebhardt¹, Rebecca Mächtel¹, Niels Zijlstra¹, Marijn de Boer², Thorben Cordes^1,2

¹Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany
²Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

Single-molecule Förster resonance energy transfer (smFRET) has evolved towards a mature toolkit for the study of distances, structures, and dynamics of biomolecules in a physiologically relevant context. There is, however, no consensus on how to derive and use quantitative distance information obtained via the FRET-ruler to derive structural models or constraints in the protein database. Recently, Hellenkamp et al. [1] presented a quantitative smFRET study of oligonucleotide ruler structures that revealed high precision, accuracy, and reproducibility of FRET-derived distances in a worldwide comparative study showing a distance uncertainty of less than 6 Å. While this establishes smFRET as a suitable technique for accurate distance measurements within static biological reference structures, we raise the question whether smFRET can also be used to accurately determine distances within proteins that exhibit conformational dynamics or allosteric modulation of their structure by an effector. Additionally, proteins are more challenging targets for site-specific fluorophore labelling as compared to oligonucleotides. We identified a suitable model system that we used here to benchmark FRET-derived distance uncertainties for proteins in the case of (i) stochastic labelling and (ii) allosteric and dynamic modulation of the structure, and show angstrom precision similar to DNA.

Anders Barth¹, Oleg Opanasyuk¹, Thomas-Otavio Peulen^1,2, Suren Felekyan¹, Hugo Sanabria^1,3, Claus A.M. Seidel¹

¹Institut für Physikalische Chemie, Lehrstuhl für Molekulare Physikalische Chemie, Heinrich Heine Universität, Düsseldorf, Germany
²Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California, USA
³Department of Physics and Astronomy, Clemson University, Clemson, S.C., USA

Solution-based single-molecule FRET in combination with multiparameter fluorescence detection (MFD) provides access to numerous spectroscopic parameters that report on the properties of the fluorophores and the structure and dynamics of the biomolecule. While a multitude of methods has been developed to study the dynamics of two-state kinetic systems on the milli- to microsecond timescale, quantitative analysis of kinetic networks involving three or more states remains challenging. Here, we present an integrated analysis workflow for multi-state kinetic systems. The starting point thereof is the use of parametric relations between the FRET efficiency and the donor fluorescence lifetime. These FRET-lines serve as visual guides that are overlaid on the two-dimensional histograms, allowing different kinetic models to be compared against the experimental data. We explore the application of FRET-lines to elucidate biomolecular dynamics of structured and disordered systems and devise procedures to decouple the local motion of the fluorophores from the global structural dynamics of the molecule. Quantitative analysis of kinetics is achieved by an integrated framework combining fluorescence decay analysis, fluorescence correlation spectroscopy and photon distribution analysis, wherein the multidimensional information provided by MFD proves crucial. Furthermore, degeneracy of conformational states may potentially be avoided by application of three-color FRET.

Maria Dienerowitz, André Dathe, Thomas Heitkamp, Hendrik Sielaff, Michael Börsch

Single-Molecule Microscopy Group, Universitätsklinikum Jena, Nonnenplan 2-4, 07743 Jena, Germany

Membrane proteins change their shape upon interacting with a substrate molecule. Identifying these conformational changes is key to understand the intricate catalysis and signalling processes of a cell. We purify individual membrane proteins to examine their conformational dynamics with single-molecule FRET. Brownian motion causes the proteins to diffuse out of the observation volume within milliseconds providing only snapshots of conformational states. Electrokinetic trapping with an ABELtrap extends the observation time up to seconds, thus enabling us to examine full working cycles of single ATP synthase proteins in solution. Time-correlated single photon counting localises the fluorescently labelled protein while an FPGA-based active feedback system generates electrokinetic forces confining the protein to the trap centre.

We investigate subunit rotations of individual E. coli F_oF₁-ATP synthase proteins in real time using single-molecule FRET. We demonstrate that changing the ATP concentration in the buffer regulates the hydrolysis rates according to Michaelis-Menten-Kinetics. Our second system inspects the oligomerisation status of the human neurotensin receptor 1 (NTSR 1) extracted from living HEK293T cells. The membrane-bound receptor resides in soluble lipid nanodiscs formed by styrene-maleic acid copolymers (SMALPs) - a detergent-free membrane protein purification technique promising superior protein stability and functioning.

Prof. W.E. Moerner

Stanford University, Departments of Chemistry and Applied Physics (Courtesy), Stanford, CA 94305, , wmoerner@stanford.edu

It is worth remembering that the first optical detection of a single molecule, a 1 nm object, arose out of an industrial research lab in the late 1980’s, while exploring the fundamentals of frequency domain optical storage (spectral hole-burning). This work led to the observations of blinking and optical switching at room temperatures, key foundations for super-resolution (SR) imaging with single molecules. SR microscopy has opened up a new frontier in which biological structures can be observed with resolutions down to 20-40 nm and below. In addition, in the “conventional” low concentration regime, single-molecule imaging and tracking informs us about dynamical processes. Current methods development research addresses ways to image in thick cells and to improve fluorescent labels. Even after 30 years, new thrusts continue to appear, for example, low temperature localizations of single labeled proteins can provide ground truth information for cryo-EM, and trapping of single molecules in solution now can be achieved without fluorescence or optical forces.

Johan Hofkens¹, Raffaele Vitale⁴, Laurens D’Huys¹, Vince Goyvaerts¹, Cyril Ruckebusch⁴, Theo Lasser², Aleksandra Radenovic^2,3

¹Chemistry department, KU Leuven, Leuven, 3000, Belgium, johan.hofkens@kuleuven.be
²Institute of Bioengineering, Ecole Polytechnique Federale de Lausanne, Lausanne, CH-1015, Switzerland
³School of engineering, Ecole Polytechnique Federale de Lausanne, Lausanne, CH-1015, Switzerland
⁴LASIR CNRS, Universite de Lille, Villeneuve d'Ascq Cedex, 59655, France

Single molecule DNA mapping has the potential to serve as a powerful complement to high throughput sequencing in metagenomic analysis. Offering longer read lengths and forgoing the need for complex library preparation and amplification, mapping stands to provide an unbiased view into the composition of complex viromes and/or microbiomes. To fully enable mapping-based metagenomics, sensitivity and specificity of DNA map analysis and identification need to be improved. Using detailed simulations and experimental data, we first demonstrate how fluorescence imaging of surface stretched, sequence specifically labeled, DNA fragments can yield highly sensitive identification of targets. Secondly, a new analysis technique is introduced to increase specificity of the analysis, allowing even closely related species to be resolved. Thirdly, we show how an increase in resolution, improves sensitivity. Finally, we demonstrate that these methods are capable of identifying species with long genomes such as bacteria with high sensitivity.

Acknowledgments: This work was supported by the Horizon 2020 Framework Programme of the European Union called ADgut [Grant No 686271]; ‘Agentschap Innoveren & Ondernemen’ in the framework of an innovation mandate [No HBC.2016.0246]; the European Union Research Council through the ERC-2017-PoC Metamapper [No 768826]

Luciano A. Masullo^1,2, Lucía F. López¹, Florian Steiner³, Philip Tinnefeld³, Fernando D. Stefani^1,2

¹Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas, (CONICET), Godoy Cruz 2390, C1425FQD, Buenos Aires, Argentina.
²Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 1 Ciudad, Universitaria, C1428EHA, Buenos Aires, Argentina.
³Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstrasse 5-13 Haus E, 81377 München, Germany

Super-resolution has revolutionized the power of optical microscopes to study biological systems at resolutions well below the diffraction limit [1]. Among the different techniques, MINFLUX nanoscopy [2] allows to achieve molecular scale resolution (~ 1 nm) by optimizing the information contained in the detected photons with spatially patterned illumination. In this presentation we introduce a novel MINFLUX experimental realization: our method combines spatial point spread function engineering with pulsed interleaved excitation to enhance the localization precision of the emitter. We will present the set-up and the first preliminary results on single-molecule samples. Our implementation is simple and robust and since it is based on time-correlated single-photon counting it is fully compatible with fluorescence life-time imaging. Therefore, it has the potential to be extended to two colors by life-time discrimination and to 3D by single-molecule metal-induced energy transfer (smMIET) localization [3].

Christian Franke¹, Urska Repnik¹, Sandra Segeletz¹, Nicolas Brouilly^1,2, Yannis Kalaidzidis^1,3, Jean-Marc Verbavatz⁴, Marino Zerial¹

¹Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany.
²Instutut de Biologie du Developpement de Marseille-Luminy, Aix-Marseille Universite, Marseille, France
³Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia
⁴Institut Jacques Monod, CNRS, Université Paris-Diderot, Sorbonne Paris Cité, 75013 Paris, France.

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which however cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional sub-domains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-colour SMLM (dSTORM & PALM) and three-dimensional electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labelled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nano-domains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.[1]
 

Markus Sauer

Department of Biotechnology & Biophysics, , Biocenter, Julius Maximilian University Würzburg, Am Hubland, 97074 Würzburg, Germany, E-mail: m.sauer@uni-wuerzburg.de

Super-resolution microscopy by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level [1]. Among all powerful high-resolution imaging techniques introduced in recent years, localization microscopy excels at it delivers single-molecule information about the distribution and, adequate controls presupposed, even absolute numbers of proteins present in subcellular compartments. This provides insights into biological systems at a level we are used to think about and model biological interactions. We briefly introduce basic requirements of localization microscopy, its potential use for quantitative molecular imaging, and discuss present obstacles and ways to bypass them. We demonstrate the advantageous use of single-molecule localization microscopy by dSTORM for quantitative imaging of synaptic proteins, the study of plasma membrane receptors, and the molecular architecture of multiprotein complexes. Finally, we outline how dSTORM can be used advantageously to improve next generation medical therapies.

Paul French

Photonics Group, Physics Department, Imperial College London

We are developing multidimensional fluorescence imaging technology with a particular emphasis on fluorescence lifetime imaging (FLIM) to contrast different molecular species and to map variations in the local fluorophore molecular environment. This includes FLIM applied to read out Förster resonant energy transfer (FRET) in order to assay protein interactions or read out genetically expressed FRET biosensors. For high content analysis (HCA) we have implemented automated time-gated FLIM for multiwell plate assays of protein interactions or cellular metabolism. We have applied FLIM FRET HCA to study signalling and disease mechanisms in 2-D and 3-D cell-based assays, including the intracellular measurement of K_D to quantify protein interactions¹. We typically use fluorescent proteins (FP) as donor/acceptor fluorophores and have analysed the impact of the slow rotational dephasing compared to their fluorescence lifetime, in order to support quantitative FRET measurements². We have explored the efficacy of FLIM/FRET applied to endogenous proteins labelled with FP and demonstrated automated FLIM FRET of endogenous (low copy number) kinetochore proteins in budding yeast labelled with FP³. We have also translated FLIM/FRET to vivo preclinical studies of murine cancer models with FLIM confocal endomicroscopy⁴.

We have realised super-resolved microscopy using stimulated emission depletion (STED) microscopy utilising a spatial light modulator (SLM) to provide programmable shaping of excitation and depletion beams for 2D and 3D STED and have introduced easySLM-STED⁵ to provide automatic co-alignment and aberration correction of excitation and depletion beams, extending the field of view and increasing the imaging speed through parallelised STED. To realise super-resolved HCA, we are developing automated easySTORM⁶, providing low-cost, large FOV single molecule localisation microscopy (SMLM) and accelerated SMLM analysis using parallelised ThunderSTORM implemented on a high-performance computing cluster⁷.

We aim to implement our multidimensional fluorescence imaging technology using open source software tools for instrument control, data acquisition, analysis and management. Our open source HCA platform utilises µManager for data acquisition and for analysis, ImageJ and FLIMfit⁸, an OMERO plug-in, for FLIM data analysis. Current open microscopy developments include a low-cost modular motorised microscope frame and an optical autofocus unit.

Matthias Simonis¹, Johannes Greiner², Idir Yahiatene¹, Christian Kaltschmidt², Barbara Kaltschmidt^2,3, Thomas Huser¹, Dietmar Manstein⁴, Simon Hennig⁴

¹Biomolecular Photonics, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany
²Department of Cell Biology, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany
³Molecular Neurobiology, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany
⁴Institute of Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str 1, 30625 Hannover, Germany

Intracellular labeling of fluorescent probes for live-cell nanoscopy remains a serious problem. Several approaches for delivery of fluorescent probes and labeling of intracellular structures with fluorescent probes exist, but most implementations are unique and tailored to a specific problem. Thus, a general strategy for labeling with the possibility for live-cell nanoscopy is missing. Electrophoretic nanoinjection unlocks a gentle and new way for the precise intracellular delivery of fluorescent probes into single living cells.

We introduce a general method called electrophoretic Nanoinjection with Points Accumulation for Imaging in Nanoscale Topography (eN-PAINT), to combine nanoinjection with live-cell nanoscopy. By continuous delivery of fluorescent probes and simultaneous detection and bleaching, we are able to visualize intracellular structures in living cells with subdiffraction resolution on a second-timescale combined with long observation-times. The unique nanopipette-based delivery is able to generate images with different fluorophore-densities ranging from sparse to very dense blinking and is thus able to optimize the imaging-conditions for several nanoscopy-techniques, unlocking the highest possible temporal and spatial resolution for live-cell nanoscopy.

The method is independent from chemical buffer conditions, and can easily be applied to living cultured, primary and stem-cells. The nanoinjection-technique is thereby cheap and simple to implement on any inverted microscope.

Christoph Spahn¹, Jonathan B. Grimm², Luke D. Lavis², Marko Lampe³, Mike Heilemann¹

¹Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438 Frankfurt, Germany
²Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, United States
³Advanced Light Microscopy Facility, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany

Image quality and resolution in stimulated emission depletion (STED) microscopy is often affected by irreversible photobleaching of fluorophores. In order to bypass photobleaching, we repurposed the use of exchangeable fluorescent probes as used in Point Accumulation for Imaging in Nanoscale Tomography (PAINT) [1] for STED microscopy, achieving pseudo-permanent labeling of target structures and permanent exchange of photobleached fluorophores. This concept allows for whole-cell, 3D, multi-color and live-cell STED microscopy [2].

Using transiently binding hydrophobic dyes and fluorophore-labeled major minor groove binders [3, 4], we visualized the nanostructure of chromatin, cell membranes and organelles in bacterial and mammalian cells in 3D. We further demonstrate that short peptides allow for STED imaging of cytoskeletal networks and use membrane-targeting, exchangeable labels to follow organelle dynamics in living cells.

Ron Tenne¹, Uri Rossman¹, Batel Rephael¹, Yonatan Israel², Alexander Krupinski-Ptaszek³, Radek Lapkiewicz³, Yaron Silberberg¹, Dan Oron¹

¹Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot 76100, Israel
²Department of Physics, Stanford University, Stanford, CA 94305, USA
³Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland

The principles of quantum optics have yielded a plethora of ideas to surpass the classical limitations of sensitivity and resolution in optical microscopy. While some ideas have been applied in proof-of-principle experiments, imaging a biological sample has remained challenging mainly due to the inherently weak signal measured and the fragility of quantum states of light. In principle, however, these quantum protocols can add new information without sacrificing the classical information and can therefore enhance the capabilities of existing super-resolution techniques. Image scanning microscopy (ISM), a recent addition to the family of super-resolution methods, generates a robust resolution enhancement without sacrificing the signal level. Here, I will introduce a new super resolution scheme, quantum image scanning microscopy (Q-ISM), that utilizes quantum photon correlations in an ISM configuration, to increase the resolution of ISM up to two-fold, four times beyond the diffraction limit. Relying solely on a quantum phenomenon, photon antibunching, as the image contrast, we were able to obtain super-resolved optical images of a biological sample stained with fluorescent quantum dots. In addition, we have shown that the z-sectioning capabilities of standard ISM are also enhanced by using the photon correlation contrast.

Thomas Schmidt

Physics of Life Processes, Leiden Institute of Physics, Leiden University, Huygens Laboratory, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands

Transcription factor mobility is a determining factor in the regulation of gene expression. Here, we have studied the intranuclear dynamics of the glucocorticoid receptor (GR) using fluorescence

recovery after photobleaching and single-molecule microscopy. First we have described the dynamic states in which the GR occurs. Subsequently we have analyzed the transitions between these states using a continuous time Markov chain model, and functionally investigated these states by making specific mutations in the DNA-binding domain. This analysis revealed that the GR diffuses freely through the nucleus, and once it leaves this free diffusion state it most often enters a repetitive switching mode. In this mode it alternates between slow diffusion as a result of brief nonspecific DNA binding events, and a state of stable binding to specific DNA target sites. This repetitive switching mechanism results in a compact searching strategy which facilitates finding DNA target sites by the GR.

Frank Mieskes¹, Fabian Wehnekamp¹, Gabriela Plucińska^2,3, Rachel Thong², Thomas Misgeld², Don C. Lamb¹

¹Ludwig Maximilians-Universität Munich, Butenandstr. 5-13, 81377 Munich, Germany
²Institute of Neuronal Cell Biology, Technische Universität München, Biedersteiner Str. 29, 80802 Munich, Germany
³Current address: Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

3D Orbital Tracking is a feedback based single particle tracking approach with high spatiotemporal resolution. As tracking uses the modulation of the fluorescence signal to lock into the moving particle, it is more robust than other tracking methods against scattering and background. Hence, it is ideal for providing deep insights in cellular and in vivo systems by following single particles with nanometer accuracy in real time. Our goal is to push our developed 3D Orbtial Tracking microscope further to gain new functionalities by extending single color experiments to multicolor systems.

In this talk, I will first present the latest results we obtained in living zebrafish embryos by tracking the mitochondrial movement over 100 µm. In the second part, I will discuss our current developments for performing two-channel single particle tracking. With the expanded setup, we can also combine the 3D Orbital Tracking approach with Förster Resonance Energy Transfer (FRET), allowing us to track in one channel while the second channel is spectrally split to measure FRET. This combination promises to be powerful tool for investigating dynamics of diffusing particles in living systems on the single molecule scale.

