Suliana Manley

EPFL, Laboratory of Experimental Biophysics LEB, Lausanne, Switzerland, suliana.manley@epfl.ch

Single molecule localization microscopies (SMLM)—such as STORM, PALM, and PAINT—occupy a special niche in the biologist's toolbox because they can achieve among the highest resolution in fluorescence microscopy. Yet, many important biological questions remain out of reach due to challenges in acquiring and handling statistically significant SMLM datasets. Previously, we created high-throughput PALM by building an automated microscope to image hundreds of bacteria cells, live, 3D, and across cell cycle. To complement this, have now constructed a novel microscope to acquire large field of view images in an automated way. To achieve this, we combined a large-detector scientific CMOS camera with an optimized laser illumination field and software developed in our group. We can now acquire multicolor PALM or STORM images of multiple eukaryotic cells, or hundreds of bacteria cells in a single image. Together with multi-field-of-view acquisitions, we can collect terabytes of data in a few hours.

This provides unique opportunities for understanding the organization of biological specimens. For example, macromolecular complexes within cells usually contain multiple protein species. Therefore, multi-color fluorescence microscopy approaches must be deployed to decipher their complex architecture and underlying assembly mechanisms. To study the organization of such complexes, particle-based analysis has proven to be powerful, but has been limited so far by difficulties in generating large multi-color particle libraries, as well as the complexity of orientational alignment. We have addressed both challenges and, as a result, present a novel framework for deciphering the 3D organization of protein complexes composed of multiple components.

Maximiliaan Huisman, David Grunwald

University of Massachusetts Medical School, 368 Plantation Street, Worcester MA 01605, USA

Wide-field microscopy can reach 10–50nm localization precision at room temperature by estimating of the center location of diffraction-limited spots. At cryogenic temperatures, localization precisions well below 1nm can be reached thanks to increased photostability of the fluorophores[1]. This improvement in localization precision effectively eliminates optics as the bottleneck of resolving power for static phenomena with sparse emitters – for a single color channel. As refractive optics are wavelength-dependent, chromatic aberrations are likely to pose the next big challenge to multi-color imaging at the nanometer scale.

Here, we present a mirror-based cryo-fluorescence microscope that promises multi-color wide-field imaging of sparse emitters with nanometer localization precision. By making use of a reflective, mirror-based objective and parabolic mirrors, dispersion by refractive elements of the microscope is eliminated, removing the main source of chromatic aberrations. In addition to the resolution improvement, fixation of the dipole moment of the fluorophore allows novel avenues of counting individual fluorophores, as well as determining distance, orientation, and stoichiometry. The ability to interrogate molecules with nanometer precision opens up a new range of macro-molecules (such as DNA, RNA and protein complexes) for optical investigation and has the potential to solve countless outstanding questions in cell and molecular biology.

Christos Karathanasis¹, Franziska Fricke¹, Sjoerd van Wijk², Ivan Dikic³, Gerhard Hummer⁴, Mike Heilemann¹

¹Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, 60438 Frankfurt, Germany
²Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, 60528 Frankfurt, Germany
³Institute for Biochemistry II, Goethe-University Frankfurt, 60590 Frankfurt, Germany
⁴Max Planck Institute of Biophysics, Department of Theoretical Biophysics, Goethe-University Frankfurt, 60438 Frankfurt, Germany

Membrane receptors are the starting point for signaling cascades in cells. They often organize into multimeric units and complex protein clusters, with the purpose to render them as functional units or starting hubs to initiate a specific cellular response.

The organization of plasma membrane proteins in such clusters is to-date inaccessible to optical super-resolution microscopy, because of the small size of the proteins and the dens packing.

We developed a tool that reports on the stoichiometry of plasma membrane proteins in clusters based on single-molecule localization microscopy (SMLM) data [1]. It builds on the available kinetic data of photoswitchable fluorescent proteins (“blinking”), which is routinely recorded in an SMLM experiment [2]. Together with a general model that describes fluorophore blinking properties, we demonstrate that we can extract the oligomeric state of proteins in small and unresolved protein clusters [3]. We will present the theoretical background of this method, SMLM data on various oligomeric proteins expressed in the cell membrane, and examples of applications to signaling-initiating membrane receptors.

Alexey Chizhik, Jörg Enderlein

Georg-August-University Göttingen, Thirst Institute of Physics, 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].

Marie-Lena IE Harwardt¹, Phoebe Young¹, Thorsten Wohland², Hartmut H Niemann³, Mike Heilemann¹, Marina S Dietz¹

¹Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Frankfurt (Main), Germany
²Departments of Biological Sciences and Chemistry, National University of Singapore, Singapore
³Department of Chemistry, Bielefeld University, Bielefeld, Germany

The receptor tyrosine kinase MET regulates diverse processes in vertebrate development as well as cell motility and proliferation. Beside its physiological ligand hepatocyte growth factor/scatter factor, MET is also targeted by the bacterial surface protein internalin B (InlB) of L. monocytogenes as a pathway for invasion of epithelial cells.

Using single-molecule photobleaching microscopy, we found that the MET receptor is partially dimeric prior to ligand binding, and that the dimer fraction increases upon activation with InlB [1]. We established a method to measure ligand affinities directly on cells [2], and determined the binding constant of InlB to MET via fluorescence correlation spectroscopy (FCS) and single-molecule imaging [1,2]. Using single-molecule tracking and imaging FCS in live cells, we found that MET receptor mobility changes following ligand binding, and we studied possible endocytosis pathways [3]. A comparison of the two methods showed their complementary information content with regard to diffusion properties of membrane molecules [4].

Jiji Chen

Advanced Imaging & Microscopy Resource, National Institute of Biomedical Imaging & Bioengineering, National Institutes of Health, Bethesda, MD 20892, United States, jiji.chen@nih.gov

Transcription factors (TF) play a seminal role in cell gene expression. Despite the extensive biochemical studies in the past, the understanding of molecular mechanisms underlying enhancer-mediated gene regulation is limited. We developed single molecule imaging in combination with multifocus microscopy, which allows imaging multiple focal planes simultaneously, to directly visualize TF dynamics behavior in 3D. Our findings reveal fundamental aspects of gene regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters. Other efforts here include developing instant TIRF structure illumination microscopy (SIM) for fast, high-contrast super-resolution imaging on the plasma membrane.

Johan Paulsson

Harvard University, Department of Systems Biology, Johan Paulsson Lab, Boston, US

Many molecules in cells are present in such low numbers that individual chemical events can have great effects. I will present some methods for accurately counting proteins in bacteria, for observing events that only occur once every ten million cell divisions, and for genotype-to-phenotype mapping of millions of strains at the level of long-term, single-cell dynamics. Applying these methods e.g. shows that many key processes in cells, from cell fate decisions to the repair of DNA, are largely driven by fluctuations in low-abundance proteins.

Bergstrand Jan¹, Lei Xu¹, Xinyan Miao¹, Nailin Li², Ozan Öktem³, Marta Lomnytska⁴, Gert Auer¹, Jerker Widengren¹

¹Royal Institute of Technology (KTH), Dept. Applied Physics, AlbaNova University Center, SE-106 91, Stockholm, Sweden
²Karolinska Univ Hospital, Karolinska Institutet, Dept. Medicine, 171 76 Stockholm, Sweden
³Royal Institute of Technology (KTH), Dept. Mathematics, 100 44, Stockholm, Sweden
⁴Karolinska Univ Hospital, Karolinska Institutet, Dept. Gynecology and Obstetrics, 171 76 Stockholm, Sweden

In previous work, we have shown that different platelet activation states, induced by thrombin and ADP, can be identified from the spatial distribution patterns of certain proteins within the platelets (pro-angiogenic VEGF, anti-angiogenic PF-4 and fibrinogen), when imaged with dual-color STED microscopy with a resolution ~40 nm [1,2].
In this work, we investigated if protein distribution in platelets can also be influenced by the presence of tumor cells, and to what extent this can be detected by fluorescence-based super-resolution imaging. For this purpose, platelets were co-cultured with tumor cell-lines representing different forms of breast- and ovarian cancer. Localization patterns of different proteins were then studied by STED microscopy with a resolution down to 20 nm. We observed that in particular the protein P-selectin localizes in circular patterns (diameter ~200-250nm) consistent with so called α–granules residing within the platelets. This circular pattern of P-selectin was more frequently observed within platelets exposed to tumor cells compared to non-exposed platelets or platelets co-cultured with normal cells. This we could also verify by image analysis using machine learning algorithms. Furthermore we investigated to what extent the observed tumor cell activation of the platelets can be linked to chemical activators such as ADP, thrombin and thromboxane A2.

Nina S. Deussner¹, Alexander Auer^2,3, Maximilian T. Strauss^2,3, Sebastian Malkusch¹, Marina S. Dietz¹, Hans-Dieter Barth¹, Ralf Jungmann^2,3, Mike Heilemann¹

¹Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt (Main), Germany
²Department of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany
³Max Planck Institute of Biochemistry, Martinsried, Germany

DNA-PAINT is an optical super-resolution microscopy method that is based on transient binding of small fluorophore labeled DNA-oligonucleotides and can visualize nano-scale molecules arrangements with excellent multiplexing capabilities [1]. Further DNA-PAINT is limited by high background signal due to freely diffusing imager strands. To minimize background, DNA-PAINT has been combined with single molecule Förster resonance energy transfer (smFRET) [2], [3]. Due to specific distance dependency of smFRET, FRET-PAINT is ideally suited to identify different targets by their FRET efficiencies.

By using FRET-PAINT we achieve multiplexed detection with sub-diffraction resolution. We establish correlated smFRET and super-resolution on DNA origami structures. They were designed with binding sequences that are targeted by two oligonucleotides labeled with donor and acceptor generating different FRET efficiencies. We demonstrate the read out of FRET efficiencies in super-resolved binding sites. This combination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging with low background, in conjunction with distance sensitive readout in the 1-10 nanometer range.

Carlas Smith

TU-Delft, Netherlands

Point spread function (PSF) engineering is used in single emitter localization to measure the emitter position in 3D and possibly other parameters such as the emission color or dipole orientation as well. Advanced PSF models such as spline fits to experimental PSFs or the vectorial PSF model can be used in the corresponding localization algorithms in order to model the intricate spot shape and deformations correctly. The complexity of the optical architecture and fit model makes PSF engineering approaches particularly sensitive to optical aberrations. Here, we present a calibration and alignment protocol for fluorescence microscopes equipped with a spatial light modulator (SLM) with the goal of establishing a wavefront error well below the diffraction limit for optimum application of complex engineered PSFs. We achieve high-precision wavefront control, to a level below 20 mλ wavefront aberration over a 30 minute time window after the calibration procedure, using a separate light path for calibrating the pixel-to-pixel variations of the SLM, and alignment of the SLM with respect to the optical axis and Fourier plane within 3 μm (x/y) and 100 μm (z) error. Aberrations are retrieved from a fit of the vectorial PSF model to a bead z-stack and compensated with a residual wavefront error comparable to the error of the SLM calibration step. This well-calibrated and corrected setup makes it possible to create complex ‘3D+λ’ PSFs that fit very well to the vectorial PSF model. Proof-of-principle bead experiments show precisions below 10 nm in x, y, and λ, and below 20 nm in z over an axial range of 1 μm with 2000 signal photons and 12 background photons.

Alex Oppermann, Dominik Wöll

RWTH Aachen University, Institute of Physical Chemistry, Landoltweg 2, 52074 Aachen

Microgels are cross-linked, swollen polymer networks with sizes in the range of 100 nm to 100 µm. They exhibit properties of both a macromolecule and a colloid. Depending on the monomer composition, microgels can show stimuli-dependent swelling and deswelling, resulting in a change in size of the polymer network. Stimuli can be, for example, temperature, pH, solvent composition or redox potential offering a wide range of possible applications for microgels.

The behavior of microgels at interfaces is of special interest for several applications. Due to their softness, the properties of microgels are drastically influenced by the presence of an interface, compared to their properties in solution. Therefore, sophisticated analytical tools are required to investigate the properties of microgel systems. However, microgels are usually swollen with more than 80-90 % solvent and therefore give only low contrast in electron microscopy.

We use singe molecule localization microscopy (SMLM) to overcome the limits of these techniques. Using SMLM we visualize the deformation of single microgels adsorbed to a water-glass interface. Furthermore, we show that poly(N-isopropylacrylamide) microgels retain their temperature sensitive behavior after adsorption to the water-glass interface.

Jérome Wenger

Chargé de recherche CNRS, Institut Fresnel CNRS UMR7249, Faculté des Sciences de St Jérome , 13397 Marseille Cedex, France, jerome.wenger@fresnel.fr

Single molecule fluorescence is receiving a widespread interest in biochemical and biophysical sciences, but its detection on optical microscopes remains limited by the diffraction phenomenon. Recent developments in nanophotonics offer new opportunities to improve single molecule fluorescence detection beyond confocal microscopy. Thanks to nanophotonics, it is now possible to manipulate light on dimensions much beyond the optical wavelength, down to the nanometer size of a single molecule. Nanophotonic devices allow to locally confine the light, inducing large absorption and diffusion cross sections and enhancing the local electromagnetic fields. This talk will review various approaches using nanophotonic devices to enhance the fluorescence detection of single molecules. We will specifically address two questions of interest for biophysicists and biochemists: how to enhance the fluorescence signal detected per molecule and how to detect a single molecule in a highly concentrated solution?

Marcelle König, Caroline Berlage, Paja Reisch, Felix Koberling, Haisen Ta, Rainer Erdmann

PicoQuant, Rudower Chaussee 29, 12489 Berlin, Germany, info@picoquant.com

Single-molecule fluorescence microscopy has been established in the Life Sciences as an essential tool to study the characteristics and dynamics of individual fluorescent emitters both in vitro as well as in vivo. Still, acquiring quantitative information from the confocal observation volume is a challenging task, whereas knowing the absolute number or concentration of proteins in, e.g., cellular structures can significantly improve our understanding of cell biology being an important step towards quantitative microscopy.

In this talk, a new quantitative analytical tool will be presented that is based on recording coincident photons. The approach, Counting by Photon Statistics (CoPS), relies on a statistical analysis of detected photon coincidences to estimate the number of independent fluorescent labels in the observation volume [1]. Hereby, CoPS exploits the photon antibunching effect: a single photon emitter can only generate one photon at a time. Originally developed for point measurements, CoPS recently has been extended to an imaging scheme [2].

Using a confocal fluorescence microscopy setup with pulsed excitation, four single-photon detectors and a parallel time-correlated single photon counting electronics (MicroTime 200, PicoQuant) we prove the applicability of the method with artificial model systems (immobilized DNA origami) and present first steps towards biological samples.

Frederike Erb¹, Christoph Müller², Fedor Jelezko³, Kay-E. Gottschalk¹

¹Institut für experimentelle Physik, Universität Ulm, Ulm
²NVision Imaging Technologies, Universität Ulm, Ulm
³Institut für Quantenoptik, Universität Ulm, Ulm

Fluorescent nanodiamonds (FNDs) offer various new imaging and metrology approaches, especially in the life sciences. Nanodiamonds containing nitrogen-vacancy centers (NV-centers) as fluorophores emit light in the near-infrared window of bioimaging. Their luminescence properties depend on the environment and thus FNDs cannot only be used for bioimaging but also may find applications as part of various biosensors, being biocompatible and non-cytotoxic.

To fully control the NV-center’s quantum state and use its potential as a biosensor, custom designed laser and microwave pulses have to be used during the experiments. We present our excitation and detection scheme to measure the T1-Time of an NV-center in nanodiamond. Our experiments use the PicoQuant MicroTime 200 setup and a custom developed control software, which enables the user to induce laser pulses of different lengths and with varying dark times onto the sample. It features an easy to use and intuitive graphical interface to set laser intensity and pulse schemes. It is also possible to trigger detection at desired steps in the pulsing scheme and collect and analyse data in SymPhoTime 64.