Jakob Schedlbauer¹, Philipp Wilhelm¹, Maria Federl¹, Florian Hinderer², Sigurd Höger², Lennart Grabenhorst³, Phillip Tinnefeld³, Jan Vogelsang³, Sebastian Bange¹, John Lupton¹

¹Universität Regensburg, Germany
²Universität Bonn, Germany
³LMU München, Germany

A new experimental approach is presented to monitor ultrafast excited state deactivation combining single molecule fluorescence spectroscopy (SMS) with a pump-probe-like excitation scheme. In the simplest way a single molecule can be described as a two level system and therefore can emit only one single photon after a given excitation pulse, a phenomenon referred to as photon antibunching. By introducing a second excitation pulse after a variable time lag the probability for reexciting the system and generating a second photon becomes a function of the excited state lifetime. A Hanbury Brown-Twiss-based detection setup enables us to measure the probability for the emission of multiple photons after a given excitation cycle.

This technique overcomes the limitation of conventional fluorescence-based lifetime measurements such as time-correlated single-photon counting, which is constrained by finite detector instrument response function. We resolve dynamical single molecule processes like the intramolecular energy transfer in a donor-acceptor-donor system (13 ps) as well as ultrafast enhanced PL of Cy7 dyes coupled to a plasmonic antenna structure (20 ps).

Toshio Yanagida

Osaka University

How muscle works has been one of the biggest target of single molecule study. However, no one has directly visualized the force generation of muscle myosin and the dynamic features remained poorly understood compared with single-molecule studies on processive molecular motors like kinesin, dynein, unconventional myosins and nucleic acid motors. One reason is that muscle myosin is non-processive and works as a group in the highly-structured sarcomere. This design and the non-processivity makes it difficult to directly observe the dynamics of the rapid and minute displacements of muscle myosin.

Here, we engineered thick filaments composed of DNA origami and recombinant human muscle myosin, and directly visualized the myosin head during force generation using nanometer-precision single-molecule imaging. We found that when the head diffuses, it weakly interacts with actin filaments and then strongly binds preferentially to the forward region as a Brownian ratchet. Upon strong binding, the head two-step lever-arm swing dominantly halts at the first step and occasionally reverses direction. These dynamic features of myosin based on the Brownian ratchet can explain all mechanical characteristics of muscle contraction. This is the first answer of how muscle works based on the direct experimental evidence.

(http://biorxiv.org/cgi/content/short/634287v1)

Irene Perez-Pi, David A. Evans, Mathew H. Horrocks, Nhan T. Pham, Karamjit S. Dolt, Joanna Koszela, Tilo Kunath, Manfred Auer

School of Biological Sciences and Edinburgh Medical School: Biomedical Sciences, University of Edinburgh, The King’s Buildings, Edinburgh, United Kingdom

α-Synuclein fibrils are considered a hallmark of Parkinson’s disease. Small oligomers that are formed during the early stages of α-synuclein aggregation are thought to be the main toxic species causing disease. A novel 2 colour bead-based aggregation assay for monitoring the earliest stages of α-synuclein oligomerization, α-Synuclein–Confocal Nanoscanning (ASYN-CONA), was developed. The α-synuclein A91C single cysteine mutant is labelled with tetramethylrhodamine, and attached to microbeads using a triple tag chemistry. Beads with bound TMR-labelled α-synuclein are then incubated with a Cy5-labelled variant of α-synuclein A91C, and aggregation is induced. On-bead TMR-labelled α-synuclein and aggregated Cy5-labeled α-synuclein from the solution are quantitatively monitored in parallel by detection of fluorescent halos or “rings”. α-Synuclein on-bead oligomerization results in a linear increase of red bead ring fluorescence intensity over a period of 5 h. Total internal reflection single molecule fluorescence microscopy was performed on oligomers cleaved from the beads, and it revealed that (i) oligomers are sufficiently stable in solution to investigate their composition, consisting of 6 ± 1 monomer units, and (ii) oligomers containing a mean of 15 monomers bind Thioflavin-T. Various known inhibitors of α-synuclein aggregation were used to validate the ASYN-CONA assay for drug screening.

Alessandro Rossetta^1,2,3, Eli Slenders¹, Giorgio Tortarolo¹, Marco Castello¹, Mauro Buttafava⁴, Federica Villa⁴, Alberto Tosi⁴, Alberto Diaspro^3,5, Giuseppe Vicidomini¹

¹Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Genoa, Italy
²DIBRIS, Università degli Studi di Genova, Genoa, Italy
³Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy
⁴DEIB, Politecnico di Milano, Milan, Italy
⁵DIFI, Università degli Studi di Genova, Genoa, Italy

Understanding the complex biomolecular processes at the base of Life can be considered the holy grail of cell biology research. Fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence/lifetime spectroscopy are among the most important tools to observe biomolecular processes – both from the structural and the dynamics perspectives – within living cells. Most of FCS and time-resolved fluorescence spectroscopy implementations rely on single-point/element detectors (i.e., PMT, APD, SPAD or Hybrid detectors) since they have superior temporal resolution (< μs) – with respect to conventional detector arrays (CCD or CMOS) – and provide the single photon-timing information. However, in stark contrast to detector arrays, single-element detectors lose the information about the spatial distribution of the fluorescent light focused on the detector sensitive area.

Recently, novel detector arrays with temporal characteristics similar to single-element detector have been introduced – namely the Airyscan [1] and the SPAD array [2]. The Airyscan has been used to implement FCS in a beam-scanning microscopy architecture, showing the great benefitsobtained introducing the spatial information provided by the detector array in the FCS analysis [2]. The SPAD array has everything in its favor to combine this enhanced FCS analysis with timeresolved fluorescence spectroscopy. However, the full benefits of this synergetic combination would be possible only when advanced data acquisition and processing platforms, able to manage the massive flux of information delivered by the SPAD array, will be developed. Indeed, each element (e.g., 25 in our implementation) of the SPAD array detector, fires – independently and parallelly – a precise (< 200 ps) and high-frequency voltage pulse (tens of MHz) every time that aphoton is registered.

Here we introduce our new time-resolved data acquisition platform, designed to implement FCS and time-resolved fluorescence spectroscopy with our SPAD array detector. We discuss the current and future main characteristics of the platform and we show few examples of its application.

We envisage that this novel platform can revolutionize the observation of biomolecular processes within live-cells, triggering a new class of fluorescence spectroscopy methods able to unlock the secrets carried by each photon emitted by the sample.

Gerhard Schütz

Vienna University of Technology, Institute of Applied Physics - Biophysics, Vienna, Austria

T-cells recognize via their T cell receptors (TCRs) even lowest numbers of pMHC on the surface of antigen presenting cells (APCs). The mechanisms underlying this phenomenal sensitivity, however, have remained elusive. Several studies suggested mechanical forces to be instrumental. To address their role most directly we engineered a calibrated FRET-based force sensor, which allowed for quantitative visualization of molecular forces exerted via the TCR within the immunological synapse between T cells and functionalized supported lipid bilayers. We could for the first time quantify the T cell-exerted tensile forces, peaking around 5 pN both under activating and non-activating conditions. Interestingly, forces were only detectable when T cells were adhered to gel-phase lipid bilayers, whereas fluid bilayers did not yield pulling forces above our detection threshold of ~2pN. The data hence suggest that exerted forces act tangentially to the plane of the immune synapse.

Gregor Jung

Biophysical Chemistry, Saarland University, Campus B2 2, 66123 Saarbruecken, Germany

Single-molecule research revolutionized life sciences, but applications in chemical reaction dynamics are sparse. We established a wealth of fluorescent probes which undergo a change of the emission color during a chemical transformation and, hence, can detect single reaction events by TIRF-microscopy.^[1] Up to three emission colors can be obtained from one fluorescent substrate molecule.^[2] One big issue, however, is transferring the results from single-molecule research to the conditions of chemical synthesis. Especially the immobilization of molecules on surfaces can modify the reactivity with respect to homogeneous solution conditions. The impact of the local surrounding on the reaction kinetics also remains hidden as each substrate molecule reacts only once.

In my presentation, I will show how we tackle these challenges. A two-step immobilization strategy on silica surfaces creates reaction centers for the palladium-catalyzed Tsuji-Trost reaction which kinetically behave as pristine molecules in solution.^[3] Differences in the kinetics between the fluorogenic approach and two-color imaging can be traced back to photochemical side reactions. Photochemical proton transfer reactions from so-called photoacids,^[4] on the other hand, allows for repeatedly monitoring one individual reaction thus giving access to the influence of the local environment.

Kunihiko Ishii^1,2, Miyuki Sakaguchi¹, Tahei Tahara^1,2

¹Molecular Spectroscopy Laboratory, RIKEN
²RIKEN Center for Advanced Photonics

We have developed a new method that applies independent component analysis (ICA) to single-molecule fluorescence data for resolving overlapped signals from multiple species in the sample. ICA is a data processing method used in combination with multichannel signal detection to decompose an overlapped signal into independent components by examining the high-order correlations of the signal fluctuations. The developed method, which we name independent fluorescence component analysis (IFCA), has advantages over existing methods. First, it is model-free and does not rely on fitting to an arbitrary theoretical model. Second, it can be applied to a relatively highly concentrated sample. Lastly, the analysis result does not depend on the temporal binning width of the photon data, so that IFCA can be applied to study transient species with a lifetime of microseconds. In order to realize IFCA, we developed a numerical procedure to analyze the third-order correlation tensor of multichannel photon signals. The photon signal was obtained with a time-resolved smFRET setup which utilizes the time-correlated single photon counting and the pulsed interleaved excitation scheme. We will demonstrate the potential of IFCA by applying the developed procedure to photon data obtained from FRET-labeled DNA oligonucleotides.

Johan Hummert, Klaus Yserentant, Theresa Fink, Dirk-Peter Herten

Herten Lab, Dept. of Physical Chemistry, Heidelberg University, Germany

Quantitative fluorescence microscopy offers unique possibilities to investigate complex protein assemblies, such as nuclear pore complexes (NPCs) or receptor nanoclusters in the immune response of T-cells in situ. However, these two examples are representative for two very different sets of challenges. While many NPC copies with a well-defined structure are present in a single cell, the formation of receptor nanoclusters is subject to stochastic variation.

We explore the limits of fluorescence microscopy methods for either situation by combining (i) different methods for molecular counting (ii) a newly developed method to determine the degree of labelling and (iii) bayesian statistical analysis and Monte-Carlo simulations. The experimental methods investigated in the scope of this work are counting by photon statistics (CoPS), by emission intensity, and by a newly developed photo bleach step analysis.

We evaluate the different quantification methods, as well as our theoretical framework, on targets with known and unknown stoichiometry. This combination enables us to define the requirements to ascertain statistically significant information on protein copy numbers. The evidence shows that a high and well-known degree of labelling is crucial especially in the case of complexes with variable stoichiometry.

Jean Comtet, Evgenii Glushkov, Vytautas Navikas, Jiandong Feng, Aleksandra Radenovic

Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, 1015 Lausanne, Switzerland, Aleksandra.radenovic@epfl.ch

In this talk, I will detail our strategy on how to translate nanoscopy techniques into the field of materials science. We have developed and applied different modalities of nanoscopy techniques that provide unique insights about the type and density of defects together with the spectral characterization (1) at locations determined with nanometre-scale precision (2). We focus on defects hosted in two classes of 2D materials: hexagonal boron nitride (h-BN) (3) and transition metal dichalcogenides (TMDs), such as MoS2, WS2, MoSe2, WSe2, and MoTe2. Defects hosted in 2D materials such as h-BN and TMDs are particularly interesting due to their single photon emission. SP emitters (3) are stable concerning transfer onto other substrates, opening the possibility of integrating them into more complex nanophotonic devices and paving the way for future semiconductor quantum information processing technologies.

Transmission electron microscopy and scanning probe microscopy can provide atomic resolution. However, both techniques require strict sample preparation protocols and are not optimal for fast in-situ operation or applications requiring the characterization of large areas. In contrast, Nanoscopy can operate in –situ under ambient conditions and is compatible with the probing of defect chemistry and dynamics in different pH environments and under different solvents (2). We also demonstrated high-content characterization of 2D materials using silicon nitride waveguides as imaging platforms that allow integration of more complex nanophotonic circuits.

Rudolf Rigler

Karolinska Institutet, Stockholm rudolf.rigler@ki.se

I will present a recollection of the time spanning from the first PicoQuant Meeting in 1995 where we demonstrated the detection of single molecules in aqueous solution at room temperature in the wide field (Dapprich et al.) until now.

Fluctuations of single molecules,scattering or emitting photons can be analysed by correlation spectroscopy and a variety of examples have been demonstrated (Rigler & Elson,2001). A more recent summay is found in (Rigler, 2010)

I will treat the ergodicity of single molecule average and ensemble averages. Analysis of single molecule catalysis exemplified by horse raddish peroxidase exhibits a complex rate matrix in substrate turnover. Using higher order correlation functions (Edman & Rigler,2000) we are able to demonstrate the existences of (non Markovian) memory states and their time dependence. Enzymes apparently can switch from a resting stage with exponential kinetics with NMF equals 0 to power law kinetics with time dependent NMF (non Markov function). Details of this analysis will be demonstrated and its result will be discussed. New data on DNA polymerase In their action of replicating DΝΑ strands will be shown.

Widengren Jerker, Tornmalm Johan, Joachim Piguet, Elin Sandberg

Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology (KTH), Albanova Univ. Center 106 91 Stockholm, Sweden

In transient state imaging (TRAST), blinking kinetics of fluorescent molecules in a sample are determined from how the time-averaged fluorescence intensity varies upon differently modulated laser excitation [1,2], without requiring time-resolved or single-molecule detection conditions. Transient dark states, such as triplet, photo-isomerized and photo-oxidized/reduced states, are long-lived (µs-ms), and therefore sensitive to the local environment. Via the kinetics of these states additional information can thus be provided, beyond that from regular fluorescence parameters. Here, we report on two categories of applications, where TRAST can offer such additional information.

First, TRAST was used to monitor transient, low-frequency interactions between proteins (GPCRs) and lipids in live cell membranes [3], via the quenching of long-lived fluorophore triplet states by spin labels (labeled to lipids and the GPCRs, respectively). These interactions are too infrequent to occur within the fluorescence lifetime of fluorophores, and to be readily followed by fluorescence quenching.

Second, we imaged metabolic states of cells via dark state transitions in NAD(P)H using laser-scanning confocal microscopy and two-photon excitation, in parallel with regular fluorescence lifetime imaging [4].

These studies suggest that imaging of highly environment-sensitive dark states can provide useful biological information beyond traditional fluorescence readouts, and is widely applicable.

Don C. Lamb

LMU Munich, Department of Chemistry, Butenandtstrasse 5-13, 81377 München

Based upon the idea of Alternating Laser EXcitation (ALEX) in Kapanidis et al1, Pulsed Interleaved Excitation2(PIE) was developed. Both ALEX and PIE has proven to be a very powerful methods for single-pair Förster Resonance Energy Transfer (spFRET) experiments. The fast alternating of pulses on the nanosecond timescale for nanosecond ALEX (nsALEX)3and PIE has advantages for fluorescence correlation spectroscopy (FCS) measurements where spectral cross-talk can be completely removed or the influence of FRET within a construct can be compensated for. We have also combined PIE with several correlation methods including Raster Image Correlation Spectroscopy (RICS) and Number and Brightness analysis4. However, one of the real strengths of PIE/nsALEX is the fluorescence lifetime information. In this presentation, I will give a brief introduction into PIE, provide an overview of its development and discuss some of its more recent applications. In the later part, I will stress the additional capabilities and advantages that the lifetime information provides.  

Abhinaya Anandamurugan¹, Philipp Wortmann¹, Aprile Garcia Fernando², Ritwick Sawarkar², Ben Schuler³, Thorsten Hugel¹

¹Institute of Physical Chemistry, University of Freiburg, Albertstr. 23a, 79104 Freiburg, Germany
²Max-Planck-Institute for Immunobiology and Epigenetics, Stübeweg 51, 79108 Freiburg, Germany
³Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

Quantifying dynamics in their native milieu provides an exciting alternative to in vitro methodologies in understanding the complexity of macromolecular interactions. The Hsp90 machinery, with two distinct major conformational states provides a good system to re-examine conformational changes in vivo using a modified TIRF (namely HILO - Highly inclined Laminar Optical Light sheet) and smFRET approach.
Here we developed a combinatorial approach of using smFRET with HILO, where smFRET provides an edge over ensemble measurements in distinguishing global state changes with nanometer precision. HILO microscopy enables us to detect dynamics of site-specifically labelled molecules within the timescale of milliseconds to seconds within the cell. The bottleneck in studying externally labelled protein molecules are limited by transfection efficiency, background introduced by the selective method and molecular properties of fluorescent dyes used for the experiment. Furthermore, molecules are in Brownian movement when introduced into cells which poses an additional challenge as single molecules have to be trapped or tracked.
First single molecule fluorescence traces with DNA and with Hsp90 demonstrate the high potential of our approach. We anticipate that the results of our study can be extended to other molecular machineries operating within similar timescales.

Ashwin Balakrishnan¹, Jan-Hagen Krohn¹, Susobhan Choudhury¹, Katherina Hemmen¹, Mike Friedrich¹, Julia Wagner¹, Martin Lohse², Katrin Heinze¹

¹Rudolf Virchow Center, University of Würzburg, Josef-Schneider-Str 2, 97080 Würzburg, Germany
²Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str 10, 13125 Berlin, Germany

G-protein coupled receptors encompass the largest superfamily of membrane receptors. They transduce ligand signals to downstream effects thereby mediating complex cell responses, in turn making them prominent drug targets and a major focus of functional studies. Their activation dynamics in particular has been subject to many biophysical and structural studies leading to the hypothesis that adrenergic receptors could involve multiple intermediate steps occurring in the ms to μs timescales [1, 2, 3]. In this work, we probe dynamics in these timescales in the β₂-Adrenergic Receptor (β₂-AR) using Fluorescent Correlation Spectroscopy (FCS) and Time-resolved Anisotropy in live cells. β₂-AR constructs conjugated to fluorescent protein and organic dyes let us understand the difference in dynamics of the fluorophore as opposed to the relevant receptor dynamics. Our results show the presence of diffusion times arising from membrane bound receptors and from inside the cell helping us to understand the strengths and weaknesses of each method. Our aim now is to further investigate micro- and nanosecond dynamics using continuous wave (cw)-FCS and perform measurements in the presence of agonists and antagonists, in essence gaining knowledge about dynamic effects imparted by different fluorophores and the fast dynamics of the protein in tandem.