Jerker Widengren

Exp. Biomol. Physics / Applied Physics, Royal Inst. Technology (KTH), Albanova Univ Center, 106 91 Stockholm, Sweden, e-mail contact: jwideng@kth.se

First, this presentation will emphasize that fluorescence blinking, a major problem in single molecule studies (reducing molecular brightness and S/N) and a prerequisite for all forms of super-resolution imaging, can also provide additional, to-date largely unexploited, information about biomolecules, their interactions and their immediate environment. By two major approaches, where the transient state information is obtained either from fluorescence fluctuation analysis (FCS) or by recording the time-averaged fluorescence response to a time-modulated excitation (transient state (TRAST) imaging/spectroscopy), it is possible to combine the detection sensitivity of the fluorescence signal with the environmental sensitivity of the long-lived transient states. Proof-of-principle experiments, advantages, limitations and applications will be discussed, including live cell transient state (TRAST) imaging of cell membrane fluidity and cellular metabolism.

Second, it will be shown how diffraction-unlimited imaging of cellular protein distribution patterns using Stimulated Emission Depletion (STED) nanoscopy can potentially provide new diagnostic parameters on the level of individual cells, and also give further insights into underlying disease mechanisms. Examples including cultured cells, clinically sampled breast cancer cells and platelets will be given.

Jan Sykora³, Zbynek Prokop^1,2, Avisek Ghose³, Piia Kokkonen^1,2, David Bednar^1,2, Mariana Amaro³, Sarka Bidmanova^1,2, Jiri Damborsky^1,2, Martin Hof³

¹Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic
²International Clinical Research Center, St. Anne's University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic
³J. Heyrovsky Institute of Physical Chemistry of the ASCR, v. v. i., Dolejskova 3, 182 23 Prague 8, Czech Republic

Haloalkane dehalogenases (HLDs; EC 3.5.1.8) catalyze conversion of different halogenated alkanes, alcohols and carboxylic acids to their non-halogenated analogues. Their broad substrate specificity and their usefulness for biocatalysis, biodegradation and decontamination have drawn enormous attention in recent years [1]. Motivation of this study was to elucidate the role of the access tunnel dynamics in enzymatic catalysis of a member of HLD family, LinB. Specifically, LinB86, a mutant with a superior enzymatic activity [2], was investigated by means of the single molecule experiments utilizing the PET-FCS approach [3]. We observed (sub)-millisecond conformational dynamics which was dependent on the presence of product Br-. Comparable results were obtained with transient kinetic data and molecular dynamics simulations, which brings a strong indication that the LinB86 dynamics can be the crucial factor in its enhanced enzymatic activity.

Acknowledgement:

Czech science foundation is greatly acknowledged via 16-06096S.

Mariano Gonzalez Pisfil^1,2, Marcelle König¹, Benedikt Krämer¹, Paja Reisch¹, Felix Koberling¹, Matthias Patting¹, Rhys Dowler¹, Andreas Herrmann², 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 essential tool for understanding the dynamics of complex cellular processes. By applying the approach know as scanning FCS (sFCS), significant improvements can be obtained when studying slow moving molecules as is often the case in cell membranes. In sFCS, the excitation volume is scanned rapidly, which reduces the time required to record a high number of transits.

The shorter residence times lead to lower photon doses experienced by each detected molecule and thus reduce the risk of photobleaching. This is especially important for sensitive fluorophores or when performing Stimulated Emission Depletion (STED) FCS measurements. The additional information gained by recording the focal position during measurement can be used to determine the shape and size of the confocal volume without requiring an additional calibration step. This information can also be used to determine parameters related to flow and other active transport mechanisms.

Using the unique pattern matching analysis method [1], multiple species can be excited with the same laser and later discriminated while ensuring that the excitation volume remains identical for all species. This approach allows for a high degree of separation even for labels with very close emission wavelengths.

Johan Tornmalm¹, Elin Sandberg¹, Heike Hevekerl¹, Mihailo Rabasovic², Erik Norberg³, Jerker Widengren¹

¹Royal Institute of Technology (KTH), Dept. Applied Physics, AlbaNova University Center, SE-106 91, Stockholm, Sweden
²Univ Belgrade, Inst Phys, Pregrevica 118, Belgrade 11080, Serbia
³Karolinska Institutet, Dept. Physiol. and Pharmacol., 171 77 Solna, Sweden

Fluorophore blinking patterns, caused by transitions to long-lived, non-luminescent states such as triplet, oxidized or isomerized states, can provide sensitive information about the molecule’s local environment. Specifically, the long lifetime of such dark states allows sensing of low frequency interactions on the micro- or milli-second timescale. This forms an additional fluorescence readout, complementary to more traditional parameters such as intensity, lifetime, polarization and wavelength.

Transient state (TRAST) monitoring uses modulated excitation to map out the dynamics of such blinking patterns, without relying on time resolved detection[1,2]. The time dependent excitation can be implemented by for instance direct modulation of the laser, or by scanning the laser across the sample.

We have used TRAST to characterize the autofluorescent coenzymes NADH and FAD[3] as well as the amino acid tryptophan. Solution measurements revealed several dark states and their sensitivity to biologically relevant factors such as oxygen and anti-oxidants was demonstrated. We also constructed electronic state models suitable for analysing these compounds with TRAST.

Finally, we could analyse NADH/NAD+ redox balances in cell lysates [4], image NADH in live cells using two-photon scanning TRAST and track conformational changes in spider silk protein by UV TRAST on native tryptophan residues[5].

Nicolas Plachta

Institute of Molecular and Cell Biology, A*STAR, Singapore, plachtan@imcb.a-star.edu.sg

Our goal is to reveal how mammalian cells resolve their fate, shape and position within a living organism. Because fixed specimens cannot capture cell dynamics, we use live imaging methods to study cells directly in the developing mouse embryo. We recently combined photoactivatio and FCS techniques to show how transcription factors bind to DNA within single cells of the living embryo to control cell fate. We also discovered that cells extend a new type of filopodia protrusions to pull their neighboring cells closer, revealing a mechanism for embryo compaction. Finally, we uncovered how cells reorganize their actin and microtubule networks to regulate the first cell lineage segregation, create the pluripotent inner mass, and establish the first forms of epithelial organization during development. Combined, our findings reveal some of the key dynamic mechanisms that regulate how cells function in real time and in vivo.

Fabian Port, Lydia Rebehn, Lucia Cajal de la Macorra, Kay-E. Gottschalk

Institut für Experimentelle Physik, Universität Ulm, Ulm

Cell adhesion to the extracellular matrix does not only function as an anchor, it also enables cells to sense their environment [1]. The focal adhesion complex, which is responsible for these adhesions, is a complex structure consisting of a multitude of different proteins.  Despite this important role, its structure remains difficult to resolve [2]. Knowing the exact position of these proteins in the focal adhesion complex is necessary to understand this working principle.

For a detailed analysis of the focal adhesion architecture, a method to measure distances with nm precision is needed. A technique which meets this challenge is Metal Induced Energy Transfer (MIET) [3]. Here we show a first analysis of the distance between various focal adhesion proteins and the underlying surface in different cell lines and demonstrate the usefulness of MIET for analyzing molecular structures close to the basal membrane.

Max J. Schnepf^1,2, Tobias A. F. König^1,2

¹Leibniz Institute of Polymer Research (IPF), Institute of Physical Chemistry and Polymer Physics, Hohe Str. 6, 01069 Dresden, Germany
²(2) Cluster of Excellence Center for Advancing Electronics Dresden (cfaed), Technische Universität Dresden, Germany

We present a colloidal based system consisting of a fluorescent emitter coupled to various systems, such as a dielectric or metallic interface as well as a metallic cavity to study their radiative decay processes. Supported by finite-difference time-domain (FDTD) simulations, we correlate the non-radiative and radiative decay rates with the absorption and scattering cross section efficiencies, respectively. On a single particle level, we use atomic force microscopy (AFM), scanning electron microscopy (SEM), scattering spectroscopy, fluorescence life time imaging (FLIM) and time-correlated single photon counting (TCSPC) to evaluate the enhanced fluorescence decay at the same location. The spectral overlap between the localized surface plasmon resonance (LSPR) of the metallic particles and the fluorescence emission results in energy transfer due to near-field interactions. We demonstrate enhancement of spontaneous emission and significant reduction of decay rates. Moreover, the presented gain material can be integrated cost efficiently in existing self-assembled plasmonic lattices, to study their coherent energy transfer. In comparison to top-down lithographic methods, the colloidal synthesis and metallic film-coupled cavities are an inherently inexpensive and flexible platform for large-scale applications such as quantum information systems and ultralow-power switches.