Aleksandr Barulin, Jean-Benoit Claude, Satyajit Patra, Jerome Wenger

Aix Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, 52 Avenue Escadrille Normandie Niemen, 13013 Marseille, France

Label free single protein detection would allow monitoring molecular dynamics without the issues related to external fluorescent labelling. The naturally fluorescent aminoacids tryptophan and tyrosine present in a vast majority of proteins can be excited and detected in the 260-350 nm UV range. However, these fluorescent molecules feature low absorption cross section, low quantum yield and limited photostability in the deep UV, that currently hinders protein autofluorescence detection at the single molecule level. On the route towards label free single protein autofluorescence analysis, we developed a time-resolved UV confocal microscope featuring 266 nm pulsed laser excitation and 300-400 nm fluorescence detection for TCSPC and FCS analysis. In this contribution, we report the influence of oxygen scavengers and antifading agents to improve the photostability of proteins and single tryptophan molecules. To further increase the fluorescence brightness, we detail the use of aluminum zero-mode waveguides (ZMW) taking advantage of nanophotonic fluorescence enhancement. However, focusing UV light on aluminum ZMW resulted in unexpected accelerated aluminum corrosion with water buffers. We discuss several approaches to overcome this issue. Altogether, this work explores new directions towards label-free single protein detection.

Clara Bodner¹, Jonathan Javitch², Wesley Asher², Gerhard Schütz¹, Mario Brameshuber¹

¹Institute of Applied Physics, TU Wien, Vienna, Austria
²Department of Pharmacology, Columbia University, New York

In membrane science, the stoichiometry of cell-surface proteins is fundamental to cellular signalling and function. Many membrane receptors including G-protein-coupled-receptors (GPCRs) have been proposed to form dimeric or higher order oligomeric complexes in order to achieve certain functional states. We present single-molecule studies of three GPCRs: the metabotropic glutamate receptor, the secretin receptor and the μ-opioid receptor. Results based on their dimerization will be used in future experiments to characterize the unknown oligomeric state of the Dopamine receptor D2s [1], which mediates the physiological function of the neurotransmitter dopamine. Abnormalities in dopaminergic neurotransmission are associated with various neurodegenerative disorders, e.g.Parkinson's disease. Previous results suggested that D2s forms dimeric or higher-order oligomeric complexes with distinctive signaling profiles and functions.

In order to characterize GPCRs at physiologically high surface densities, we utilized the in-house developed single-molecule method TOCCSL (thinning out clusters while conserving stoichiometry of labeling)[2]. In a subregion of the plasma membrane the surface density of fluorophores is diluted by photobleaching and subsequent imaging at the onset of the recovery process. A two-color TOCCSL approach [3] was utilized in order to identify the subunit stoichiometry of the receptors in live cell membranes based on co-localization analysis of the two different fluorophores. 

Richard Börner¹, Melodie C.A.S. Hadzic¹, Fabio D. Steffen¹, Danny Kowerko², Sebastian L.B. König¹, Susann Zelger-Paulus¹, Marc Ritter³, Roland K.O. Sigel¹

¹Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
²Department of Informatics, TU Chemnitz, Straße der Nationen 62, 09111 Chemnitz, Germany
³University of Applied Sciences Mittweida, Technikumplatz 17, 09648 Mittweida, Germany

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique to probe biomolecular structure and dynamics. A popular implementation of smFRET consists in recording fluorescence intensity time traces of surface- or vesicle immobilized, chromophore-tagged molecules, such as nucleic acids or proteins [1].

We developed MASH-FRET, a MATLAB-based Multifunctional Analysis Software for Handling smFRET data that allows to analyze and simulate camera-based single-molecule videos (SMV) [2-6]. In brief, our software extracts fluorescence trajectories from SMVs, allows sorting the molecules according to their dynamics and photophysics and analyzes the resulting FRET or fluorescence intensity state populations both thermodynamically and kinetically [2,4]. To validate experimental distributions of FRET states and their interconversion rates, MASH-FRET additionally allows the user to simulate realistic SMV [3]. The software is freely available for download on GitHub at https://github.com/RNA-FRETools/MASH-FRET and documented with the help of step-by-step tutorials that are available at https://rna-fretools.github.io/MASH-FRET/.

Here, we provide a presentation of our software package with a standard analyzing strategy for SMV. We further explain the basic concepts of smFRET and how to get the most out of your intensity-based smFRET data in terms of thermodynamics and kinetics.

Financial support by the Swiss National Science Foundation (RKOS), the UZH Forschungskredit (MCASH, DK, FDS, SLBK, SZP and RB) and the University of Zurich (RB and RKOS) is acknowledged.

Joshua Botha¹, Tjaart Krüger¹, Rienk van Grondelle²

¹Department of Physics, Faculty of Natural and Agricultural Sciences, University of Pretoria, Private bag X20, Hatfield 0028, South Africa
²Department of Physics and Astronomy, Faculty of Sciences, VU University, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands

Photosynthetic organisms have developed to regulate light harvesting by switching photoprotection on and off. We have shown that LHCII, the main light-harvesting complex of plants, exploit fluorescence blinking for photoprotection by controlling the dynamic equilibrium between on and off states. The blinking dynamics of these complexes do not follow a binary on-off behaviour but exhibit numerous intermediate intensity levels, some of which are accessed reversibly and some irreversibly. The intermediate levels may also be related to photoprotection. We used an agglomerative hierarchical clustering algorithm to show that the intermediate intensity levels represent a statistically significant population of the accessed fluorescence states of single, isolated LHCII antennae and their possible importance is argued. We found that the occurrence of reversibly accessed intermediates states decreases when the in vivo conditions establishing photoprotection are mimicked, whereas the occurrence of irreversibly accessed intermediates increases. The former behaviour suggests that the photoprotective state of LHCII involves decreased conformational disorder, whereas the latter behaviour suggests that partial protein denaturation contributes to establishing the photoprotective state.

Aneesh Chandrasekharan, Santhosh Kumar TR

Rajiv Gandhi Centre for Biotechnology,Bio-Innovation Centre,Kinfra Park, Kazhakoottam, Thiruvanathapuram, Kerala, India

Calcium ions control almost every aspect of cellular life and death decisions.  Imaging of Endoplasmic Reticulum (ER) and Mitochondrial calcium is a key to understand cell cycle and cell death signaling.  Eventhough ER and mitochondrial targeted FRET probes of calcium   showed excellent performance in real-time ratio imaging in confocal and wide-field imaging, their potential utility in determining the minute changes in calcium and calcium heterogeneity within an unperturbed cell is difficult to achieve. Similarly their utilities in 3D tumor models are yet to be described.   We developed cancer cell lines stably expressing calcium probe at ER and mitochondria and developed tumor spheres to study the dynamics of calcium heterogeneity in a growing tumor spheres simultaneous with cell cycle probe.  We show the life time imaging in 3D is much superior in demonstrating the small changes and calcium heterogeneity between cells within a single cell derived clone. The studies using these probes reveal the potential application of life time imaging for calcium heterogeneity detection in growing tumors that is difficult to demonstrate with other methods.

Tao Chen¹, Fengxia Tong², Jörg Enderlein¹, Zhaoke Zheng²

¹III. Institute of Physics–Biophysics, Georg-August- Universität, 37077 Göttingen, Germany
²State Key Laboratory of Crystal Materials, Shandong University, Jinan 250100, China

The catalysis of industrial metals such as Pd, Pt, Ru and Rh enhanced by the localized surface plasmon resonance (LSPR) of the plasmonic metals brings new opportunities to increase the catalytic efficiency and tune catalytic selectivity. Understanding the underlying kinetic mechanism is of importance for bimetal nanostructure design and intelligent utilization of light to tailor the catalytic activity and selectivity. By charactering the product formation and dissociation processes at a single particle level with a single molecule methods, we revealed a variation of the underlying catalytic kinetic mechanisms of a bimetal nanostructure (Au-Pt) and obtained the corresponding kinetic parameters at different light illumination condition. The results show that energetic charge carriers induce the kinetic model from a competitive reactant adsorption type to a noncompetitive adsorption type, which leading to the avoidance of the catalytic rate decay at high reactant concentration. We show the variation of kinetic parameters of the product formation process and product dissociation process as a function of power intensity at different photon energy. This work helps us deeply understand the plasmon-enhanced catalysis of bimetal nanostructures and points us in a direction toward enhancing catalytic activity of the traditional heterogeneous catalysis by introducing plasmonic metals and light.

Eugenia Butkevich¹, Navneet Verma², Ingo Gregor¹, Christoph Schmidt¹, Chayan Nandi², Jörg Enderlein¹, Alexey Chizhik¹

¹Georg-August-University Göttingen, Thirst Institute of Physics, Göttingen, Germany
²School of Basic Sciences, Indian Institute of Technology Mandi, Kamand, Himachal Pradesh 175001, India

We all got used to buying fluorophores from manufacturers that are believed to produce any kind of dye our experiments may ever require. The reverse of the medal is high price, often impossibility of any chemical modification of the dye or even unknown chemical structure, or even improper characterization of the fluorophore’s physico-chemical properties.

In 2004, Scrivens and co-workers accidentally found a way around it, probably even haven’t been realizing it first [1]. They obtained fluorescent carbon-based impurities as a result of purification of carbon nanotubes. The publication was followed by a tsunami of works, where researchers reported on cheap and simple synthesis of various fluorophores that consisted mostly of carbon nanoparticles and numerous types of surface chemical groups. It turned out that thermal treatment of basically any organic substance leads to generation of fluorescent carbon nanoparticles, which have been often called “carbon dots”.

In recent years, a lot of efforts have been made to understand the mechanism of their fluorescence and to develop more advanced ways of synthesis in order to achieve high monodispersity of particles and homogeneity of their properties. We present new results of our study of this intriguing and promising type of label [2-4].

Anna Chizhik¹, Daja Ruhlandt¹, Ingo Gregor¹, Florian Rehfeldt¹, Ralf Kehlenbach², Andreas Janshoff³, Jörg Enderlein¹, Alexey Chizhik¹

¹Georg-August-University Göttingen, Thirst Institute of Physics, Göttingen, Germany
²Universitätsmedizin Göttingen, University of Göttingen, Department of Molecular Biology, GZMB, Göttingen, Germany
³Institute of Physical Chemistry, University of Göttingen, Göttingen, Germany

Ultra-high resolution fluorescence microscopy is one of the key tools that allowed one to look beyond the diffraction limit in bio-imaging. This allowed for discerning tiny intracellular structures in their natural condition. A simple method that can be used using conventional fluorescence microscopes and that has a capability to reach the resolution of the order of the size of molecular structures is high on the wish list of many researchers.

We introduce the metal-induced energy transfer (MIET) method that allows one to achieve 1 nm axial resolution while keeping the lateral resolution within the diffraction limit [1]. The method is based on modulation of excited state lifetime of a fluorophore by a thin semitransparent metal film deposited on the sample surface. Besides that, one needs just a standard confocal microscope with FLIM extension. This makes MIET accessible to the wide community of life science researchers.

We show our new results on live cell imaging that have been obtained using MIET: three-dimensional profilometry of cell membrane [1], cell-substrate dynamics of the epithelial-to-mesenchymal transition [2]. Using dual-color MIET, we did three-dimensional reconstruction of nuclear envelope architecture [3] and structure of stress fibers anchoring at focal adhesions [4].

Daja Ruhlandt, Jörg Enderlein, Alexey Chizhik

Georg-August-University Göttingen, Third Institute of Physics, Göttingen, Germany

We present new results of absolute quantum yield measurements using a plasmonic nanocavity. The method is based on measurement of excited state lifetime of a fluorophore as a function of the cavity length [1]. Changing the distance between the cavity mirrors modifies the local density of states of the electromagnetic field and thus, the radiative transition rate of the emitters. By modeling the cavity-induced modulation of radiative rate and measuring the total de-excitation rate (that is, excited state lifetime), we determine absolute value of the emitters’ quantum yield.

Using a plasmonic nanocavity, we measure absolute quantum yield of fluorophores in a mixture of different types of fluorophores (dye molecules and semiconductor nanocrystals) [2,3]. We show that quantum yield measurements can be performed in an attoliter volume, both in liquid and solid phases, even if both types of chromophores absorb and emit light in the same spectral range. We show results of measurements of quantum yield of dye molecules placed inside a single supported lipid bilayer [4] or even of a single dye molecule [5].

Michael Isselstein¹, Lei Zhang¹, Viktorija Glembockyte², Philip Tinnefeld², Thorben Cordes¹

¹Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany
²Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, Haus E 81377 München, Germany

Single-molecule fluorescence spectroscopy and super-resolution techniques are widely used tools for studying structural and dynamical properties of (bio)chemical systems with good contrast, high spatial and temporal resolution. Limiting factors in these techniques are both the signal duration and quality, as well as the control over functional properties of the dyes (e.g., photoswitching or specific biolabelling). To achieve optimal performance, the addition of photostabilizing and photoswitching compounds to buffer systems remains the standard procedure within various scientific communities. Recently, the concept of self-healing dyes, which was originally proposed by Lüttke and co-workers in the 1980s[2], has been revived and keeps prospering.[3,4] In self-healing dyes, the covalent linkage of photostabilizers allows quenching of reactive states such as triplets or radicals for improved imaging performance. In this contribution, we review the achievements made over the past years by various groups[3-7] and outline potential routes for improvement. The contribution includes information on our current understanding of the mechanisms underlying self-healing fluorophores and their applications in single-molecule spectroscopy, super-resolution microscopy[7] and general imaging. We also outline still existing limitations of self-healing dyes including photostability efficiency, consequences of environmental effects and influence of solution-based stabilizers[7], and an overview of the currently available bioconjugation chemistry.

Quinten Coucke¹, Guillermo Solis^1,2, Susana Rocha¹, Johan Hofkens¹

¹KU Leuven, Molecular Imaging and Photonics (MIP), Chemistry Dep., Celestijnenlaan 200F, 3001 Heverlee BEL
²Instituto de Salud Carlos III, Carretera de Majadahonda , 28220 Majadahonda, Madrid, ESP

Mechanical forces play an undisputed elementary role in the interactions between cells and the surrounding extracellular matrix (ECM)⁽¹⁾. Not only are these forces essential for the cell migratory behavior, they also influence proliferation (including tumor growth) and differentiation^(2-4). These forces are transferred across focal adhesions (FAs) which connect ECM and cell skeleton through patches of activated integrin proteins. Since the origin of exerted cellular forces lies in these FAs, they are the ideal starting point for characterization of mechanotransduction pathways.

Consequently, studying how the properties of the ECM affect the cellular forces is key in understanding how cells connect to their environment and alter their behavior appropriately.  While the scientific field of cellular mechanosensation has been studied for years, recent developments in imaging techniques and force sensor development enable us to dig deeper.

A home-built confocal Fluorescence Lifetime Imaging Microscopy (FLIM) microscope is used to measure Förster Resonance Energy Transfer (FRET) in FAs. Photon timing data is analyzed with the phasor approach presented by M. A. Digman et al. ⁽⁵⁾.

Nader Al Danaf¹, Berenice Jahn¹, Arjan Pol², Huub Op den Camp², Lena J. Daumann¹, Don C. Lamb¹

¹Ludwig-Maximilians-Universität München, Department Chemie, Butenandtstr. 5-13, 81377 München, Germany.
²Department of Microbiology, Institute of Wetland and Water Research, Radboud University, Nijmegen, The Netherlands

Lately, it has become clear that rare earth elements (REE) are biologically relevant. Certain microbes like the thermoacidiphilic Methylacidiphilum fumariolicum SolV is strictly dependent on REE for its growth.[1] In the metalloenzyme methanol dehydrogenase (MDH), lanthanides are found to be necessarily central metal ions in the active site of the Pyrolloquinoline quinone (PQQ)-containing protein found in the strain  SoIV. The MDH is responsible for oxidizing methanol to formaldehyde and further to formic acid. Although challenging to purify, the first europium(III)-containing MDH derivative was successfully obtained,[2]  to provide a luminescence based method that monitors and investigates the features of the MDH active site. Here, we provide a presently developed assay that utilizes both the phosphorescence of europium(III)[3] and the fluorescence of PQQ.[4] A fluorimeter with the capabilities of time correlated single photon counting was used to study the spectral and temporal properties of the europium(III) and PQQ bound to the MDH active site under different conditions. Eu bound to MDH active site revealed a different phosphorescence behavior compared to free Eu. Furthermore, we validated the presence of PQQ and its proximity to Eu at the active site, which was monitored by the spectral and lifetime changes of both, PQQ and Eu.

Frederike Erb, Kay-E. Gottschalk

Institute of Experimental Physics, Ulm University, Ulm, Germany

Nanodiamond particles offer various new imaging and metrology approaches, especially in the life sciences. Nanodiamonds that contain nitrogen-vacancy centres (NV-centres) emit fluorescent light in the near-infrared window of bioimaging and are thus called fluorescent nanodiamonds (FNDs). As their fluorescence properties depend on the environment, FNDs cannot only be used as labels for bioimaging but also find application as part of various biosensors. A nanodiamond particle can be smaller than 50 nm in diameter and read-out optically in biological samples without contact. As they are also biocompatible and non-cytotoxic, they can be used for experiments in vivo.

We present experiments using the NV-centre in nanodiamond as a detector for magnetic field. Gd³⁺ ions in the surrounding of a nanodiamond introduce magnetic field fluctuations affecting the NV’s spin relaxation time T₁ [1]. Reading-out this T₁-Time with a commercial confocal microscope gives a measure of the Gd³⁺ concentration in the sample.