Soheil Mojiri¹, Arindam Ghosh¹, Ashvini Purohit², Dominik Wöll², Ingo Gregor¹, Narain Karedla¹, Jörg Enderlein¹

¹Third Institute of Physics, University of Göttingen, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany
²Institute of Physical Chemistry, RWTH Aachen University, Landoltweg 2, D-52056 Aachen, Germany

We present a novel approach to localize single molecules with nanometer accuracy in all three dimensions. This is done by bringing two techniques together, sm-MIET (single-molecule Metal-Induced Energy Transfer) [1] and defocused imaging [2], which provides axial distance of a fluorophore from a metal surface, and molecular orientation respectively. Measurement is done using a home-built confocal microscope where the emission light is split into two different detection pathways, a single-photon sensitive avalanche photodiode (τ-SPAD) and a camera. Excited-state lifetime of the dye is measured in the τ-SPAD detection channel and the obtained values are used to calculate the distance from the surface using the theory of smMIET [3]. On the other hand, defocused intensity patterns of the rotating fluorophore are recorded in the camera, which is later matched with computed patterns to obtain orientation information. We have estimated the theoretical lower bounds for lateral and axial localization accuracy recently [4]. Here, we provide proof-of-concept results by probing rotational diffusion of the dye- terrylene diimide (TDI) embedded in a thin polymer (pBMA) film spin-coated on a gold surface containing a dielectric SiO2 spacer of 30 nm thickness. These experiments are exclusively directed to test whether the rotational diffusion (local viscosity) is height dependent or not. Additionally, we would like to answer if the polymer show a viscosity gradient along the axial direction.

Adai Colom^1,2, Saeideh Soleimanpour^2,3, Emmanuel Derivery¹, Caterina Tomba^1,2, Naomi Saka^2,3, Marcos González-Gaitán^1,2, Stefan Matile^2,3, Aurélien Roux^1,2

¹Biochemistry Department, University of Geneva, CH-1211 Geneva, Switzerland.
²Swiss National Centre for Competence in Research Programme Chemical Biology, CH-1211 Geneva, Switzerland.
³School of Chemistry and Biochemistry, University of Geneva, CH-1211 Geneva, Switzerland

Cells are delimited by a lipid bilayer that is highly deformable, a property essential to many cell processes such as motility, endocytosis, cell division. During these deformations, lipid membranes experience stretch causing membrane tension. Membrane tension is thereby a major regulator of membrane remodeling cell processes, but has proved very hard to measure in vivo. FliptR (for Fluorescent LIPid Tension Reporter) can monitor changes of membrane tension by changing its fluorescence lifetime as a function of the twist between its fluorescent groups. We show that fluorescence lifetime depends linearly on membrane tension within cells, allowing for an easy quantification of membrane tension by fluorescence lifetime imaging microscopy (FLIM) and Fast-FLIM. We further showed using model membranes that this linear dependency between lifetime of the probe and membrane tension relies on a membrane-tension dependent lipid phase separation. We obtained a calibration curves that allow to measure accurately membrane tension using FLIM and Fast-FLIM in vivo. FliptR thus tremendously facilitates membrane tension measurements, opening new possibilities of studying membrane tension in cell processes and developing tissues.

Maria F. Garcia-Parajo

ICFO-Institute of Photonic Sciences, Castelldefels (Barcelona), Spain. & ICREA- Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain, maria.garcia-parajo@icfo.eu

The quest for optical imaging of biological processes at the nanoscale has driven in recent years a swift development of a large number of nanoscopy techniques based on far-field optics. These, so-called super-resolution methods are providing new capabilities for probing biology at the nanoscale by fluorescence. In parallel, and within the nanophotonics field, photonic antennas have emerged as excellent alternative candidates to break the diffraction limit of light by enhancing electromagnetic fields into regions of space much smaller than the wavelength of light. I will describe our efforts towards the fabrication of different nanoantenna probe configurations as well as in-plane 2D antenna arrays for applications in nano-imaging and spectroscopy of living cells with unprecedented resolution and sensitivity. In terms of applications, we have taken advantage of the superior optical performance of in-plane 2D-antennas arrays together with their extreme planarity to enquire on the nanoscale dynamics of multicomponent lipid bilayers. Our results reveal the coexistence of cholesterol-enriched fluctuating nanoscopic domains on mimetic and living cell membranes, in the microsecond scale and with characteristic sizes below 10nm. These nanoscale assemblies might represent lipid raft precursors that in the absence of proteins and/or other molecular stabilizing factors, are poised to be highly transient.

Giorgio Tortarolo, Marco Castello, Mauro Buttafava, Takahiro Deguchi, Federica Villa, Sami Koho, Paolo Bianchini, Colin J. R. Sheppard, Alberto Diaspro, Alberto Tosi, Giuseppe Vicidomini

Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Via Morego 30, 16163, Genoa, Italy

Confocal laser scanning microscopy (CLSM) established as a pivotal technique in live cell imaging thanks to its combination with different spectroscopy methods, e.g., fluorescence lifetime (FL), and its optical sectioning capability, i.e., photons from out-of-focus planes are discarded by positioning a pinhole in front of the detector. Whereas closing the pinhole grants a spatial resolution improvement, it also lowers the number of photons registered, de facto reducing the signal to noise ratio (SNR) of the final image. We recently upgraded [1] the detection unit of a confocal microscope replacing the traditional single-point detector with a novel SPAD (single photon avalanche diode) array of 5 x 5 elements: this approach represents the most straightforward implementation of image scanning microscopy (ISM) [2], allowing to overcome the above-mentioned tradeoff between resolution and SNR. We have shown through FRC analysis [3] that the desired resolution can be achieved with a 10-fold reduction of the excitation intensity, a feature that is significant for live-cell imaging. Additionally, we exploited the single-photon detection ability (< 200 ps photon-jitter) of the SPAD array combining ISM with FL to obtain an image with higher resolution and better FL precision with respect to the confocal counterpart. To investigate fast molecular dynamics otherwise hidden by the scanning nature of CLSM, we are exploring the possibility to use SPAD array to implement a feedback-based single particle tracking (SPT) system and to perform fluorescence correlation spectroscopy (FCS). This system will pave the way for a novel method able to correlate the dynamics of a biomolecule with its structural and/or environmental information, via fluorescence resonance energy transfer (FRET) analysis.

Dominik Brox¹, Andreas Haderspeck¹, Felix Braun¹, Klaus Yserentant¹, Philipp Werther², Richard Wombacher², Johan Hummert¹, Dirk-Peter Herten¹

¹Institute for Physical Chemistry, Heidelberg University, Germany
²Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Germany

Fluorescent probes have long been used in a broad range of applications, e.g. as indicators in analytical chemistry, as labels in biochemistry or markers in microscopy. Over the last decades, they gained more importance in microscopy as their photo-physical properties were driving developments with improved sensitivity and resolution enabling different methods of single-molecule tracking as well as super-resolution microscopy. Over the past years, we implemented a new approach in super-resolution microscopy based on reversible reactions switching between a non-fluorescent off-state and a fluorescent on-state. [1] Chemical control of spectroscopic states also enables completely new approaches in microscopy, like chemical multiplexing. [2] Based on these notions, we have synthesized new fluorescent switches based on reversible complexation with metal cations that can be tagged to various cellular targets enabling advanced fluorescence microscopy applications. Beyond this, generalisation of the approach of controlling spectroscopic states by specific chemical reactions led to fluorogenic probes enabling even no-wash (super-resolution) fluorescence microscopy experiments.

Andrea Schulze¹, Gerti Beliu¹, Dominic Helmerich¹, Jonathan Schubert¹, Laurence Pearl², Chrisostomos Prodromou², Hannes Neuweiler¹

¹Department of Biotechnology and Biophysics, Julius-Maximilians-University Würzburg, Am Hubland, 97074 Würzburg, Germany
²Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, UK

The 90-kDa heat shock protein Hsp90 is a molecular chaperone that activates a large number of structurally and functionally diverse client proteins by changing their conformation. The enigmatic chaperone machinery acts as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. We engineered fluorescence probes based on contact-induced fluorescence quenching of an oxazine label by the amino acid tryptophan through photoinduced electron transfer (PET) into yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of local structural rearrangements at remote positions that acted cooperatively. PET in combination with fluorescence correlation spectroscopy (PET-FCS) uncovered that critical structural elements that rearranged upon ATP binding were mobile on a sub-millisecond time scale. PET fluorescence quenching emerges as powerful tool to probe conformational changes in complex protein machineries at one-nanometer spatial and with nanosecond temporal resolution.  

Erik D. Holmstrom², Mark Nueesch², Daniel Nettles², Ben Schuler^1,2

¹Department of Physics, University of Zurich, 8057 Zurich, Switzerland
²Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland

Single-stranded nucleic acids (ssNAs) are critical components in many biological processes ranging from telomere maintenance to RNAi. These polymers also play an important role in a wide range of technological applications like nucleic acid origami and DNA PAINT. Although extensive research efforts have helped advance our understanding of structured ssNAs, little is known about the conformational dimensions and dynamics of unstructured ssNAs. Here we use single-molecule FRET to measure the end-to-end dimensions of six different 19-nucleotide homo-polymers as a function of ionic strength, denaturant concentration and temperature. We observe that the dimensions of the ssNAs are largely dominated by charge interactions. At low ionic strengths these polymers sample conformations that are indistinguishable from those associated with their double-stranded counterparts and stabilized by an extended helix of enthalpically-driven base-stacking interactions. At high ionic strengths the ssNAs sample conformations that are significantly more compact than those associated with the duplex, likely resulting from less persistent back-stacking. Using nanosecond fluorescence correlation spectroscopy, we see that the characteristic reconfiguration time of these short oligos is approximately 10 ns and largely insensitive to ionic strength. However, unlike polypeptides, these conformational dynamics scale perfectly with the solvent viscosity indicating that ssNAs experience very little internal friction.

André Dathe, Maria Dienerowitz, Thomas Heitkamp, Michael Börsch

Single-Molecule Microscopy Group, Jena University Hospital, Friedrich-Schiller-University, 07745 Jena,Germany

Analyzing the conformational changes of biological nanomachines like enzymes, transporters or receptors with confocal single-molecule FRET in solution is limited by Brownian motion through the small detection volume. We have built an Anti-Brownian Electrokinetic trap (ABELtrap, developed by A. E. Cohen and W. E. Moerner at Stanford University, [1-2]) using FPGA-based feedback controls and simultaneous data recording by time-correlated single photon counting. Employing a laser pattern generated by fast electro-optical beam deflectors focused in a flat microfluidic chip, the fluorescent or scattering objects are detected and localized by single photons. Their estimated Brownian motion in the field-of-view is cancelled by electrokinetic forces in real time with microsecond feedback algorithms. Therefore, observation times of single molecules in solution are achieved up to seconds. We observed the ATP concentration-dependent kinetics of the membrane enzyme FoF1-ATP synthase determined by its experimentally detected conformational changes. Additionally, changing diffusion properties and the electrokinetic mobility of individual DNA-origami, nanoparticles and other nanostructures in the ABELtrap will be presented [3].

Julian Folz, Thomas Peulen, Oleg Opanasyuk, Claus Seidel

Heinrich-Heine University, Duesseldorf

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 consider which data should be acquired and what information is contained in the data with what accuracy. We compared data of fluorescence lifetime and fluorescence intensity experiments for the sake of FRET based-structural integrative modelling though dye models. Different models of the fluorescent dyes used to model observables of FRET experiments will be introduced and discussed concerning their accuracy. Exploiting the solvatochromism of fluorescent dyes in time-resolved fluorescence experiments, we probe experimentally fully solvated dyes and dyes bound to protein surfaces. Such data will be presented for a network of labeling sites of a large GTPase and may be utilized for refined coarse-grained dye models for high-speed integrative structural modelling at high accuracy.

Salina Quack¹, Ramasubramanian Sundaramoorthy², Tom Owen-Hughes², Jens Michaelis¹

¹Ulm University, Institute of Biophysics, Ulm, Germany (www.uni-ulm.de/biophys)
²University of Dundee, College of Life Sciences, Center for Gene Regulation and Expression, Dundee, Scotland

We have developed the so called nano-positioning system (NPS)  to determine the relative position of dye molecules attached site specifically to macromolecular complexes [1]. Via Bayesian parameter estimation dye positions for the investigated system are estimated and the respective experimental uncertainty is visualized by densities. Together with additional structural data this can be used for monitoring dynamic states in transient complexes. 

In this project we use NPS to study the structural rearrangement of chromatin-remodeler Chd1 (Chromodomain-Helicase-DNA-binding protein 1) upon ATP-dependend nucleosome remodeling. Chd1 is a single-subunit chromatin remodeler consisting of three characteristic domains, a tandem-chromodomain, a helicase-like ATPase domain and a DNA-binding domain. It has been shown to re-position endpositioned mononucleosomes to a central position in vitro and to associate with actively transcribed regions in vivo. 

By designing different experiments in the presence of ATP and its analogues AMP-PNP and ADP-BeFx we study the conformation of Chd1 as well as its dynamics. In addition combining our results with cryo-electron-microscopy (cryoEM) allows us to model the activity of Chd1 on a mononucleosome [2].  

Christian Eggeling

Professor of Super-resolution Microscopy, Institute of Applied Optics, Friedrich‐Schiller‐University & Department of Biophysical Imaging, Leibniz Institute of Photonic Technology, Jena, Germany, Professor of Molecular Immunology, MRC Human Immunology Unit & Scientific Director, Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, christian.eggeling@rdm.ox.ac.uk

Understanding the complex interactions of molecular processes underlying the efficient functioning of the human body is one of the main objectives of biomedical research. Scientifically, it is important that the applied observation methods do not influence the biological system during observation. A suitable tool that can cover all of this is optical far-field fluorescence microscopy. Yet, biomedical applications often demand coverage of a large range of spatial and temporal scales, and/or long acquisition times, which can so far not all be covered by a single microscope and puts some challenges on microscope infrastructure. Taking immune cell responses and plasma membrane organization as examples, we outline these challenges but also give new insights into possible solutions and the potentials of these advanced microscopy techniques, e.g. for solving long-standing questions such as of lipid membrane rafts.

Dr. Elke Hebisch^1,2, Dr. Martin Hjort^1,2, Dr. Christelle N. Prinz^1,2

¹Solid State Physics, Lund University, 22100 Lund, Sweden
²NanoLund, Lund University, 22100 Lund, Sweden

The specific arrangement of membrane lipids and proteins in a living cell at the interface to high-aspect ratio nanostructures (nanowires and nanostraws) is still unknown – as are the dynamic structural adaptations and molecular rearrangements of living cells in the vicinity of such nanostructures. To elucidate these questions, STED nanoscopy is the ideal technique because it is live-cell compatible, target-specific, and offers a lateral resolution on the protein level. 

We present STED based investigations of the live-cell membrane and the cytoskeletal Actin signal in the presence of hollow Alumina nanostraws. We find that the cellular membrane wraps tightly around the nanostraws. On the other hand, the Actin cytoskeleton forms intricate, coil-like nanometric structures around the nanostraws. Further, STED images of living cells stained for both membrane and Actin signal reveal a significant degree of co-localization at the apical cell membrane further away from the nanostraws. This co-localization is lost at the basal membrane close to the nanostraws due to a strongly reduced Actin signal.