Julian Folz, Milana Popara, Suren Felekyan, Claus Seidel

Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany

Fluorescence spectroscopy and imaging are important biophysical techniques to study dynamics and function of biomolecules in vitro and in live cells. The use of more than one fluorophore per molecule opens additional opportunities arising from photon densities, coincidences and dipolar coupling by Förster Resonance Energy Transfer (FRET) to study the stoichiometry and structure of biomolecular systems.

Before performing FRET measurements, one needs to know the detection properties of the setup which depends on the photophysical properties of used dyes and characteristics of optical components. In the classical universal calibration via stoichiometry [1] these are mixed in one γ factor. Comparing multiple manifold γ factors leading to same FRET efficiencies raises the question which factor is the correct one. To answer this question and to improve accuracy we calculate the detection efficiencies using emission spectra of used dyes and spectra of every optical component in the setup. We used a rational determination to estimate the calibration factors using DNA samples and compared this to lifetime based analysis. This leads to a unique calibration that is valid for many molecules of interest with distinct conformers that are studied with the same donor/acceptor dye pair combination.

Mattia Fontana^1,2, Mark Roosjen², Simon Lindhoud², Willy van den Berg², Dolf Weijers², Johannes Hohlbein¹

¹Laboratory of Biophysics. Wageningen University & Research. Wageningen, The Netherlands
²Laboratory of Biochemistry. Wageningen University & Research. Wageningen, The Netherlands

Auxin signalling plays a role in regulating almost every aspect of plant growth and development. The transcriptional response to auxin is mediated mainly via proteins belonging to the Auxin Response Factor (ARF) family [1].

Structure-based model of ARF’s DNA-binding-domain (DBD) shows that regulation of target genes requires both protein-DNA interaction as well as protein dimerization [2]; nevertheless information about the dynamics of these interactions is still missing.

We developed fluorescence-based assays to quantify the dynamics of these inter-molecular interactions at the single-molecule level. We used fluorescently labelled dsDNA (~40bp) containing variations of the auxin response element (AuxRE); we then applied techniques such as single-molecule Förster resonance energy transfer (smFRET) alone and in combination with protein induced fluorescence enhancement (smPIFE-FRET [3]) to characterize the binding kinetics of ARF-DNA complexes.

Anna Fucikova

Charles University, Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Ke Karlovu 3, 121 16 Praha 2, Czech Republic

The silicon nanocrystals (Si-NCs) exhibit efficient room-temperature photoluminescence, the quantum yield can reach up to 60 %. The mechanism behind Si-NCs photoluminescence is still not clear today; latest experiments suggest that quantum confinement and its interplay with trap states are the origin of luminescence. The emission of individual Si-NCs is not only influenced by their size, but is also affected by their shape, embedded impurities, surface passivation and the surrounding environment.

The Si-NCs were prepared by one step synthesis. They have Si crystalline core and thin surface shell which is similar in composition to silica ceramics. The individual Si-NCs, studied by single dot spectroscopy, exhibited significantly narrower emission peak at room temperature (lowest linewidth ~ 17 meV) compared to Si-NCs embedded in a silicon oxide shell (150 meV) [1] . These extremely narrow emission peaks are observed for the first time for Si-NCs at room temperature and are even narrower than that of single CdSe quantum dots (>50 meV). The luminescence from produced nanocrystals covers a broad spectral range from 530-720 nm (1.7-2.3 eV) suggesting strong application potential in for solar cells and LEDs or as a florescent marker in biology.

The properties of these nanoparticles have been studied with our micro-spectroscopy combined with AFM setup allowing us to study mechano-optical properties of individual nanoparticles. In the future we would like to use this setup to study influence of organic molecules on individual nanoparticle mechano-optic properties.

Subhabrata Ghosh¹, Anna M. Chizhik¹, Gaoling Yang², Narain Karedla³, Ingo Gregor¹, Dan Oron², Shimon Weiss^3,4,5,6, Jöerg Enderlein¹, Alexey I. Chizhik¹

¹Third Institute of Physics, Georg August University Göttingen, Göttingen 37077, Germany
²Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot 76100, Israel
³Department of Physics, Institute for Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 52900, Israel
⁴Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California 90095, United States
⁵California NanoSystems Institute, University of California Los Angeles, Los Angeles, California 90095, United States
⁶Department of Physiology, University of California Los Angeles, Los Angeles, California 90095, United States

Over the last few decades, semiconductor nanocrystals attract considerable attention due to their tunable photophysical properties. Generation and recombination of excitons in semiconductor quantum dots play an important role to understand their photophysics. While many studies have been focused on type-I semiconductor nanocrystals, the photophysics of type-II nanorods, where the hole is located in the core and the electron is located in the shell of the nanorod, remain largely unexplored. Such nanoparticles are of great interest for local voltage sensing at the nanometer scale.

We present the results of measurements of the dimensionality and orientation of both emission and excitation transition dipoles of single type-II nanorods [1]. Dimensionality of excitation pattern of an individual nanorod is obtained by scanning the single nanorod through the focal region of an azimuthally or radially polarized laser beam. The emission transition dipole of the same single nanorod is measured by defocused imaging technique. By comparing the measured excitation and emission patterns with the theoretical model, we unambiguously determine the dimensionality and orientation of single type-II semiconductor nanorods. The results show that in contrast to previously studied quantum emitters, the particles possess a 3D degenerate excitation and a fixed linear emission transition dipole.

Joelle Goulding^1,4, Alexander Kondrashov^2,4, Sarah Mistry^3,4, Nurul Yusof^2,4, Nguyen Thi Ngoc Vo^2,4, Chris Denning^2,4, Stephen Briddon^1,4, Stephen Hill^1,4

¹Division of Physiology, Pharmacology & Neuroscience, School of Life Sciences, University of Nottingham, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK
²School of Medicine, University of Nottingham, University Park, Nottingham NG7 2QR, UK
³School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2QR, UK
⁴Centre of Membrane and Protein and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK.

Fluorescence correlation spectroscopy (FCS) is an ideal technique to study membrane protein receptor dynamics (expression level and diffusion speed) in endogenous systems in which expression levels are significantly lower than can be detected by standard imaging techniques [1]. The Beta2-adrenergic receptor (B2AR) is a clinically relevant G protein-coupled receptor which displays two non-synonymous single nucleotide polymorphisms (SNPs) within its N-terminal region capable of altering its regulation [2]. Characterisation of SNP specific effects on membrane protein organisation and regulation could lead to pharmacogenetic targeting of the B2AR within respiratory and cardiovascular diseases.

We created overexpressed (incorporating an N-terminal Snap-tagged B2AR) and CRISPR engineered (HUES7) stem cell lines, which displayed one of the 4 potential haplotypes that arise from SNPs at amino acid positions 16 (Glyine/Arginine) and 27 (Glutamate/Glutamic acid). Stem cell lines were differentiated into both fibroblasts and cardiomyocytes and used for FCS studies utilising either a SNAP-Alexafluor-488 (overexpressed lines) or a novel B2AR selective fluorescent ligand (CRISPR lines) to detect the B2AR. FCS readings were taken on a Zeiss LSM510NLO Confocor 3 microscope.

We were able to quantify receptor expression level and diffusion speed of the B2AR in both differentiated cell types allowing haplotype comparison at very low expression levels.

Lennart Grabenhorst¹, Birka Lalkens², Philip Tinnefeld¹

¹Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 München, Germany
²Laboratory for Emerging Nanometrology, Technische Universität Braunschweig, Langer Kamp 6 a/b, 38106 Braunschweig, Germany

Förster resonance energy transfer (FRET) is the method of choice for the vast majority of experiments probing dynamic changes of biomolecules such as proteins or nucleic acids. Its sensitivity for distance changes in the low nanometer range allows for the acquisition of valuable information on conformational changes. When it comes to very fast processes, such as transition paths in folding or binding and unbinding, the achievable photon count rate of available fluorophores is usually the limiting factor for an experiment.

We therefore propose self-assembled plasmonic nanoantennas as a way to increase photostability and photon count rates in single-molecule FRET experiments. We employ the DNA origami technique to selectively place entities into the plasmonic hotspot formed by two gold nanoparticles. In the hotspot, electric field strength and radiative and non-radiative processes are enhanced by severalfold. We show that this enables MHz count rates for several seconds in single-molecule FRET transients. Furthermore, we demonstrate that we can utilize our system to probe conformational changes of biomolecules on the microsecond timescale.

Mathias Hammer¹, Alessandro Rigano¹, Farzin Farzam¹, Maximiliaan Huisman¹, Koray Kirli¹, Carlas Smith², Burak Alver¹, Caterina Strambio-De-Castilla¹, David Grunwald¹

¹UMass Medical School, 368 Plantation Street, Worcester, MA 01605
²TU Delft, Mekelweg 5, 2628 CD Delft, Netherlands

Adequate recordkeeping is essential for most experiments as it is necessary in order for the results to be evaluated, data to be shared and reused, and experiments to be repeated. Keeping notes on microscopy experiments should be relatively unchallenging in this regard, as the microscope is a machine equipped with a limited number of known parts and settings. Nevertheless, to this date no widely adopted set of metadata descriptors to be recorded or published with imaging data exists. Metadata automatically recorded by microscopes from different companies vary widely and pose a substantial challenge for microscope users to create a good faith record of their work. Similarly, the complexity and aim of experiments using microscopes varies leading to different reporting requirements from the simple description of a sample to the need to document the complexities of sub-diffraction resolution imaging in living cells and beyond.

Here we present a tiered system of guidelines for describing and documenting microscopy experiments developed by the 4DN Imaging Standards Working Group, a comprehensive list of metadata key-value pairs that should be recorded for each tier and a detailed explanation of why these values matter.

Anoushka Handa¹, Edita Bulovaite², Alexander R. Carr¹, Seth Grant², Steven F. Lee¹

¹Department of Chemistry, Lensfield Rd, Cambridge CB2 1EW
²Centre for Clinical Brain Sciences, Chancellor's Building, Edinburgh BioQuarter, 49 Little France Crescent, Edinburgh EH16 4SB

Quantitative imaging in complex biological samples such as brain tissue requires techniques such as super-resolution imaging to better understand the morphology and stoichiometry of proteins, specifically synaptic proteins below the diffraction limit. The 3D double helix-point spread function (DH-PSF) is a technique which has an increased depth of field (~4 μm) and is capable of isotropic resolutions of 25 nm. This makes it highly compatible to investigate sub-synaptic diversity in a physiologically relevant environment. Here we demonstrate postsynaptic density 95 (PSD95) genetically expressed to mEos2 imaged in brain tissue using the 3D DH-PSF.

PSD95 is known to form nanoclusters which make up the basic structural unit of an excitatory synapse. We have been able to optimise this imaging technique and have performed cluster analysis on our results to better understand the role of these nanoclusters in different regions of the hippocampus. To do this we have developed quantitative imaging analysis algorithms to compare the role of inter and intra synaptic diversity in the mouse brain during development.

A deeper understanding of how PSD95 nanoclusters form and how mutations occur in these synapses in the hippocampus contributes knowledge to understanding how this can lead to schizophrenia, learning disabilities and autism.

Paul D Harris, Vlad Raducanu, Fahad Rashid, Hubert Piwonski, Samir Hamdan, Satoshi Habuchi

4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia

The dynamics of single stranded breaks are of great interest for structural DNA nucleases, as many of them bind DNA in a highly bent conformation. In order to obtain the highest time resolution possible, we applied the photon by photon Hidden Markov algorithm of Pirchi for microsecond resolution dynamics of our FRET labeled DNA substrates. We confirmed our ability to detect transitions with Holiday junction, and then investigated our DNA structures of interest. Under a range of magnesium concentrations, nicked and 1 nucleotide gap, and a 6 nucleotide flap DNA showed were best fit to two state models where one state corresponded to bleached or missing acceptor fluorophore. The other state, reflective of the conformational state of the DNA constructs corresponded to an extended conformation of the DNA, nowhere close to the bending seen in nucleases like FEN1. Backed up by previous single molecule and computational work, we conclude that in the absence of protein like FEN1 DNA structures with single strand breaks have very small structural fluctuation remaining in a largely duplex like configurations, due to the strong entropic cost of breaking the stacking between nucleobases.

Stefano Stella², Pablo Mesa², Johannes Thomsen¹, Bijoya Paul², Simon B Jensen¹, Bhargav Saligram², Matias E Moses¹, Guillermo Montoya², Nikos S Hatzakis^1,2

¹Department of Chemistry & Nanoscience Centre, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark
²Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences

Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks(1, 2). Here, we combined cryoEM structures and single molecule FRET to provide a complete mechanistic understanding of endonuclease structural dynamics role in function and resetting(3). Cryo-EM readout provided the structures of intermediates of the cleavage reaction, and identified protein regions that sense the crRNA-DNA hybrid assembly triggering catalytic activation. Combined with our single molecule readout (4-7)and specifically smFRET allowed us to directly observe the protein conformational dynamics along the entire reaction pathway. Parallel single molecule imaging provided the directionality of conformational transitions as well as the complete thermodynamic and kinetic characterisation of the conformational activation leading to function. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity degrading ssDNA molecules after activation and how other crRNAs displace the R-loop inside the protein after target DNA cleavage terminating indiscriminate ssDNA degradation. We proposed a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will recycle Cas12a specifically targeting new DNAs.

Maria Hoyer¹, Alvaro H Crevenna², Radoslav Kitel³, Kherim Willems⁴, Don C Lamb¹

¹Department of Chemistry, Center for NanoScience, Nanosystems Initiative Munich (NIM) and Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians University Munich, Munich, Germany
²Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
³Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland
⁴imec, Kapeldreef 75, B-3001 Leuven, Belgium

Actin filament dynamics underlie key cellular processes. Although actin filament elongation has been extensively studied, the mechanism of filament nucleation remains unclear. High background from the micromolar concentrations needed for filament formation have prevented direct observation of nucleation dynamics using conventional methods. To overcome this limitation, we have used the attoliter excitation volume of zero-mode waveguides (ZMW) to directly monitor the early steps of filament assembly.

Here, we present data obtained from ZMW measurements, investigating the binding of individual molecules to form filaments.  Additionally, we show analysis tools for this type of single-filament data. We used the results to determine mechanistic differences between the nucleation processes of gelsolin, formin and spire.

Kristina Hübner¹, Mauricio Pilo-Pais², Florian Selbach¹, Tim Liedl³, Philip Tinnefeld¹, Fernando Stefani^4,5, Guillermo Acuna²

¹Department of Chemistry and Center for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Butenandtstr. 5-13 Haus E, 81377 München, Germany
²Department of Physics, Photonic Nanosystems, University of Fribourg, Chemin du Musée 3, 1700 Fribourg, Switzerland
³Faculty of Physics, Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, 80539 München, Germany
⁴Center for Bionanoscience Research (CIBION, CONICET), National Scientific and Technical Research Council (CONICET), Godoy Cruz, 2390 Ciudad Autonoma de Buenos Aires, Argentina
⁵Department of Physics, Faculty of Exact and Natural Sciences (FCEN), University of Buenos Aires, Güiraldes, 2620 Ciudad Autonoma de Buenos Aires, Argentina

Optical antennas (OA) are able to control light at the nanoscale and manipulate the photophysical properties of single photon emitters.¹ OAs can focus freely propagating light in the antennas hotspot or conversely direct the emission of fluorescent dyes placed in the hotspot.² Approaches for OA fabrication by lithography techniques faced the difficulty to position single emitters at the near field of OAs.³ The DNA origami approach⁴ enabled a self-assembly of colloidal nanoparticles onto a DNA oriagmi structure and the positioning of single emitters with nanometer precision and stoichiometric control.

We study how an OA can mediate the emission of a single molecule tailoring the emission pattern. We self-assemble OAs based on two gold nanoparticles and a single fluorophore positioned in the hotspot using the DNA origami technique. The emission pattern of a fluorophore coupled to a dimer structure can be visualized by defocused wide-field fluorescence imaging⁵. The obtained pattern resembles that of a fixed dipole and indicates that the fluorophores emission is dominated by the coupling to the OA. Additionally rotating the excitation polarization proofs that both exciation and emission are co-aligned. This work is the basis for experiments to manipulate the emission of single molecules based on self-assembled OA.

Maximiliaan Huisman¹, Carlas Smith², David Grunwald³

¹UMass Medical School, 368 Plantation Street, Worcester, MA 01605
²TU Delft Mekelweg 5, 2628 CD Delft, Netherlands
³UMass Medical School, 368 Plantation Street, Worcester, MA 01605

Abstract: Fluorescence microscopy has become a more and more sensitive and versatile tool for many branches of science, thanks to many advances in fluorescent labelling as well as microscope technology and image processing. As we continue to push the limits of what is technically possible, the quality of data obtained through fluorescence microscopy is increasingly determined by factors that are often not be readily visible in the image: the image acquisition settings, microscope properties and data-processing steps often contribute significantly to the experimental outcome and therefore need to be known and understood for proper interpretation and comparison.

Accurate metadata collection and optical calibration of the microscope go a long way towards allowing imaging data to be properly evaluated and compared; however, there are certain crucial pieces of information that simply are not captured in even the most rigorous and precise routines for record-keeping and calibration, as they simply cannot be measured without the aid of (often costly, cumbersome and complicated) external devices. Here, we present an inexpensive, easy-to-use calibration device that, among other things, allows the user to measure excitation power and perform basic detector calibration routines. In doing so, the “MetaMax” tool provides crucial meta-data to evaluate potential photo-toxicity and allows current and future model-based data processing tools to get as much quantitative information as possible out of the images.

Ilanila IlangumaranPonmalar¹, Ramesh Cheerla², KGanapathy Ayappa^1,2, JaydeepKumar Basu³

¹Center for Biosystems Science and Engineering, Indian Institute of Science, Bengaluru-560012, India
²Department of Chemical Engineering, Indian Institute of Science, Bengaluru-560012, India
³Center for Physics, Indian Institute of Science, Bengaluru-560012, India

Listeriolysin O (LLO) is a pore forming protein that forms pore by binding to cholesterol, followed by oligomerization and insertion inside the membrane bilayer[1]. Studies also suggest that LLO transitions through an inactive intermediate pre-pore state[2]. Although LLO has been widely studied, there is very little information that connects its implication on membrane lipid dynamics during pore formation[3]. LLO induced dye leakage of Giant Unilamellar Vesicles (GUVs), revealed two distinct population of vesicles: leaked and unleaked. Interestingly, LLO preferentially binds to the L_D region of GUVs.  FRET between Alexa488-tagged-LLO and DiI-labelled-lipid was used to observe pore states in leaked vesicles whereas it was rarely observed in unleaked vesicles. Interestingly, lipid diffusivities as measured from FCS, also showed corresponding difference between leaked and unleaked vesicles. Leaked vesicles demonstrated enhanced lipid diffusivity in comparison to the unleaked vesicles. These results are attributed to the different structural changes that happen during the pore formation by linking with the FRET data. All-atom molecular dynamic simulations on lipid bilayers in the presence of LLO monomer revealed a strong correlation between cholesterol bound to LLO and lowered diffusivities. Based on our results, lipid dynamics can potentially be used as a marker to distinguish between oligomeric states.

Sebastian Isbaner, Roman Tsukanov, Narain Karedla, Arindam Ghosh, Jan Christoph Thiele, Ingo Gregor, Jörg Enderlein

III. Institute of Physics, University of Goettingen, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany

Superresolution microscopy today is able to resolve structures on the order of a few nanometers, far below the optical diffraction limit. In particular, single-molecule localization methods are used routinely to resolve intricate biomolecular structures. However, most of these techniques are limited to the sample plane and offer no or limited resolution along the axial direction. Single molecule Metal-Induced Energy Transfer (MIET) is able to localize several emitters along the optical axis with nanometer precision [1]. However, this works for a small number of emitters only because step-wise bleaching was previously used to distinguish between emitters. Here, we use the transient binding of short fluorescently labeled oligonucleotides to the target structure (DNA-PAINT). This allows to collect an in principle unlimited amount of photons and simplifies the data analysis for MIET. Since DNA-PAINT is commonly used for lateral superresolution imaging, the combination with MIET is a step towards an isotropic nanometer localization accuracy.

Hongje Jang, Soheil Mojiri, Steffen Mühle, Sebastian Isbaner, Ingo Gregor, Jörg Enderlein

III. Institute of Physics – Biophysics, Georg August University, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany

We have present advanced three-dimensional image deconvolution methods for microscopy data recorded with our recently developed multiplane 3D microscope [1]. Because the microscope is an extension of a conventional wide-field microscope, three-dimensional reconstruction of the sample from the data is highly non-trivial, due to the well-known missing cone of spatial frequencies in the Optical Transfer Function. Moreover, we are not only interested in a faithful three-dimensional reconstruction of a sample, but also in tracking fast sample motions. Thus, the problem is a four-dimensional in nature: using deconvolution of time-lapse data for obtaining the three-dimensional temporal evolution of a sample. We have adopted and tested several approaches to deconvolution, including standard Richardson-Lucy deconvolution, but also some recent methods such as SPIDER [2], and have applied these techniques to data recorded with our rapid multi-plane 3D microscope. We present two biological examples where we used out technique to follow fast motion in 3D: following the rapid actin dynamics in and cell morphology changes of chemotactically stimulated cells of the slime mold Dictyostelium discoideum, and following the rapid three-dimensional beating of demembrated cilia (bare axonemes).

Ija Jusuk, Mario Raab, Andrés Vera Gómez, Philip Tinnefeld

Department Chemie, Ludwig Maximilian University of Munich, Butenandtstr. 5 – 13, 81377 Munich

Fluorescence microscopy and spectroscopy on the single molecule level is a powerful method revealing informational details of complex biological systems that remain hidden in the ensemble measurements due to the averaging effects [1]. On the other hand, because of the diffraction-limited optics, samples are diluted to lower nanomolar concentrations to enable single molecule detection. Thus, bimolecular reactions that only occur to an increasing degree at micromolar concentrations cannot be visualized. One of the prominent approaches that overcome this concentration barrier is the so-called “single-molecule real-time sequencing” technique (SMRT) [2]. It is based on observing a single DNA polymerase enzyme during the synthesis of DNA by incorporation of fluorescently labeled deoxyribonucleotides (dNTPs). In order to discriminate single-molecule incorporating events from freely diffusing dNTPs, nanometric apertures, so-called zero-mode wave guides are applied, to create a confined optical observation volume, thus significantly reducing the fluorescence background.

Here, we present a novel strategy to break the concentration barrier in SMRT. Based on the DNA origami nanotechnology we developed a plasmonic nanoantenna [3], which is capable of high fluorescence enhancement between two metallic nanoparticles and nanometer precise positioning of single enzyme molecules in this antenna hotspot. We present the first steps towards single-enzyme assays in plasmonic hotspots.

Aditya Katti, Joerg Enderlein

Third Institute of Physics, University of Goettingen, Friedrich-Hund-Platz 1, 37077 Goettingen, Germany

FCS is a technique used to measure diffusion coefficients of fluorescently labeled molecules in nanomolar and picomolar concentrations. Fluorescence fluctuations as and when fluorophores move in and out of the observation volume are recorded and correlated to find the translational diffusion coefficients. However, in scattering and aberrating media like PAA and agarose, the observation volume is distorted which gives inaccurate values. A more robust method to measure diffusion coefficients would be to measure rotational diffusion coefficients. FCS with polarized detection is advantageous in this case as the measurements are dependent on the polarization of the emitted photons and not the shape of the observation volume.[1] Flat gels of PAA with different monomer and cross-linker concentrations were prepared with enhanced green fluorescent protein (EGFP) in the solution. Fluorescence anisotropy is a method used when the rotational diffusion time and fluorescence emission lifetime of the fluorophore are comparable. Using beads filled with Europium (Eu³⁺) chelate ions, which have a long lifetime in a bead of suitable size, anisotropy measurements are performed determining the rotational diffusion time of the beads. Dark-field microscopy is helpful as it circumvents all issues associated with fluorescence measurements as scattered light is used as the information. Using gold nanorods of suitable sizes, dark field illuminated defocused images can be used to analyze rotation of these nanorods using a high-speed camera and hence rotational diffusion time can be measured. [2,3] All these techniques can provide an insight into measurement of pressure indirectly using rotational diffusion time as a measure. [4]

Xenia Knigge^1,2, Christian Wenger^3,4, Frank F. Bier², Ralph Hölzel^1,5

¹Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics, and Bioprocesses (IZI-BB), Potsdam, Germany.
²Universität Potsdam, Germany
³IHP - Leibniz Institute for Innovative Microelectronics, Frankfurt (Oder), Germany
⁴Brandenburg Medical School Theodor Fontane, Neuruppin, Germany
⁵Freie Universität Berlin, Germany

One goal in bioanalysis is the use of individually addressable biomolecules as bioreceptors in biosensors, as this would result in the highest possible sensitivity and a maximum level of miniaturization. These bioreceptor molecules need to be immobilized, so it would be helpful to find a way to immobilize individual molecules in a highly parallel manner. A promising approach is the use of regular arrays of nanoelectrodes that are activated by alternating currents. The sharp tips of these electrodes produce strongly inhomogeneous electric fields that lead to an immobilization of dissolved molecules on top of these electrodes without any chemical modifications. The underlying phenomenon – dielectrophoresis – can be controlled by proper choice of, e.g., field strength and frequency. These arrays consist of thousands of vertical nanoelectrodes with tip diameters ranging from 500nm down to about 1nm. This system has been optimized by using nanospheres of different sizes as a model system. We have found that immobilization of single objects on each electrode can be ensured by electrodes with tip diameters of half the particle size [1]. This result was transferred to the successful dielectrophoretic immobilization of autofluorescent R-phycoerythrin proteins as few or singles on each electrode.

Julian Koch, Jan-Hendrik Budde, Annemarie Greife, Qijun Ma, Elisabeth Kravets, Nora Steffens, Suren Felekyan, Daniel Degrandi, Klaus Pfeffer, Claus A. M. Seidel

Julian.Koch@hhu.de

While recent studies of liquid-liquid phase behavior often focused on intrinsically disordered proteins, well-structured proteins like murine guanylate binding proteins (mGBPs) can also be capable of phase transition and separation. Here, we characterized the phase separation properties of fluorescently labeled mGBPs in cellulo via FRET, super-resolution and confocal microscopy. Recently, we showed that mGBPs largely interact with each other in vesicle-like structures and at the PVM (parasitophorous vacuole membrane) after infection with the parasite T. gondii. Super-resolution imaging and super-resolution spectroscopy revealed a highly specific, diverging phase behavior between mGBP2, mGBP3 and mGBP7, likely caused by differences in protein sequence and structure. While mGBP7 and mGBP3 form a mixed phase containing minor heterogeneities, mGBP7 becomes encapsulated within the mGBP2 phase. FRET-measurements show close interactions for the mixed mGBP7/mGBP3 phase, but none for the separated mGBP7/mGBP2 phase. Phase separation and dynamics of the miscible and immiscible mGBPs were confirmed by colocalization analysis and FRAP-experiments. FRET-based affinity determination yielded high affinity for the mGBP7-exclusive phase, medium affinity for the mGBP7/mGBP3 mixed phase and none for the separated mGBP7/mGBP2 phases. Apart from extending methodical knowledge, we can conclude that the analyzed mGBP proteins, although not disordered, clearly show hallmarks of liquid-liquid phase behavior.

Sergei Kopanchuk¹, Edijs Vavers^2,3, Santa Veiksina¹, Kadri Ligi¹, Liga Zvejniece², Maija Dambrova^2,3, Ago Rinken¹

¹University of Tartu, Institute of Chemistry, Ravila 14a, 50411, Tartu, Estonia
²Latvian Institute of Organic Synthesis, Aizkraukles Str. 21, Riga, LV-1006, Latvia
³Riga Stradins University, Dzirciema Str. 16, Riga, LV-1007, Latvia

The dynamics of Sigma-1 receptor fluorescent protein conjugate (S1R-YFP) was studied in human ovarian adenocarcinoma (SKOV3) cells. The cells were transfected with S1R-YFP and with fluorescent marker of endoplasmatic reticulum (ER), KDEL-mRFP or marker of mitochondria mito-mKate2 using Bac-Mam technology. Binding of [³H](+)-pentazocine to the membranes of SKOV3 cells with S1R-YFP was with high affinity and could be inhibited with PRE-084.

The expressed S1R-YFP located mainly in nuclear envelope and in tubular structures of endoplasmic reticulum (with utmost abundance in vesicular parts), but not in plasma membranes. The colocalization of S1R-YFP was detected only with KDEL-mRFP. Using SRRF approach with HILO illumination fluorescence microscope displayed that the intensity spots of S1R-YFP and KDEL-RFP have only partial overlap and activation of S1R with 100 nM pentazocine decreased this overlap. The Statistical Object Distance Analysis (SODA) workflow reveal the decrease of coupling more than 10%, at 100 nm distance, with half-life 19 min.

Obtained data indicate that intracellular vesicular ER are involved in the dynamics of activation of S1R causing changes in colocalization with vesicular ER marker proteins, but the mechanism of this actions is topic for further studies.

This work was supported by the Estonian Research Council grants (PSG230 and IUT20-17)

Michal Gwizdala^1,2, Tjaart Krüger¹

¹Department of Physics, University of Pretoria, South Africa
²Department of Physics and Astronomy, Vrije Universiteit Amsterdam, the Netherlands

Phycobilisome (PB), the main light-harvesting antenna of cyanobacteria and many algae, is an enormous multi-subunit pigment-protein complex, containing nearly 400 pigments. Despite its size it exhibits single-step fluorescence blinking even at physiological light intensities [1]. Using Stark spectroscopy and single molecule spectroscopy, involving simultaneously measured fluorescence intensities, lifetimes and spectra, combined with kinetic modelling, we have identified that charge-transfer states are correlated with fluorescence blinking in PBs, the formation of which is controlled by the incident light intensity to serve a vital functional purpose [1]. In vivo, this mechanism could serve as a novel type of photoprotection that can be rapidly accessed before the orange carotenoid protein (OCP) is photoactivated and bound to PB to induce further photoprotection. Furthermore, we investigated the interaction between a single PB and an OCP and managed to reversibly switch the fluorescence of PB off and on [2]. This study revealed the presence of quasistable intermediate states during the binding and unbinding of OCP to PB, with a spectroscopic signature indicative of transient decoupling of some of the PB subunits during docking of OCP. Real-time control of emission from individual PBs has the potential to contribute to the development of new superresolution imaging techniques.

Alessandro Valeri¹, Suren Felekyan¹, Stanislav Kalinin¹, Markus Richert¹, Stefan Marawske¹, Enno Schweinberger¹, Oleg Opanasyuk¹, Ivan Rech², Angelo Gulinatti², Ralf Kühnemuth¹, Claus A.M. Seidel¹

¹Lehrstuhl für Molekulare Physikalische Chemie, Heinrich-Heine-Universität, Universitätsstrasse 1, Geb. 26.32, 40225 Düsseldorf, Germany
²Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, piazza Leonardo da Vinci 32, 20133 Milano, Italy

Holliday junctions, i.e. DNA four-way junctions, play essential roles in DNA replication, repair, and recombination. In addition they are key building blocks in DNA nanotechnology. Previous studies have shown that in the presence of Mg2+ ions Holliday junctions switch between two stacking conformations with rates strongly dependent on the ion concentration. The molecular mechanism for this is unclear. We performed FRET studies using confocal single-molecule detection (SMD) with diffusing molecules or evanescent excitation of immobilized single molecules as well as ensemble fluorescence lifetime studies. We used the full dynamic time range of fluorescence from ns to s to capture the complex Mg-dependent kinetics. The minimal model that describes the observed kinetics consists of four states, with only two distinct FRET levels. All methods and analysis techniques consistently reveal continuous interconversion also in absence of Mg2+ whereas addition of Mg2+ “locks” the junctions in the respective states. Our results shape a new view on structural properties of four-way DNA junctions, identifying a dynamic equilibrium instead of an accumulation of a single open structure at low Mg2+. DNA four-way junctions do not adopt static conformations but the exchange of Mg2+ ions determines their effective dynamic and structural features in their distinct environments.

Seoungjun Lee, Marie-Therese Salcher-Konrad, Sarah Mizielinska

Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, 5 Cutcombe Road, Camberwell, London, SE5 9RX

Recent studies taking advantage of single molecule dynamics have been highlighted in nucleocytoplasmic transport. Studies have investigated transport dynamics using super-resolution microscopes, such as TIRF, STORM and PALM. Most of the recent work has been done with digitonin permeabilized cells to allow the delivery of labelled cargo at an appropriate level for single molecule imaging. We set out to study nucleocytoplasmic transport dynamics in the human cell line SH-SY5Y at the single molecular level in intact cells, using fluorescently-tagged dextran by highly inclined and laminated optical sheet (HILO) microscopy (near-TIRF).

Our results suggest that TIRF bleaching and HILO imaging (TBHI) can observe single molecule tracking at 2 µm above the substrate, using 53fps (images per 18 ms, Andor iXon EMCCD) in a Nikon TIRF microscope. We are monitoring how dextran cargoes are transported across the nuclear envelope and whether they are successfully transported into the nucleus (import) or cytoplasm (export). We also compared passive transport in SH-SY5Y cells overexpressing TDP-43 and found different transport dynamics through the nuclear pore complex.

We have developed our technique using TBHI microscopy to further analyse the transport dynamics of single molecule cargo/receptors through the inner and outer domains of individual nuclear pores and the impact of disease-associated proteins in neurodegeneration.

Yichen Li¹, Eliza M. Warszawik², Jochem H. Smit¹, Andreas Herrmann^2,3, Thorben Cordes^1,4

¹Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
²Department of Polymer Chemistry, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
³DWI – Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52056 Aachen, Germany
⁴Physical and Synthetic Biology, Faculty of Biology, Ludwig Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany

Aminoglycoside antibiotics are therapeutically important polycationic bacteriocides widely applied to treat infections^[1]. Understanding the fundamental mechanisms by which aminoglycosides cross the cell wall and find their ribosomal targets is important for overcoming bacterial resistance. Although the details of aminoglycoside action on Escherichia coli (E. Coli) are still obscure, it has been proposed that there is a three-step model of accumulation of these antibiotics^[2]. Initial ionic binding to cells is followed by transport into the bacterial cytosol. This results in the generation of misfolded proteins, which are inserted into the membrane – a process that ultimately leads to cell death. Here, we designed a series of fluorophore-labeled aminoglycoside conjugates to study the uptake process in time and space. Since uptake is driven by the electrostatic interactions^[3], we first investigated how charges influence the process. For this, we varied fluorophores and aminoglycoside scaffold. For the analysis, we used a highly automated imaging system for live-cell fluorescence microscopy that visualizes cellular processes with high temporal-spatial resolution and high statistics. For data analysis, we used an open-source data analysis package that we have developed allowing systematic and quantitative analysis of fluorescence signal distributions^[4].

Haichun Liu¹, Qiuqiang Zhan², Baoju Wang², Hans Ågren¹, Sailing He²

¹Department of Theoretical Chemistry and Biology, KTH Royal Institute of Technology, S-10691 Stockholm, Sweden
²Centre for Optical and Electromagnetic Research, South China Academy of Advanced Optoelectronics, South China Normal University, 510006 P.R. China

MOTIVATION Lanthanide-doped upconversion nanoparticles (UCNPs) have been developed as an important group of luminescent biomarkers. However, efficient optical modulation of UCNPs has not been well established, hindering their use in advanced imaging techniques, such as stimulated emission depletion microscopy. In this work, we devote our efforts to establish an efficient optical depletion approach for UCNPs and to implement super-resolution microscopy.