Conclusively, STED based investigations of the behavior of single living cells cultured on nanostraws reveals a strong response of the cellular membrane and the Actin cytoskeleton – two of the main structure-giving features of the cell.

Iztok Urbančič^1,2, Falk Schneider¹, Erdinc Sezgin¹, Silvia Galiani¹, Francesco Reina¹, Dominic Waithe^1,3, Ana Mafalda Santos¹, Simon Davis¹, Christian Eggeling^1,3,4,5

¹MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK
²"""Jozef Stefan""" Institute, Ljubljana, Slovenia
³Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, UK
⁴Institute of Applied Optics Friedrich‐Schiller‐University Jena, Germany
⁵Leibniz Institute of Photonic Technology e.V, Jena, Germany

Nanoscale architecture of the membranes in living cells plays crucial roles in numerous vital processes, including the activation of T-cells as the onset of the adaptive immune response. However, the detailed nature and function of membrane heterogeneities have remained largely unclear, as the experimental techniques with appropriate spatiotemporal resolution are only now becoming available. 
To gain further insights into the molecular mechanisms of lipid reorganisation in activating T-cells, we combined two fluorescence-based spectroscopic techniques that probe complementary properties of the membranes at the molecular level: STED-FCS to reveal the detailed picture of the diffusion of the lipids, and spectral STED imaging with polarity-sensitive membrane probes [1] to map differences in the local molecular order within the lipid bilayer. For the most sensitive description of the latter, we thoroughly compared the established methods for spectral analysis approaches, i.e. generalized polarization, lineshape fitting, and spectral phasors. 
Correlating lipid diffusivity and order with the locations of the key membrane proteins responsible for triggering the activation of T-cells revealed that the T-cell receptor and its phosphatase, which spatially segregate upon activation, reside in clearly distinct membrane environments.

Jong-Chan Lee^1,2, Ye Ma³, Kyu Young Han⁴, Taekjip Ha^2,3,5

¹Department of New Biology, DGIST, Daegu 42988, Republic of Korea
²Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, Maryland
³Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA
⁴CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, Florida, USA
⁵Howard Hughes Medical Institute, Baltimore, Maryland

Stimulated emission depletion (STED) super-resolution microscopy (or nanoscopy) offers significant enhancement of optical resolution compared to conventional microscopy [1]. To achieve resolution beyond the diffraction-limit, STED nanoscopy uses orders of magnitude (roughly ~10^5) more photons than the conventional confocal microscopy. Those additional ‘STED’ photons, which are designed to deplete the fluorescence at the periphery of focus, can induce unintended background noise. Increased low spatial frequency background noise decreases the signal-to-background ratio (SBR) and deteriorates the image quality by masking the high spatial frequency, super-resolved signal.

Here, report a simple and easy-to-implement method, which we call polarization switching STED (psSTED), that can efficiently suppress the low spatial frequency background appearing in STED images. In psSTED, we switch the STED beam polarization between two different circularly polarized states to record a regular STED image and a background noise image. A simple, unambiguous subtraction process between these two images accomplishes a background-free super-resolved image. With both simulation and experimentation, we demonstrate psSTED works universally for different STED conditions. Finally, we compare the performance of psSTED with other state-of-the-art background subtraction methods and highlight its capability of efficient background suppression with a much simpler hardware implementation [2-4].

Hagen Hofmann

Department of Structural Biology, Weizmann Institute of Science, Herzl St. 234, 76100 Rehovot, Israel

Protein networks are huge biological communication systems with complex architectures. Yet, the stabilities of its constituents are often secondary for their behavior. We show that the opposite is true for a network of intrinsically disordered proteins (IDPs). Polymer interactions in five homologous IDPs from a cell-cycle network cause tenfold affinity-variations within physiological ionic strength limits. In the network, however, the dimer distribution is unaffected by ions due to a built-in buffer that is rooted in the homologous physics of the IDPs. The buffer capacity depends on the network architecture and increases with network size, which allows an efficient suppression of global extrinsic noise in large networks. We argue that sequence homology in IDP-networks is an effective blueprint for creating robust regulatory networks.

Ganesh Agam, Anders Barth, Don C. Lamb

Department of Chemistry, Center for NanoScience, Nanosystems Initiative Munich (NIM) and Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians University Munich, Munich, Germany.

Every protein needs to undergo a folding process after it’s synthesis in the cell. Most in vitro protein folding studies are carried out on small proteins. To understand complex folding processes in large proteins, we studied maltose binding protein (MBP) as a model system. MBP is a two domain, 42 kDa protein natively folds within a minute. With the two mutations V8G and Y283D, the spontaneous folding process is slowed down to half an hour.
Here, we apply single molecule three-color FRET which allows us to monitor three distances at a time. With the method in hand, we asked the question whether the two domains fold together or independently. For three color FRET, MBP is labeled with three fluorophores at specific positions to have three coordinates that monitor the folding of the N-terminal (NTD) and C-terminal (CTD) domains simultaneously.

Three color FRET measurements as well as control measurements on double labeled MBP revealed that both domains folds simultaneously in a cooperative fashion.

Caroline Berlage, Marcelle König, Paja Reisch, Felix Koberling, Haisen Ta, Rainer Erdmann

PicoQuant, Rudower Chaussee 29, 12489 Berlin, Germany, info@picoquant.com

Quantifying molecules in subdiffraction-sized structures is crucial for understanding various fundamental biological processes. Ideally, a non-destructive measurement would result in an image of a cellular structure along with the number of fluorescent molecules in each region of interest. Recently, a considerable amount of work has gone into developing such methods.

Counting by Photon Statistics (CoPS) is a promising new technique based on photon antibunching, which exploits the fact that a single molecule can only generate one photon at a time [1]. The number of independent fluorescent emitters can be determined by measuring photon coincidences on a time-resolved confocal microscope with four single-photon detectors (e.g., MicroTime 200, PicoQuant) [2]. Both a molecular brightness and the spatial density of fluorophores per image pixel are estimated. By summing over all corresponding pixels, the number of molecules in a structure can be calculated. The only calibration required is to determine the instrument’s Point Spread Function (PSF) size.

We investigated imaging with CoPS under a wide range of experimental conditions. The applicability of the method both to artificial samples such as immobilized DNA origami, as well as to biological samples is explored. We also discuss sample preparation requirements and practical guidelines for performing CoPS measurements.

Anna M. Chizhik¹, Syamantak Khan², Ingo Gregor¹, Jörg Enderlein¹, Chayan Nandi², Alexey I. 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].

Alexey I. Chizhik, Jörg Enderlein

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 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].

Jelmer Cnossen

TU-Delft, Netherlands

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. We benchmarked the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also used the pipeline to extend our understanding of TtAgo by measuring the protein's binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.

Gustavo H. R. Soares¹, Guilherme A. M. Jardim², Eufrânio N. da Silva Júnior², Luiz A. Cury¹

¹Instituto de Ciências Exatas, Departamento de Física,Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, Minas Gerais, Brazil
²Instituto de Ciências Exatas, Departamento de Química,Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, Minas Gerais, Brazil

Dropcast film of phenazine-based 1,2,3-triazole phosphorescent probe molecules, blended with an optically inert matrix of Zeonex, was investigated by steady-state fluorescence. The very diluted blend solution, with a ratio about 1.25 x 10-13 in mass of the probe to the Zeonex molecules, enabled us to obtain an inhomogeneous dropcast film containing relatively small domains of probe molecules distributed all over the Zeonex matrix. The observed different steady-state emission characteristics were associated to different probe molecular conformations, revealing the existence of singlet monomer and dimer fluorescent states, as well as, phosphorescent triplet states with different levels of aggregation. As a complementation, micro-fluorescence measurements coupled to time correlation single photon counting are planned to be performed in spin-coating blended films in order to get more resolved results.

Arindam Ghosh, Akshita Sharma, Sebastian Isbaner, Ingo Gregor, Narain Karedla, Jörg Enderlein

III:Institute of Physics, University of Göttingen, Friedrich Hund Platz 1, 37077, Göttingen

We present a new technique to localize single molecules along the axial direction with ängström scale accuracy. The method exploits the near-field interaction between a graphene monolayer and a fluorescent emitter resulting into a distance-dependent quenching of the fluorophore’s brightness and reduction in its excited-state lifetime. A fluorescent emitter, which can be typically well described by an ideal oscillating electric dipole, can efficiently couple to electronic excitations (plasmons) in graphene via a distance-dependent electromagnetic near-field coupling. The effect is similar to recently introduced single-molecule metal-induced energy transfer (smMIET) [1] [2] where the energy transfer takes place from a dipole emitter to a thin metal film. However, in contrast to smMIET, here we observe a stronger distance-dependent modulation of a dye’s fluorescence properties (intensity, angular distribution of emission, excited-state lifetime). As a result, one can increase the localization accuracy by nearly tenfold of smMIET. We demonstrate the power of the method by axially localizing single dye smolecules and by measuring lipid bilayer thickness values with ängström precision. Remarkably, we were also able to separate the inter-leaflet distance of supported lipid bilayers (SLB) of two different lipid compositions which differ only 9 Å- 12Å in thickness.

Fabian R. Goßler^1,2, Tobias A.F. König^1,2

¹Institute of Physical Chemistry and Polymer Physics, Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Str. 6, 01069 Dresden
²Cluster of Excellence Centre for Advancing Electronics Dresden, TU Dresden, Germany

Colloid-to-film coupled nanocavities are highly appealing systems for studies on fundamental light-matter interactions. Due to their strong nearfield enhancement upon excitation, they are able to confine electromagnetic modes into sub-wavelength volumes. We present a scalable approach to create high quality plasmonic cavities using directed self-assembly methods of uniform plasmonic building blocks. Novel silver-indium-sulfide (AgInS) quantum emitters are used as emitters to probe that the plasmonic nanocavities can enhance fluorescence over a wide spectral range. Time-resolved confocal fluorescence microscopy, dark field scattering spectroscopy and finite-element simulations were employed to study the quantum-electrodynamic properties of the system. We observed hybridized light-matter states of the plasmon resonances which indicates strong plasmon-exciton interactions and a mean fluorescence enhancement factor of over 1000.  

Enrico Gratton¹, Lorenzo Scipioni¹, Alberto Diaspro², Luca lanzano²

¹3210 Natural Sciences II, Laboratory for Fluorescence Dynamics
²IIT, Genova, Italy

The Airyscan detector is used as a new concept in fluctuation correlation spectroscopy using superresolution.  This detector which acquires data simultaneously on 32 detectors arranged in a hexagonal array is used as a nanocamera with frame rate in the range of one million fps.  We exploit this detector for fluctuation methods based on time correlation at single points or at a number of points simultaneously, as well as methods based on spatial correlation in the area covered by the detector.  We developed a Comprehensive Correlation Analysis of molecular fluctuations that allows the large number of users of the ZEISS LSM 880 to access current fluctuation methods in one single platform.

Johan Hummert, Wioleta Chmielewicz, Klaus Yserentant, Dirk-Peter Herten

Single-Molecule Spectroscopy, Dept. of Physical Chemistry, Heidelberg University, Germany

Quantitative microscopy aims to directly access the number of participating molecules in complex biological systems. In counting by photon statistics (CoPS) [1] this number is estimated based on the distribution of coinicidence detection events. This approach has been successfully demonstrated for optically separated molecular clusters [2]. Recently the technique was extended to the evaluation of scanning nanoscopy data [3].

Currently, the application of CoPS to intracellular protein clusters strongly depends on the quality of sample preparation, limiting its application to samples with only minor fluorescent background. In samples with high cluster density (also in different z-sections) or high fluorescent background the current approach of modelling the background as a weak emitter is insufficient. Therefore, we aim to gauge the impact of background signals of varying intensity and origin on the CoPS counting result by simulating single photon data. Depending on the nature of the simulated sample, e.g. cytosolic or membrane anchored clusters, different strategies of background treatment will be necessary. Thus, the analysis of simulated data is crucial to evaluate the current analysis routines. It is furthermore essential to improve the analysis of 2d CoPS data and to further develop CoPS towards a quantitative imaging technique for biological systems.

Shun Qin, Sebastian Isbaner, Dirk Hähnel, Ingo Gregor, Jörg Enderlein

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

Confocal Spinning Disk (CSD) systems are widely used for 3D cell imaging because they offer the advantage of optical sectioning at high framerates and are easy to use.  However, as in confocal microscopy, the imaging resolution is limited by the diffraction of light. Image Scanning Microscopy (ISM) is a technique that improves the resolution of a confocal microscope by a factor of 2 [1]. ISM with a Confocal Spinning Disk setup (CSDISM) has been shown to improve contrast as well as lateral resolution. A minimum total acquisition time of one second per ISM image makes this method highly suitable for 3D live cell imaging [2]. Here, we present a multicolor implementation of CSDISM for the popular Micro-Manager Open Source Microscopy platform. Since changes in the optical path are not necessary, this will allow any researcher to easily upgrade their standard Confocal Spinning Disk system at remarkable low cost (~5000 EUR) with an ISM superresolution option.

Hongje Jang¹, Soheil Mojiri¹, Ingo Gregor¹, Marco Tarantola², Jörg Enderlein¹

¹Third Institute of Physics, University of Göttingen, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany
²Max Planck Institute for Dynamics and Self-Organization, , 37077 Göttingen, Germany

Rapid three-dimensional high-resolution imaging is an important challenge when investigating microscopic features and dynamics inside moving cells. In this study, we investigate the chemotactic behavior of the slime mold Dictyostelium discoideum (D.d.) using a new technique of fast 3D deconvolution imaging. As a proof of principle, we validate our approach on simulated images of dispersed point emitters, and investigate time-lapse movies of actin bundle dynamics in D.d. cells. For this purpose, actin was fluorescently labeled with the GFP-tagged actin-binding protein Lim-E (Lim-E-mGFP). To confine single cells along the optical axis, we employ a specialized microfluidic device that allows for controlled confining and squeezing of cells along the optical axis. Our 3D deconvolution imaging technique uses a multi-plane imager that splits the detected fluorescence into eight detection channels, where each channel predominantly collects light from a different focal plane. 3D image reconstruction of from acquired 3D image stack is done by a deconvolution with penalized regression. By controlling the penalty parameters, we are able to optimize the quality of the deconvolution results. We compare our results with a more conventional Lucy-Richardson algorithm, and evaluate quantitatively the improvement in algorithmic efficiency and image quality.

Aditya Katti, Joerg Enderlein

1Third Institute of Physics, Georg August University of Göttingen, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany

FCS is a spectroscopic technique that is widely used for measuring diffusion coefficients and thus the size of fluorescently labeled molecules at picomolar to nanomolar concentrations. For a correct extraction of a translational diffusion coefficient from an FCS measurement, the FCS detection volume has to be accurately known. [1] However, in scattering or aberrating media like PAA or agarose gels, the observation volume is distorted which introduces systematics errors into FCS-based translational diffusion measurements. However, FCS can be also used to measure the rotational diffusion of molecules if used in conjunction with polarized excitation and detection. Such rotational diffusion measurements are independent on the peculiar shape and size of the detection volume and would be thus insensitive to aberrations and background [1]. Here, we present a systematic study of rotational diffusion in planar gels of PAA with different monomer and cross-linker concentrations. As the fluorescent probe, we used enhanced green fluorescent protein (EGFP). In the gel, the trapped EGFP rotates more slowly than in aqueous solution. With higher monomer and cross-linker concentration, the rotational diffusion time increases. However, after a particular concentration, the rotational diffusion time does not change any further. We relate the observed rotational diffusion behavior to gel stiffness and pore size, and explore the possibility to use rotational diffusion measurements as in pressure-sensing mechanism in cells and tissues [2].