RESULTS We establish an efficient optical depletion approach for the blue upconversion luminescence at 455 nm of Tm³⁺ ions in high Tm³⁺-doped NaYF₄:Yb³⁺,Tm³⁺ UCNPs.¹ This achievement enables us to implement super-resolution luminescence microscopy.¹ We further extend the super-resolution imaging to a two-color mode using the same pair of excitation/depletion beams.¹ In addition, we implement cytoskeleton protein super-resolution imaging of immunolabeled HeLa cells using antibody-conjugated UCNPs.¹

CONCLUSIONS We implemented two-color super-resolution imaging using a single excitation/depletion laser beam pair using upconversion nanoparticles. In addition, we have achieved a successful immunolabeling of fine subcellular structures and super-resolution cellular imaging using UCNPs.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (61675071, 61405062, and 91233208) and the Swedish Research Council (2015-00160 and 2016-03804).

Steven Magennis

School of Chemistry, University of Glasgow, University Avenue, Glasgow, G12 8QQ

Fluorescence spectroscopy and imaging are powerful techniques for probing the structure, dynamics and interactions of nucleic acids, particularly when performed at the single-molecule level. In this talk I will discuss a new method for probing local and global DNA structure and dynamics using the common fluorescent dye Cy3 [1]. It takes advantage of the isomerization-induced enhancement of Cy3 emission, which occurs through the site-specific stacking of Cy3 in an adjacent nick, gap or overhang in double-stranded DNA. The DNA structure and dye position can be designed according to the particular application. It can operate at the single-molecule level and can probe both large-scale structural rearrangements that result in dye stacking (e.g. the opening and closing of a DNA hairpin) and also local changes near the site of dye stacking, as demonstrated by the ability to reveal long-range perturbations through the DNA backbone.

Emmanuel Margeat¹, Anne Marinette Cao¹, Robert Quast¹, Fataneh Fatemi¹, Philippe Rondard², Jean-Philippe Pin²

¹Centre de Biochimie Structurale - CNRS - INSERM - Université de Montpellier, France
²Institut de Génomique Fonctionelle - CNRS - INSERM - Université de Montpellier, France

Metabotropic  glutamate  receptors  (mGluRs) are multidomain proteins belonging to class C G-protein coupled receptors (GPCR). There are essential in controlling synaptic transmission, and as such are  important  drug  targets  for  the treatment of several disorders including pain, Parkinson’s disease, schizophrenia etc...

We use single molecule FRET combined with Multi-parameter Fluorescence Detection (MFD) and Pulsed Interleaved Excitation (PIE) to investigate the allosteric transitions associated with mGluR activation. We demonstrated that the isolated ligand binding (VFT) domain oscillates between a resting and an active state in a time range of 50-100 µs. Here, we extend these investigation to the full length receptor solubilized in detergent. Our result confirm the general mechanism observed for the VFT domain, and decipher the role of the transmembrane domain, that slightly slows down the receptor dynamics, while stabilizing its active state solely in the presence of a full agonist.

Koen Martens^1,2, Sam van Beljouw¹, Simon van der Els^3,4, Jochem Vink⁵, Sander Baas¹, George Vogelaar¹, Stan Brouns⁵, Peter van Baarlen³, Michiel Kleerebezem³, Johannes Hohlbein¹

¹Laboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands
²Laboratory of Bionanotechnology, Wageningen University and Research, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands
³Host-Microbe Interactomics Group, Animal Sciences, Wageningen University and Research, De Elst 1, 6708 WD, Wageningen, The Netherlands
⁴NIZO food research, Kernhemseweg 2, 6718 ZB Ede, The Netherlands
⁵Kavli Institute of Nanoscience, Department of Bionanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, Netherlands

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires the constant screening of the protospacer adjacent motif (PAM) and an upper limit for PAM screening (<30 ms) was recently found. To quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9 further, we developed an open-microscopy framework that combines straightforward installation with high spatiotemporal resolution and introduce Monte-Carlo diffusion distribution analysis (MC-DDA) [1,2]. Our analysis revealed that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging just 17 ± 4 ms per binding event. Using heterogeneous expression of dCas9, we further determined the number of cellular target-containing plasmids and modelled the expected cleavage efficiency [1]. We found that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data will facilitate further optimization of the CRISPR-Cas toolbox.

Satomi Matsuoka^1,2, Masahiro Ueda^1,2

¹Graduate School of Frontier Biosciences, Osaka University. 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
²RIKEN Center for Biosystems Dynamics Research. 6-2-3, Furuedai, Suita, Osaka 565-0874, JAPAN.

Mutually exclusive membrane localization of anterior and posterior signaling molecules, phosphatidylinositol 3,4,5-trisphosphate (PIP3) and PIP3 phosphatase (PTEN), respectively, is a hallmark of eukaryotic motile cells. The molecular mechanism leading to the spatial separation between these two molecules remains unsolved. In this study, we manipulated PIP3 levels in living Dictyostelium discoideum cells to show that PIP3, the substrate, suppresses the membrane localization of PTEN, the enzyme. In the cells with high and low PIP3 levels, we performed single-molecule imaging of PTEN and statistical analysis of membrane-association and -dissociation kinetics and of lateral diffusion. The results showed that PIP3 suppresses the PTEN binding site on the membrane that is required for stable PTEN membrane binding. We found that due to the mutual inhibition between PIP3 and PTEN, bistability that creates a PIP3-enriched/PTEN-excluded state and a PTEN-enriched/PIP3-excluded state arises, which underlies the strict spatial separation between PIP3 and PTEN. The PTEN binding site also mediates chemotactic signaling in Dictyostelium cells. These results illustrate that the PIP3-PTEN bistable system provides a cell’s decision-making for directional movement irrespective of the environment.

Siegfried Musser, Guo Fu, Li-Chun Tu, Anton Zilman, Rajdeep Chowdhury, Sandeep Dave

Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, Texas A&M University, College Station, TX 77843 USA

Systems of intrinsically disordered proteins are typically highly concentrated heterogeneous assemblies that undergo rapid internal dynamics and exchange with their environment. Both the nuclear pore permeability barrier and phase-separated condensates require fast (millisecond) imaging to accurately track particle behavior, which informs about local structural and environmental properties.  The application of polarization PALM (p-PALM) and rapid 2D and 3D super-resolution imaging of the nuclear pore and in vitro condensates of the fused in sarcoma (FUS) protein will be presented.  The p-PALM method reports on the rotational mobility of the probe, which is a proxy for the local viscosity as influenced by macromolecular crowding, and reveals spatial heterogeneity within the nuclear pore.  The characteristics of both translational and rotational motion within in vitro phase-separated states of FUS will be described. 

Andrey Naumov^1,2, Maxim Gladush^1,2, Aleksey Gorshelev¹, Ivan Eremchev¹, Juergen Koehler³, Lothar Kador³

¹Institute of Spectroscopy RAS, Moscow Troitsk, Russia
²Moscow State Pedagogical University, Moscow, Russia
³Bayreuth University, Bayreuth, Germany

The present work demonstrates, how nanoscopy based on the study of single quantum emitters (fluorescent molecules –SM; semiconductor quantum dots – QD) can be extended to perform advanced materials characterization. To this end, SMs and QDs are not only used as point light sources, but as sensitive multi-parameter local probes.

Simultaneous imaging and measurement of spectroscopic characteristics of the SMs/QDs yields the possibility of mapping materials parameters on the nanometer scale. In the present case we recover the index of refraction, n, which is one of the most important materials parameters of solids and, in recent years, has become the subject of significant interdisciplinary interest, especially in nanostructures and meta-materials. It is, in principle, a macroscopic quantity, so its meaning on a length scale of a few nanometers, i.e., well below the wavelength of light, is not clear a priori and is related to methods of its measurement on this length scale.

Here we demonstrate a novel experimental approach for mapping the effective local value n’ of the refractive index in solid films and the analysis of related local-field enhancement effects. [1] The approach is based on the imaging and spectroscopy of SMs at cryogenic temperatures, when excited state life-time limited zero-phonon lines are reachable for detection. Since the fluorescence lifetime T₁ of dye molecules in a transparent matrix depends on the refractive index due to the local density of the electromagnetic field (i.e., of the photon states), one can obtain the local n’ values in the surroundings of individual chromophores simply by measuring their T₁ times. Spatial mapping of the local n’ values is accomplished by localizing the corresponding chromophores with nanometer accuracy. We demonstrate this approach for a polycrystalline n-hexadecane film doped with terrylene. Unexpectedly large fluctuations of local-field effects and effective n’ values were found.

Finally, continuing this approach we use single colloidal semiconductor quantum dots CdSe/ZnS as nanoprobes for local field sensing by nanoscopy with direct measurements of QD luminescence kinetics.

Financial support from DFG (RTG 1640) and RFBR (17-02-00652) is gratefully acknowledged.

Oleksii Nevskyi, Weixing Li, Narain Karedla, Ingo Gregor, Jörg Enderlein

III. Institute of Physics – Biophysics, Georg‐August‐University Göttingen, Friedrich‐Hund‐Platz 1, 37077 Göttingen, Germany

Single molecule localization microscopy techniques have become one of the most successful and widely applied methods of super-resolution fluorescence microscopy. The position determination of a single emitter is the key point of the method and is usually done by fitting 2D Gaussian to the emission intensity distribution of the corresponding fluorescent molecule. However, the intensity distribution of an emitting molecule depends not only on its position in space, but also on orientation of its emission dipole. We propose a flexible cost-efficient wide-field cryo-fluorescence microscope with an exceptionally high thermal and mechanical stability, outstanding single molecule imaging quality, and possibility of a sample change at cryogenic temperatures [1]. With the help of it, a variety of fluorescent dyes have been investigated and an improvement of the photostability of these molecules by more than two orders of magnitude has been found. The improvement mentioned above corresponds to a theoretical localization precision around 0.1 nm at liquid nitrogen temperatures [2]. In the scope of super-resolution microscopy imaging at the cryogenic temperatures, the orientations of dipole emitters are fixed, which causes significant mislocalizations utilizing common fitting approaches [3]. Here we demonstrate  an  easy  experimental  solution  to  the current  problem,  based on the polarization  splitting in the detection path of the microscope.                     

David Nobis, Steven W. Magennis

WestCHEM School of Chemistry,University of Glasgow, Joseph Black Building, University Avenue, Glasgow, G12 8QQ, UK

A single molecule fluorescence study of DNA normally involves attaching a bright extrinsic fluorescent probe [1]. There would be benefits to using intrinsic probes such as fluorescent nucleobases; however, photobleaching, UV absorption and high background has so far prevented their practical detection at the single-molecule level.

We seek to solve these problems using multiphoton excitation in a new home-built setup, deploying the next generation of ultrafast Ti:Sapphire laser (135 nm FWHM) [2]. Multiphoton excitation offers reduced photobleaching as well as reduced background due to the large spectral separation of excitation and emission [3].

Multiphoton absorption requires ultra-short laser pulses for effective excitation. Using the MIIPS-method for pulse compression [4], pulses of 8fs length at the focal plane of the microscope can be achieved. Additionally, chromophore-specific phase and amplitude pulse shaping can be employed to enhance the signal to background ratio.

We will present our study of new DNA nucleobase analogs with this multiphoton setup [2]. We will show how pulse compression (via phase shaping) dramatically improves the signal and how the signal to background ratio can be further improved upon by amplitude shaping of the exciting light.

Mareike Noffke¹, Xenia Knigge¹, Christian Wenger^2,3, Frank F. Bier^1,4, Ralph Hölzel¹

¹Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany.
²IHP - Leibniz Institute for Innovative Microelectronics, Frankfurt (Oder), Germany.
³Brandenburg Medical School Theodor Fontane, Neuruppin 16816, Germany
⁴University of Potsdam, Germany

For the observation of single enzyme molecules, the molecules are immobilized or entrapped as isolated singles. In most cases this is achieved by the dilution of the enzymes before entrapment or immobilization or by the creation of a low surface concentration of acceptors for enzyme binding. Singling then follows a Poisson distribution and only a few molecules can be studied.

In this work, a new platform for a parallel, label free, deterministic singling of active enzyme molecules is developed. Enzyme molecules are immobilized on planar electrode arrays by dielectrophoresis, the force acting on a polarizable particle in an inhomogeneous electric field. The electrodes are available in different materials, shapes and tip diameters ranging from 500 nm down to about 1 nm. Thousands of electrode tips are arranged in each array, allowing to perform many experiments in parallel. So far, we successfully immobilized nanoparticles as singles deterministically [1]. Immobilization of clusters of the enzyme Horseradish Peroxidase in its active form was also successful [2]. These arrays are being characterised and optimized to reach the singling of active enzyme molecules. This kind of nanoarray will enable a higher throughput and better statistics in single enzyme molecule studies.

Roman Tsukanov¹, Shama Sograte-Idrissi^2,3, Nazar Oleksiievets¹, Sebastian Isbaner¹, Mariana Eggert-Martinez^2,3, Jörg Enderlein¹, Felipe Opazo^2,3

¹Third Institute of Physics − Biophysics, Georg August University, 37077 Göttingen, Germany
²Center for Biostructural Imaging of Neurodegeneration (BIN), University of Göttingen Medical Center, 37075 Göttingen, Germany
³Institute of Neuro- and Sensory Physiology, University Medical Center Göttingen, 37073 Göttingen, Germany

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straightforward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.

Fabian Port, Ulla Nolte, Kay-E. Gottschalk

Institute of Experimental Physics, Ulm University, Ulm, Germany

Focal adhesions function as anchoring points to the extracellular matrix, and also enable cells to sense and exert forces on their environment [1]. Focal adhesions are complex structures consisting of a multitude of different proteins.  Despite the important role of the focal adhesion complex in cellular adhesion, its structure and dynamics remain difficult to resolve [2]. Knowing the exact position of the proteins in the focal adhesion complex in live cells is necessary to understand their working principles.

For a detailed analysis of the focal adhesions dynamic architecture, we require a method to measure small distances that may be applied over a variable time scale. To meet this challenge, we use Metal Induced Energy Transfer (MIET) [3] to resolve protein positions at the nanoscale level in live cells. Here we show an initial analysis of the dynamics of focal adhesion associated actin.

Robert B. Quast¹, Anne-Marinette Cao¹, Fataneh Fatemi², Michel Kranendonk³, Philippe Rondard⁴, Jean-Philippe Pin⁴, Gilles Truan⁵, Emmanuel Margeat¹

¹Centre de Biochimie Structurale (CBS), INSERM, CNRS, Université de Montpellier, F-34094, Montpellier, France.LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, 31077, Toulouse (France)
²Protein Research Center, Shahid Beheshti University, G.C., Evin, 1983969411Tehran (Iran)
³Center for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Human Toxicology, NOVA Medical School, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1150-082 Lisboa (Portugal)
⁴IGF, CNRS, INSERM, Univ. de Montpellier, Montpellier, F-34094, Montpellier, France.
⁵LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, 31077, Toulouse (France)

Förster resonance energy transfer (FRET) can precisely report on distance changes between two fluorophores, which allows real-time monitoring of structural dynamics occurring in labeled proteins down to the single-molecule level. Nevertheless, their direct local environment can influence the biophysical properties of fluorophores and thus conjugation of dyes to proteins may alter their behavior. Therefore, a careful characterization of fluorescence parameters is required, to allow for accurate FRET measurements and actual distance calculations. In order to facilitate accurate FRET measurements we labeled human cytochrome P450 reductase, a redox-enzyme whose native amino acid composition does not allow modification by classical reactions targeting functionalities of proteinogenic amino acids. We used two distinct bioorthogonal chemistries to attach the popular Cy3/Cy5 pair in a domain-specific manner. Specific labeling was facilitated by cotranslational incorporation of two uniquely reactive non-canonical amino acids in response to simultaneous TAG and TAA stop codon suppression. Single molecule FRET measurements revealed the impact of the dye position on derived FRET values as a result of altered quantum yields of conjugated dyes. We are currently applying these and other site-specific labeling approaches to elucidate the various structural dynamics that occur in different domains throughout full-length metabotropic glutamate receptors.

Suhaila Rajab¹, Simone Schwarze¹, Hannes Neuweiler²

¹suhaila.rajab@uni-wuerzburg.de
²hannes.neuweiler@uni-wuerzburg.de

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate most of the excitatory neurotransmission in the mammalian central nervous system¹. iGluRs are crucial for brain development and function, including learning and memory¹. Accordingly, iGluRs are key targets for drug development in pharmacology. iGluRs are divided into three major groups (AMPA, kainate and NMDA receptors), each of which forms trans-membrane spanning tetrameric assemblies². The ligand-binding domain (LBD) transfers conformational change upon binding of neurotransmitter and opens the ion channel for signal transduction^1,2. Here we measured the kinetic pattern and time constants of LBD clamshell motions from iGluRs in solution at the single-molecule level on time scales from nanoseconds to milliseconds using photoinduced electron transfer (PET) fluorescence quenching in combination with correlation spectroscopy (PET-FCS)³. We engineered PET-based fluorescence probes to monitor conformational changes on the one-nanometer scale. We uncovered three kinetics of conformational motions of both the AMPA receptor GluR2 LBD and the NMDA receptor NR1 LBD, each on a sub-millisecond time scale. We found slightly slower relaxation time constants in the NMDA receptor. In the presence of the neurotransmitter glutamate, we observed stalling of motions in both receptor types, indicating reduced mobility of the LBD in its bound state.                   

Aigerim Rakhmatulina, Maged F. Serag, Bader Al Alwan, Shuho Nozue, Jasmeen S. Merzaban, Satoshi Habuchi

King Abdullah University of Science and Technology (KAUST), Biological and Environmental Science and Engineering Division, Thuwal 23955-6900, Saudi Arabia

Hematopoietic stem cells, HSCs, are widely used to treat various types of cancer through bone marrow transplantation. For a successful transplantation, HSCs should undergo migration and homing processes in the microenvironment of the bone marrow of the recipient. The homing is initiated by the interaction of endothelial E-selectin with surface ligands of HSCs such as PSGL-1 and CD44. Here we investigate the relevance of PSGL1 dynamics on tethers and slings, thin and flexible structures protruding out of HSCs formed during the homing, to the initial step of the homing, rolling of HSCs on endothelium.

Single-molecule tracking analysis of PSGL1 molecules on tethers and slings of HSCs showed diffusion coefficient twice faster than of the ones localized on microvilli. Also, mean square displacement analysis showed random diffusional motion of the molecules on tethers and slings and a confined diffusion on microvilli. We found that the observed random diffusional motion of the selectin ligands on the tethers and slings is a result of the detachment of actin cytoskeleton from the cell membrane, which occurs during the formation of the tethers. We will discuss how the diffusion behavior of PSGL-1 on the tethers and slings affect the rolling of HSCs on E-selectin.

Lydia Rebehn, Fabian Port, Ulla Nolte, Kay-E. Gottschalk

Institute for Experimental Physics, Ulm University, Ulm

Focal adhesions (FAs) are physical anchoring areas connecting cells to the extracellular matrix, as well as enabling cells to sense their environment and respond to changing circumstances [1].  These complexes consist of a multitude of different protein components, arranged in a highly regulated structure axially and laterally.  Internally, these FAs couple to actin stress fibers, which transmit the variable applied external forces into internal mechanical and biochemical signals.

Despite the importance of the focal adhesion’s roles, the structure remains difficult to resolve due to the nanoscale order of their protein components [2]. To understand cell mechanosensing mechanisms, FA structural dynamics can be discerned from the associated actin stress fiber. This type of detailed analysis requires a method of measuring the molecular locations with increased resolution in the nanometer range to be able to clarify the positions of different protein components.  A technique which meets this challenge is Metal Induced Energy Transfer (MIET) in combination with the use of micropatterns [3]. Presently, we provide an initial analysis of the angles of stress fibers to the underlying surface thereby demonstrating the usefulness of MIET for analyzing molecular structures close to the basal membrane with nanometer accuracy.

Sumeet Rohilla^1,3, Benedikt Krämer¹, Felix Koberling¹, Ingo Gregor², Andreas C. Hocke³

¹PicoQuant GmbH, Berlin, Germany, info@picoquant.com
²Georg-August-University, Friedrich-Hund-Platz 1, Göttingen, Germany
³Department of Internal Medicine / Infectiology, Respiratory and Critical-Care-Medicine, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, andreas.hocke@charite.de

Indirect labelling techniques of cellular molecules with primary and secondary antibodies for immunofluorescence-based imaging is still serving well for the needs of biologists in advancing the understanding of biological processes. However, these techniques impose a stringent selection of primary and secondary antibody pairs to avoid false-positive immunolabeling leading to misinterpretation, in particular for (co-)localization/interaction of target molecules. At best, primary antibodies raised in different species are used and combined with corresponding secondary antibodies originating in another species or, at least, different to the origin of primary antibodies. Due to this, indirect immunofluorescence is either regularly limited to two - four antigens or a particular combination of target molecule labelling can’t be carried out at all.

Here, we present a new method to use this ostensible disadvantage of cross-labelling secondary antibodies by separation of the fluorescence signals by spectral FLIM-FRET. This becomes possible since the undesirable cross-labelling among secondary antibodies leads to the generation of new characteristic FRET emission spectra including a change of the donor lifetime. To demonstrate this, we adapted a sequential labelling protocol and selected appropriate fluorophore pairs on interacting secondary antibodies (e.g. Alexa488 and Alexa546) to achieve strong FRET effects.

As a model, we labelled the target molecules pan-Cytokeratin (Alexa546-rabbit-anti-mouse), TOM20 (Alexa488-goat-anti-rabbit), and golgin (Alexa546-rabbit-anti-mouse). This led to single labelling of TOM20 with Alexa488-goat-anti-rabbit and golgin with Alexa546-rabbit-anti-mouse as well as cross-labelling of pan-Cytokeratin with Alexa488-goat-anti-rabbit and Alexa546-rabbit-anti-mouse. We used an eight channel spectrally resolved FLIM (sFLIM) detection system and acquired data for all labels excited with two lasers pulsating in interleaved mode. Data analysis was performed using the pattern-matching algorithm1 taking into account emission spectra as well as nanosecond time-resolved fluorescence decays.

The method enabled us to precisely separate all three target molecules generated by just two fluorophore species due to their cross-linking and resulting FRET interaction. Consequently, spectral FLIM-FRET together with pattern-matching analysis forms an excellent tool for use in indirect immunofluorescence by overcoming the undesirable effect of secondary antibody cross-labelling by assigning separate colour channel to cross-linked fluorophores.

Jan Schlegel¹, Felix Wäldchen¹, Tim Walter², Julian Fink², Thomas Klein³, Nurcan Üceyler³, Jürgen Seibel², Markus Sauer¹

¹Department of Biotechnology and Biophysics, Biocenter - Am Hubland, Wuerzburg, Germany
²Institute for Organic Chemistry, Julius-Maximilian University Wuerzburg, Am Hubland C1, Wuerzburg, Germany
³Department of Neurology, University of Wuerzburg, Wuerzburg, Germany

Ceramides are sphingolipids which play an important role in many cellular processes such as proliferation, differentiation, apoptosis, sphingolipidoses and cancer development. Nevertheless, detailed investigations of their distribution and dynamics in cells has been hampered by the lack of appropriate visualization tools in cellular environment. Here, we combined complementary imaging techniques to obtain precise information with increased spatiotemporal resolution. On the one hand, single-molecule localization microscopy was used to obtain accurate insights into the spatial distribution of native and azido-modified ceramides within membranes of mammalian cells and pathogenic bacteria. Combining stoichiometric and bio-orthogonal click-chemistry with single-molecule localization microscopy in living cells we were able to detect and follow ceramide dynamics in the endoplasmic reticulum (ER) membrane. On the other hand, we applied lattice light sheet (LLS) microscopy to record fast lipid dynamics within whole cell volumes and correlated their distribution with cellular structures of the ER and microtubules. Finally, we were able to combine these imaging techniques to obtain valuable information about ceramide distribution and dynamics across large cellular volumes at high spatial resolution in the context of human diseases.

Ralf Schmauder, Susanne Thon, Maik Otte, Andrea Schweinitz, Marco Lelle, Thomas Zimmer, Tina Schwabe, Klaus Benndorf

Institute for Physiology II, Jena University Hospital, Friedrich Schiller University Jena, 07740 Jena, Germany

Ligand-gated ion channels are essential in many fast signaling processes. Their multi-subunit composition allows for cooperativety and with this fine-tuning of the set points of their signaling. Here we aim to directly observe the cooperativety in single-ligand binding to the tetrameric HCN2 pacemaker channels. These cation channels are voltage activated and, in addition, modulated by cyclic nucleotides.

Studying ligand binding with fluorescence is usually limited to high affinities (picomolar to low nanomolar) or requires advanced techniques as zero-mode waveguides or STED.

To follow binding at higher concentrations (nanomolar to micromolar) we screened and optimized ligands: Molecular brightness and environmental sensitivity was evaluated with FCS and fluorescence lifetime measurements, linker chemistry was optimized and function was evaluated with confocal patch-clamp fluorometry.

Following binding of fluorescent ligands to individual HCN2-channels with TIRF microscopy directly suggests positive cooperativity. We further analyze the single molecule binding data to test for possible heterogeneities in the binding of the ligands to the different binding sites of the receptors. Signal processing is optimized to follow the levels and the dwell times of the unitary binding events, ultimately to increase the understanding of the channel dynamics in terms of Hidden Markov Models.

Magdalena Schneider, Andreas Arnold, Florian Baumgart, Gerhard Schütz

Institute of Applied Physics, TU Wien, Vienna, Austria

Observations using single-molecule localization microscopy have led to the belief that the majority of tested membrane proteins are organized in clusters at sizes below the diffraction limit. These nanoclusters are thought to be important for cellular signaling. However, concerns about their existence have been fueled by the notion that virtually all fluorescent probes show complex blinking behavior including long-lived dark states. This results in localization clusters due to repeated observations of molecules. Existing post-processing approaches commonly struggle to reliably distinguish real molecular clustering from blinking artifacts.

Here, we present a novel analytical method using information from two-color STORM experiments for overcoming the erroneous detection of clustering due to fluorophore blinking. Targeting the same protein species with different labels allows to calculate distance distributions between localizations from both color channels. Molecular clusters exhibit a characteristic bias towards shorter distances. Applying toroidal shifts to the data breaks correlations between the color channels, thus providing realizations of the null hypothesis of independence (randomly distributed molecules). This allows for statistical significance tests without the necessity of additional calibration. Monte-Carlo simulations showed the reliability and robustness of the proposed method. Moreover, the method was validated with experiments on both clustered and randomly distributed membrane proteins.

Jonathan Schubert¹, Andrea Schulze¹, Hannes Neuweiler²

¹jonathan.schubert@uni-wuerzburg.de
²hannes.neuweiler@uni-wuerzburg.de

The molecular chaperone Hsp90 is a central node of protein homeostasis¹ and involved in several cellular events including signal transduction and regulatory processes. Its mode of function is therefore of great interest, not at last because many oncogenic proteins are clients of this chaperone². The remarkably slow ATPase rate (0.2 ATP per minute) drives the chaperone through a conformational cycle that is accompanied by several local rearrangements. So far, three of these motions have been successfully investigated in bulk experiments using photoinduced electron transfer (PET) fluorescence quenching of an extrinsic label by the amino acid tryptophan³. By transferring the PET technique to fluorescence imaging microscopy, we developed a powerful tool to investigate details of the conformational cycle explicitly in single Hsp90 molecules. We analysed each of the local motions individually in single molecules over a period of minutes by utilizing established PET reporter systems employing the fluorophore AttoOxa11. Single-molecule immobilization was realized on passivated glass surfaces using Biotin‑NeutrAvidin biochemistry. We established a control experiment making use of oxidation of tryptophan by molecular oxygen in order to distinguish between photobleaching and PET events. We found alternative fluorophores that show either better photostability or less fluorescence fluctuations compared to AttoOxa11.

Jaime Segura-Ruiz¹, Julie Villanova¹, Remi Tucoulou¹, Joel Eymery², Gema Martinez-Criado³

¹ESRF: The European Synchrotron. Grenoble, France
²University Grenoble Alpes, CEA, INAC. Grenoble, France
³Instituto de Ciencia de Materiales de Madrid (ICMM). Madrid, Spain

The ID16B beamline at the European Synchrotron Radiation Facility (ESRF)[1] is a nano-probe that provides a focused (down to 50x50 nm²), intense (up to 10¹² ph/s), hard X-rays (up to 33 keV) beam. ID16B offers several characterization techniques: X-ray fluorescence (XRF), X-ray diffraction, X-ray absorption spectroscopy (XAS), X-ray excited optical luminescence (XEOL) and 3D phase contrast imaging, among others. The various methods available, the possibility of using them simultaneously and the capability to install different types of samples environments, make this beamline a unique characterization tool. In this talk, I will detail the main characteristics and the experimental setup of the beamline ID16B. I will then present the results obtained from the multi-technique characterization performed on InGaN coaxial nano-LEDs. The combined use of XRF and XEOL techniques available on ID16B provided unique information on the composition and optical properties of these nano-devices at the nanometer scale. The use of the optical spectral information allowed us to further increase the spatial resolution provided by the nano X-ray beam. Doing this, nanostructures with sizes smaller than 100 nm could be easily identified and their optical properties studied. This same approach can be extended to other types of systems, for instance biological samples with light emitting nanoparticles.

Tarcio de Castro Silva⁵, Hssen Fares³, Robson da Silva⁸, Douglas Franco⁷, Anna Fucikova¹, Jan Valenta⁴, Marcelo Nalin⁶, Sidney J. L. Ribeiro²

¹anna.fucikova@email.cz
²sjlribeiro@gmail.com
³fares.hssen@gmail.com
⁴jan.valenta@mff.cuni.cz
⁵tarcioquimica@gmail.com
⁶marcelo.nalin@unesp.br
⁷frazafranco@yahoo.com.br
⁸robsilva31@gmail.com

Materials containing silver molecular nanoclusters (Ag_mⁿ⁺) are promising candidates in modern spectroscopy owing their attractive tunable optical properties [1]. Combined with rare earth ions RE³⁺, new materials have been developed for numerous applications like luminescent layers for silicon solar cell, white light generation, light frequency converter and others [2-5]. Herein, we report the synthesis of silver-conatining fluorophosphate glass via a facile melt-quenching method. Stabilization and optical properties of silver species, such as Ag⁺ and Ag_mⁿ⁺ were investigated. Further improvement aiming to obtain white light emission was achieved doping the sample with Tm³⁺ and Mn²⁺ ions. By changing the excitation wavelength, a reversible energy-transfer process between silver nanoclusters and Tm³⁺/Mn²⁺ ions was explored and was correlated with the size of Ag_mⁿ⁺. The markedly behavior exhibited by the different species reflects in their emission properties and gives rise a bright white emission with varying tint tuned from cold white through the red-white to the warm white edge. Moreover, quantum yield efficiency (QY) results indicate that our glass system may be a possible candidate for white light-emitting-diodes (W-LED) devices. Last but not least, single Ag_mⁿ⁺ spectroscopy for biological applications will be under investigation.

Soheila Rezaei Adariani

Institut für molekulare physikalische Chemie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany

For a mechanistic understanding of the function of proteins it is important to capture time-resolved structures. Fast dynamics is typically studied by MD or NMR, the techniques that have high temporal resolution and yield per-residue information, but inadequate force fields, high energy barriers and low populated states render excited states invisible and prohibit a deep understanding of proteins in action. We demonstrate how fluorescence spectroscopy captures of large scale conformational transitions and dynamics using the γ-aminobutyrate type A receptor-associated protein (GABARAP) as an example. Here, FRET spectroscopy revealed important structural features of GABARAP in solution, which not captured by X-ray crystallography and NMR derived models, with possible functional impact. We modeled possible conformational dynamics of GABARAP with FRET-guided MC simulations. We tested the influence of GABARAP lipidation and anchoring to the membrane, which may provide an additional insight into the mechanism of function of this protein it its native conditions.

Oliver Stach, Sebastian LB König, Andrea Holla, Daniel Nettels, Benjamin Schuler

Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

Many organisms use double-stranded RNA binding proteins (dsRBP) to specifically recognize double-stranded RNAs (dsRNA) in the context of posttranscriptional gene regulation. These dsRBP usually consist of multiple copies of RNA binding domains separated by long unstructured linker regions. Furthermore, RBPs remain remarkably dynamic in their RNA bound state. In this study, we used a variety of single molecule fluorescence techniques to resolve the binding mechanism of the dsRBP TRBP to dsRNA. We were able to characterize the kinetics of interconversion of different states and could furthermore resolve the fast diffusion time of TRBP by using photon by photon-based analysis. These results demonstrate the ability of single molecule fluorescence techniques to resolve the complex conformational landscape of functional protein-RNA-complexes on a variety of timescales.

Florian Steiner¹, Viktorija Glembockyte¹, Kateryna Trofymchuk¹, Lennart Grabenhorst¹, Florian Selbach¹, Sarah Ochmann¹, Carolin Vietz², Birka Lalkens², Guillermo P. Acuna³, Philip Tinnefeld¹

¹Department Chemie and Center of Nanoscience, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 München, Germany
²LENA, Institute of Semiconductor Technology, Langer Kamp 6a/b, 38106 Braunschweig, Germany
³Department of Physics, University of Fribourg, Chemin du Museé 3, Fribourg CH-1700, Switzerland

The main requirements for new point-of-care diagnostic applications are quick and absolutely reliable sensing assays at low concentrations. For this reason, the originally low signals of single molecules have to be amplified by orders of magnitude. Our approach to amplify the optical signal of single fluorescent dyes is the use of self-assembled plasmonic nanoantennas based on DNA nanotechnology. Two metallic nanoparticles are attached to a DNA origami which build an antenna that directs the electric field of light strongly focused into a zeptoliter volume inside the gap. By placing a fluorescent dye precisely into this plasmonic hotspot, the fluorescence is enhanced by two to three orders of magnitude.[1-3] Due to these large enhancement factors it will be even possible to detect the fluorescence of single dyes with low-cost detectors, such as the camera of a smartphone.

This contribution shows that we are able to use the strong enhancement of fluorescence to detect a Zika virus nucleic acid by placing a molecular beacon into the plasmonic hotspot of the DNA origami nanoantenna.[4] Furthermore, we will also show our recent developments on a handheld smartphone-based microscope to detect this strongly enhanced fluorescence via an inexpensive and portable system.

Dmitry Tabakaev, Geraldine Haack, Robert Thew, Hugo Zbinden

Group of Applied Physics - Quantum Technologies, Chemin de Pinchat, 22, Carouge, 1227, Switzerland.

Two-photon absorption is a well-studied process, also well-known for its quadratic dependence of the absorption rate on the input flux, and thus for its inefficiency – typical two-photon absorption cross-section values for different materials are about 10⁻⁵⁰ cm⁴ s photon⁻¹, requiring high power laser pulses to compensate it. It automatically excludes samples with the low damage threshold from consideration.

The concept of entangled two-photon absorption (ETPA) predicts linear dependence [1,2] of its rate on the pair’s flux in the low-power regime, and provides a tool to overcome this obstacle – the linear process is obviously more efficient than quadratic, although presenting possible difficulties  for detection [3,4]. To show this signature, the ETPA induced fluorescence intensity of Rh6G ethanol solution was measured as a function of 1064 nm spontaneous down-converted (SPDC) photon pair flux and Rh6G concentration to obtain Rh6G ETPA cross-sections.

The developed methods have possible applications in sensing, spectroscopy, imaging and fluorescence microscopy, especially for biological objects in vivo, that could be susceptible to damage from intense laser schemes. It also combines a unique combination of sharp temporal response and penetration depth, typical for pulsed two-photon absorption systems, and high spectral resolution inherent to continuous-wave systems [2].

Daniel Thédié¹, Elke De Zitter², Matthew Jessop¹, Clarissa Liesche¹, Ninon Zala¹, Virgile Adam¹, Irina Gutsche¹, Dominique Bourgeois¹

¹Institut de Biologie Structurale, Grenoble, France
²Katholieke Universiteit Leuven, Belgium

Over the last decade, PALM (Photoactivated Localisation Microscopy) has been used in a variety of applications exploiting the single-molecule information inherent to the technique. One of them is quantitative PALM (qPALM), which can be used to determine the stoichiometry of protein complexes in-cellulo. This technique typically uses fluorescent proteins, which have the major advantage of being genetically encoded and of providing a one-to-one labelling of the protein of interest. Several qPALM methods have been developed so far, but all suffer from the incomplete maturation and slow blinking processes (i.e. the reversible entry into non-emissive states for up to tens of seconds) that are characteristic of fluorescent proteins. Here, we provide a solution to the slow blinking problem. Our recent mechanistic studies indeed provide a clue to largely suppress slow blinking by virtue of weak 488-nm light illumination. The use of this simple technique was first demonstrated in the case of spt-PALM, and we now show its value for qPALM. To this aim we developed a decameric construct based on the lysine decarboxylase LdcI, as a nanotemplate for counting. This strategy will be demonstrated and discussed in this presentation.

Jan Christoph Thiele, Oleksii Nevskyi, Jörg Enderlein

Third Institute of Physics – Biophysics, Georg August University, Göttingen, Germany

Localisation based super-resolution microscopy techniques like dSTORM¹ and PALM² usually rely on wide field or TIRF illumination and wide field detection. This allows for simultaneous acquisition of the whole field of view but comes with the limitations of a camera based detection. Instead, we use a confocal setup with pulsed excitation, single photon detection and a fast laser scanner. We evaluate different dyes and conditions to achieve a slow blinking and a high number of photons per event. We show that with the combination of confocal scanning and dSTORM blinking events can be localised and a super resolved image reconstructed. The huge advantage of a single photon detection is that each localisation contains information about the fluorescence lifetime. This is required to combine dSTORM with metal induced energy transfer (MIET), a distance depended modulation of the lifetime of a fluorophore by a thin metal film. MIET enables axial localising single fluorophores with a precision below 5 nm.³ Our goal is to achieve a high, isotropic 3D-localisation accuracy by combining the high lateral precision of dSTORM with the high axial precision of MIET.

Ashish Tiwari¹, Navneet C Verma², Anup Singh³, Chayan K Nandi², Jaspreet K Randhawa¹

¹School of Engineering, Indian Institute of Technology Mandi, Mandi, India
²School of Basic Sciences, Indian Institute of Technology Mandi, Mandi, India
³Center for BioMedical Engineering, Indian Institute of Technology Delhi, Delhi, India

Superparamagnetic iron oxide nanoparticles (SPIONs) have shown great potential as magnetic resonance (MR) contrast agents. However, lack of fluorescence in SPIONs restricts their applications in bioimaging. Herein, we present a single step synthesis of carbon coated core–shell multifunctional fluorescent SPIONs with excellent magnetic susceptibility, inherent fluorescence, multicolor emission and high photostability. MR imaging shows significant potential of SPIONs as a contrast agent in cancer diagnosis. Single particle fluorescence imaging revealed that SPIONs show photon counts as high as ∼90 000 and a single step photobleaching with a value of ∼65 sec than the Cyanine dye. Further, SPIONs show a higher photostability than the Rhodamine dye when illuminated with same laser power for 400 sec. Real time single particle measured photobleaching results show that the optical response of the SPIONs is much better than commonly used Rhodamine or Cyanine dyes. Multifunctionality of SPIONs suggests their great potential in multimodal imaging in theranostic applications.  

Roman Tsukanov, Arindam Ghosh, Steffen Mühle, Jan Christoph Thiele, Sebastian Isbaner, Ingo Gregor, Narain Karedla, Jörg Enderlein

Third Institute of Physics − Biophysics, Georg August University, 37077 Göttingen, Germany

Recent Metal-Induced Energy Transfer (MIET) experiments have shown the axial localization and co-localization of single molecules with nanometer precision. Here, we present Dyna-MIET (Dynamics using Metal-Induced Energy Transfer), a novel approach for detailed investigation of fast dynamic processes in polymer chains and biomolecules.

The method relies on the electrodynamic coupling between the excited-state of a fluorescing molecule (donor) and surface plasmons propagating along a thin metal film (acceptor). As a result, the intensity of the fluorophore alias donor is increasingly quenched, and its excited state lifetime reduced, with decreasing distance between the fluorescing molecule and the acceptor. We provide proof-of-concept results on loop opening and closing dynamics of prototypical DNA-hairpin molecules occurring at millisecond time-scale. Furthermore, we use Dyna-MIET to quantify nanosecond diffusion of the opened state of the hairpin in a harmonic potential well which cannot be easily done with existing techniques.

Luke Ugwuoke¹, Farooq Kyeyune¹, Tomas Mancal², Tjaart Kruger¹

¹Department of Physics, University of Pretoria, Private Bag X20, Lynnwood Rd, Hatfield 0028, South Africa
²Faculty of Mathematics and Physics, Charles University, Ka Karlovu 5, 121 16 Prague 2, Czech Republic

Serving as the main light-harvesting complex of plants and green algae, LHCII is the most abundant membrane protein on earth. This pigment-protein complex balances two vital functions: fast and efficient energy transfer in a noisy environment as well as effective photoprotection in demanding environments. The switch governing the dynamic equilibrium between the two functional states is still poorly understood, partly due to LHCII’s fluorescence quantum yield of only 0.26. We show, using both a theoretical approach based on a modified version of the Gersten-Nitzan model, and experiment, that chemically synthesized gold nanorods can enhance the fluorescence brightness of single LHCII complexes by two orders of magnitude and simultaneously decrease the fluorescence lifetime of the complexes by two orders of magnitude. This is caused by the combined effect of incident field enhancement, due to excitation of localized surface plasmons of the nanorod, and changes in the fluorescence quantum yield of LHCII, as a result of the modifications of its radiative and non-radiative decay rates near the nanorod, respectively. In addition, we report model parameters at which we obtained optimal Purcell factors in the single LHCII-nanorod system.

Farzaneh Vaghefikia¹, Alexandros Katranidis², Jörg Fitter^1,2

¹RWTH Aachen University, I. Physikalisches Institut (IA), 52074 Aachen, Germany
²Forschungszentrum Jülich, Institute of Complex Systems (ICS-5), 52428 Jülich, Germany

Most of our general knowledge in the field of protein biochemistry arises from experiments in highly diluted conditions. However, the complexity of processes inside the cell is affected by a wide variety of macromolecules present in the cytosol. To mimic the properties of cellular crowding, specific crowding agents can be dissolved in high concentrations, with crowder volume fractions of up to 20% for the prepared solutions. In our study we employed crowding agents with different molecular masses and of different structural compactness, like the synthetic polymers Polyethylene glycol and Ficoll, or  double-stranded DNA. With these crowder solutions we studied the translational diffusion of biological tracer molecules of different sizes (e.g., several proteins and 70S ribosomes) by employing fluorescence correlation spectroscopy (FCS). Studies as a function of crowder volume fraction revealed for several crowding conditions, that the diffusion coefficient of larger tracer molecules was reduced in a way that cannot be explained by the Stokes-Einstein relation, which indicates a molecular sieving effect [1]. Motivated by this observation we will analyze the activity of ribosomes in cell-free protein synthesis assays [2] under crowded conditions to gain information about the ribosome activity for more cell-like conditions.

Johannes Vandaele¹, Boris Louis¹, Kaizheng Liu², Paul Kouwer², Johan Hofkens¹, Rafael Camacho¹, Susana Rocha¹

¹Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, KU Leuven, Leuven, Belgium
²Institute for Molecules and Materials, Radboud University, Nijmegen, The Netherlands

It is well known that cells respond to mechanical properties of their local environment. Moreover, the tuning of the mechanical properties of synthetic scaffolds will influence cellular behaviour, more specifically, how cells degrade and remodel their surroundings. In order to design better biomimetic scaffolds, it is crucial to understand how the cell dynamically alters its surrounding.

Classically, structural characterization of materials is performed with electron microscopy or scanning probe microscopy. Despite the high spatial resolution achievable with these techniques, they are unable to measure dynamics ‘in situ’ and sample preparation can be a laborious process. In contrast, optical microscopy has the potential to unravel the dynamics in complex heterogeneous systems. The various possibilities of fluorescence microscopy to probe dynamics and heterogeneities, with molecular resolution, for a wide range of time scales makes it an ideal tool to address many topics of polymer science.

Here we use fluorescence microscopy to characterize the structure of synthetic hydrogels based on polyisocyanopeptides (PICs). Previous reports have shown that PIC polymers assemble into bundles, giving rise to a 3D structure similar to what is found in biopolymers such as fibrin and collagen. Although PICs, and biomaterials scaffolds in general, provide initially well-defined microenvironments for 3D culture of cells, less is known about the changes that occur over time. A quantitative description of local matrix remodelling by the cell and how the mechanical changes of the polymer network influence cell behaviour is lacking. By using fluorescence microscopy we aim to unravel and quantify the dynamics of cell-induced structural changes, at the micrometre scale.

Leonie Vollmar, Anastasia Holovchenko, Thorsten Hugel

Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstraße 23 a, 79104 Freiburg

Dynamic interactions between proteins and protein conformational changes are essential to regulate cellular processes. Single molecule FRET (smFRET) allows us to study those interactions and their dynamics in real time. Often proteins are immobilized in microfluidic chambers and imaged in total internal reflection (TIR) geometry with a high-end camera. EMCCD and scientific CMOS (sCMOS) cameras are two rivalling state-of the-art technologies for single molecule detection.

Here we compare the performance of EMCCD and sCMOS cameras for single molecule FRET dynamic analysis. Such an analysis is carried out on a pixel-by-pixel basis over many frames and hence requires a very high degree of stability and reproducibility which can only be achieved by excellent signal to noise characteristics for every pixel. To find the camera which suits these experiments best, both cameras were incorporated into an objective-type TIRF setup for a quantitative comparison. The fluorescence signal is split by a beam splitter to allow simultaneous imaging of one and the same single fluorophore. First dynamic single molecule FRET traces were obtained on both cameras. The quantification and discussion of our results will help scrutinizing which camera type to best use for what application.

Nicolaas T.M. van der Voort¹, Jan-Hendrik Budde¹, Nina Bartels², Cornelia Monzel², Claus A.M. Seidel¹

¹Heinrich Heine Universität, Molekulare Physikalische Chemie (MPC), Lehrstuhl II Prof. Dr. Claus Seidel, Gebäude 26.32, Etage 02, Universitätsstraße 1, 40225 Düsseldorf, Te.: +49 211 81-15881
²Heinrich Heine Universität, Mathematisch-Naturwissenschaftliche Fakultät, Experimentelle Medizinische Physik,Building 25.33, Room 01.24, Universitätsstraße 1
,40225 Düsseldorf

Single-molecule spectroscopy using Förster Resonance Energy Transfer (FRET) is a well-established tool to achieve structural resolution below the diffraction limit. FRET spectroscopy is able to resolve distances from 4 to 10 nm, making it ideally suited to study the structure and dynamics of proteins and protein clusters. Stimulated emission depletion (STED) microscopy is a spatial super-resolution technique that provides information on the spatial organization of fluorescently labelled proteins in cells  with a resolution of 40 nm and below. Together, STED microscopy and FRET spectroscopy synergize to provide structural and spatial information simultaneously in live cells. Here, we report our progress on the application of FRET in STED imaging. We present strategies to maximize the photons available for FRET and report on the maximum achievable STED resolution of our system. The STED-FRET approach is applied to the CD95 protein, a regulator of cell-death. CD95 exists in two conformational states and forms small (< 100 nm) clusters on the cellular membrane. Early results on the dynamic clustering properties of the CD95 protein will be presented.

Tianjin Yang, Benjamin Schuler

Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

Single-molecule Förster resonance energy transfer (FRET) is an ideal methodology for investigating the behavior of intrinsically disordered protein (IDP) by resolving heterogeneity between and within molecules, tackling many levels of complexity. However, the range from milliseconds to seconds required for probing rapid kinetics of their interactions or structure formation is notoriously difficult to access in single-molecule experiments. Especially for highly positively charged proteins, adhesion to the device surfaces has prevented the use of rapid microfluidic mixing by hydrodynamic focusing, one of the key techniques for resolving fast biomolecular kinetics at the single-molecule level.

We developed a generally applicable novel droplet-based microfluidic system that encapsulates the proteins in droplets and thus prevents protein adhesion to the channel surface. The droplets are mixed by chaotic advection in the sub-millisecond range, and then decelerated by orders of magnitude to enable confocal single-molecule detection. With this approach, biomolecular kinetics on timescales from milliseconds to seconds can be probed. We use the droplet-based microfluidic system with single-molecule FRET measurement to investigate the rapid binding and dissociation kinetics of IDPs, such as ACTR and NCBD or prothymosin α and linker histone H1.0.

Hua-Wei Yi^1,3, Wei-Ping Zhang², Chun Tang^1,3,4

¹CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key, Laboratory of Magnetic Resonance and Atomic Molecular Physics, National, Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and, Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province, 430071, China
²Department of Pharmacology, Institute of Neuroscience, Key, Laboratory of Medical Neurobiology of Ministry of Health of China, and, Zhejiang Province Key Laboratory of Mental Disorder’s Management, Zhejiang, University School of Medicine, Hangzhou, Zhejiang Province 310027, China.
³University of Chinese Academy of Sciences, Beijing, 100049, China.
⁴Wuhan National Laboratory for Optoelectronics, Wuhan, Hubei Province, 430074, China

         The interaction between K48-linked ubiquitin (Ub) chain and ubiquitin receptors, namely Rpn1, Rpn10 and Rpn13, on proteasome is the first step in the degradation of proteins by the ubiquitin-proteasome system (UPS). We and other showed recently that Ser65 phosphorylation triggers a pH-dependent conformational switch in phosphorylated ubiquitin (pUb), so we explore here if phosphorylation affects the structure of the K48-linked ubiquitin chain and further affect the degradation of substrate proteins by proteasome. With confocal single-molecule imaging platform (equipped with an A1 confocal microscope (Nikon), two picosecond-pulsed lasers (PicoQuant), two SPCMAQRH detectors (Excelitas) et al.), we found that K48-diUb fluctuates among three distinct conformational states, and phosphorylation makes K48-diUb more compact, which weakens binding to the ubiquitin receptor Rpn1, Rpn10 but increases binding to the ubiquitin receptor Rpn13. In addition, using total internal reflection (TIRF) imaging system, we found that phosphorylation can reduce the dwell time of the K48-linked ubiquitin chain on proteasome. Finally, through in vitro degradation experiments, we demonstrated that phosphorylation of K48-linked ubiquitin chain can inhibit the degradation of proteasome to its substrates. Phosphorylation can affect the structure of the K48-linked ubiquitin chain and thereby inhibit the ubiquitin-proteasome system, which opens a new window for modulating proteasomal function.

Olessya Yukhnovets¹, Henning Höfig^1,2, Jörg Fitter^1,2

¹I. Physikalisches Institut (IA), RWTH Aachen University, 52074 Aachen, Germany
²Institute of Complex Systems ICS-5, Forschungszentrum Jülich, 52428 Jülich, Germany

In most biological processes the strength and the extent of intermolecular binding are crucial. By attaching fluorophores of different colors to each of the binding partners, simultaneous dual-color fluorescence detections techniques can be employed to measure binding interactions. Single molecule two-color coincidence detection (TCCD) is a powerful tool for molecular binding characterization¹. It utilizes confocal fluorescence microscopy principles and single-molecule burst analysis to determine the fraction of bound and unbound molecules diffusing through the confocal volume. Recently we validated the use of two-color coincidence detection for biological applications^2,3. Due to the mismatch of confocal volumes for two excitation wavelengths and differences in emission efficiency of fluorophores, TCCD suffers from an underestimation of coincident events. Here we applied brightness-gated TCCD to overcome this drawback. Furthermore, we considered concentration effects on chance coincidence events and identified most reasonable conditions for TCCD measurements. For this purpose, we used custom designed single- and double-labeled double-stranded DNA model samples as positive and negative controls for precise determination of molecular binding fraction.

Daniel Zalami¹, Oliver Grimm^2,3, Felix H. Schacher^2,3,4, Uwe Gerken¹, Jürgen Köhler^1,5,6

¹Spectroscopy of soft Matter, University of Bayreuth, Universitätsstraße 30, 94557 Bayreuth, Germany
²Institute for Organic Chemistry and Macromolecular Chemistry (IOMC), Friedrich-Schiller-University Jena, Lessingstraße 8, 07743 Jena, Germany
³Jena Center of Soft Matter (JCSM), Friedrich-Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
⁴Center for Energy and Environmental Chemistry (CEEC), Friedrich-Schiller-University Jena, Philosophenweg 7a, 07743 Jena, Germany
⁵Bavarian Polymer Institute, Universitätsstraße 30, 94557 Bayreuth, Germany
⁶Bayreuth Institute of Macromolecular Research (BIMF), University of Bayreuth, Universitätsstraße 30, 94557 Bayreuth, Germany

Porous media with cavities in the nanometer range are of great importance for drug delivery or filtration. Typically, the pore structure of such media is characterized using high-resolution techniques such as electron microscopy or atomic force microscopy. However, these techniques are restricted to the surface of the material and/or are highly invasive. We applied single-particle orbit tracking (SPOT) [1,2] for investigating the three-dimensional pore size distribution of nanoporous polystyrene-block-polyisoprene-block-poly(N-isopropylacrylamide) (PS₄₃-b-PI₄₀-b-PNIPAAm₁₇) triblock terpolymer membranes under liquid filled conditions. In order to do so, we monitor the diffusion behaviour of single dye-labeled particles of different diameter with a spatial precision of about 10 nm, thereby mapping out the cavity structure of the membrane. Using fluorescent tracers with a diameter of about 10% of the relevant void structures, the tracking experiments yielded results that were comparable to those obtained from reference experiments employing  environmental scanning electron microscopy (eSEM).  [3].