Ma Alejandrina Martinez-Gamez¹, Araceli Romero², Abel Moreno³, Mayra Cuéllar-Cruz²

¹Centro de Investigaciones en Optica AC, Loma del Bosque 115, Col. Lomas del Campestre, 37150 León, Gto, México
²Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta S/N, Col. Noria Alta, C.P. 36050, Guanajuato, Guanajuato, México.
³Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Av. Universidad 3000, Ciudad Universitaria, Ciudad de México, 04510. México

ABSTRACT
Most of the beauty of nature can be reflected as much in the existence of gems as in the attractive minerals such as calcite, quartz, pyrite, galena, which have grown for millions of years deep under Earth’s surface. However, the most extraordinary minerals in terms of physico-chemical properties are those forming complex structures, namely those composed of minerals and biological macromolecules, usually called biominerals, these are present in living organisms. These biominerals are usually formed by a combination of chemical, biochemical and biophysical processes. Microorganisms like Candida are capable of biomineralization, forming nanocrystals (NCs) in the presence of heavy metals [1]. In this work, NCs of CdPbS are synthesized in vivo by Candida species. These NCs were characterized using fluorescence techniques. Our results show that, in the presence of Pb2+ and Cd2+, Candida cells are able to replicate and form extracellular CdPbS NCs. This shows that these Candida species adapt to the environment differentially, counteracting the effect of the heavy metals. The synthesis of NCs is a form of homeostasis and adaptation. To our knowledge this is the first report in which the biosynthesis of CdPbS NCs has been shown in several species of Candida, which is differentially regulated in each of these pathogens, and allows them to adapt and survive in different physiological and environmental habitats. 

Keywords: Nanocrystals of lead and cadmium, Crystal Growth, Crystal Structure, Candida species.

*Corresponding Author: E-mail: mcuellar@ugto.mx

Willem Melching

Tu-Delft, Netherlands

Knowing the exact model of your optical system can be a powerful tool in microscopy, and can be used for a lot of purposes. An interesting part of the optics to model is the Point Spread Function (PSF). The PSF describes the image on the sensor generated by a point light source. In STORM microscopy (Stochastic Optical Reconstruction Microscopy) small fluorophores are used that bind to specific spots on a molecule. These fluorophores are so small they can be approximated by a point source. By collection a video of these fluophores attaching and de-attaching to different spots in a cell the structure of the molecule can be reconstructed. By using a a model of the PSF of a single fluorophore we can construct an optimization problem. We try to find the molecular structure that with the given PSF model best predicts the acquired images. This can also be used to reconstruct 3D information from a 2D image by introducing a know aberration (such as a cylindrical lens) and including this in the PSF model[1].Constructing a model of your optics can be done in different ways. Thestraightforward way to do this by using the pupil function and doing a two-dimensional Fourier transform. However, this is requires a lot of computations,and quickly becomes infeasible to do multiple times for each iteration of your optimization algorithm.

Mehri Moradi, Chunchu Deng, Michael Sendtner

Versbacher straße 5, 97078 Würzburg, Institute of Clinical Neurobiology, University Hospital of Würzburg, Germany

Axonal mRNA translocation and local protein synthesis are crucial for axonal guidance, branching, synaptic maturation and maintenance in motoneurons in vivo and in vitro. In a previous study, we revealed that intra-axonal synthesis of the three actin isoforms; α-, β- and γ-actin, in axonal branch points and growth cones contributes to the cytoskeletal dynamics required for axonal maturation in motoneurons. Furthermore, we demonstrated that both axonal trafficking and local translation of these actin mRNAs are impaired when Smn protein is deficient in a mouse model of SMA.

Therefore, impaired axonal mRNA trafficking and translation might contribute to the pathogenesis of various neurodegenerative diseases including SMA and other motoneuron diseases. In a current study, we found that axonal transport of mRNAs coding for active zone proteins such as Munc13-1 and 13-2 is impaired in Smn-depleted motor axons. Moreover, our preliminary results using super resolution microscopy show that the distribution and clustering of Munc13 proteins and Ca2.2 channels are impaired in neuromuscular endplates in SMA mice. Our goal is to investigate the specific role of locally synthesized active zone components such as Munc13 proteins in the control of synaptic function at neuromuscular endplates and the potential defects in SMA mouse models.

Franziska Zosel¹, Davide Mercadante¹, Daniel Nettels¹, Benjamin Schuler^1,2

¹Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
²Department of Physics, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

The interactions of intrinsically disordered proteins (IDPs) with their molecular targets are essential for the regulation of many cellular processes. IDPs can perform their functions while partly or fully disordered, and they may fold to one or more structured conformations on binding. Here we show that the conformational flexibility of IDPs can entail pronounced kinetic heterogeneity. By single-molecule spectroscopy, we identified a conserved proline residue in NCBD (the nuclear coactivator binding domain of CBP) whose cis/trans isomerization in the unbound state modulates the association and dissociation rates with its binding partner, ACTR. As a result, NCBD switches on a timescale of tens of seconds between two populations that differ in their affinities to ACTR by about an order of magnitude. Molecular dynamics simulations indicate that the cis isomer causes distortions in the loop containing the proline that propagate throughout the structure and lead to reduced packing of the complex. Given the prevalence of proline residues in IDPs, especially in phosphorylation motifs, peptidyl-prolyl cis/trans isomerization may be a common mechanism for regulating IDP interactions.

Nazar Oleksiievets, Arindam Ghosh, Ingo Gregor, Joerg Enderlein

Friedrich-Hund-Platz 1

Molecular diffusion in biomembranes is central to a number of signaling and trafficking pathways (citation needed). Conventional fluorescence microscopy and spectroscopy techniques fails to shed light on these dynamic processes, in particular for sub-diffraction limited structures like synaptic vesicles and nanoscopic membrane domains. Additionally, the influence of membrane curvature on viscosity, diffusion speed, or lipid composition becomes dominant at this scale. In this regard, Small Unilamellar Vesicles (SUVs- diameter 30 to 200 nm) serve as an ideal mimicking system in vitro to investigate diffusion-based dynamic processes. We report DynaMIET-FCS by combining Fluorescence Correlation Spectroscopy (FCS) and Metal-Induced Energy Transfer (MIET) (citation) to probe spherical diffusion-based dynamics of a labelled lipid particle in surface-bound SUVs. The underlying physical basis of MIET is that the intensity of a fluorescent molecule becomes increasingly quenched and excited-state lifetime gets reduced when approaching a metal surface, due to electro-dynamic coupling of the excited state of the dipole emitter to surface plasmons on a thin metal film [1].  The core idea here is to use a fluorophore-labelled lipid head group in SUVs and correlate it’s intensity fluctuations over time along the axial direction to extract the dynamic timescales involved and diffusion coefficient from the decay curves. In conclusion, DynaMIET-FCS can be successfully applied to address relevant biological questions governed by diffusion based dynamic processes on vesicular structures.

Filiz ÖZTEKİN, Demet KAYA AKTAŞ

Istanbul Technical University, Department of Physics, Maslak ISTANBUL - TURKEY

Time resolved fluorescence technique was employed for studying swelling of poly(acrylamide-co-acrylic acid)  (P(AAm-co-AAc)) composite anionic hydrogels for different pH. Disc shaped composite hydrogels were prepared by free-radical crosslinking copolymerization of P(AAm-co-AAc). N, N'- methylenebis (acrylamide) (BIS) and ammonium persulfate (APS) were used as crosslinker and initiator, respectively.   Pyrene (Py)was introduced as a fluorescence probe. P(AAm-co-AAc) composite hydrogelsdried before using for swelling experiments.  Fluorescence lifetime of Pywas monitored during in situ swelling processes of composite hydrogels. It was observed that fluorescence lifetime values decreased as swelling is proceeded. Li-Tanaka equation was used to determine the swelling time constants, τ and cooperative diffusion coefficients, D from intensity variations during the swelling processes. Gravimetrical experiments for swelling process were also performed.  It was shown that swelling time constants, τ decreased, diffusion coefficients for swelling process, D increased  as the pH values  are increased.

Mariano Gonzalez Pisfil^1,2, Marcelle König¹, Benedikt Krämer¹, Paja Reisch¹, Felix Koberling¹, Matthias Patting¹, Andreas Herrmann², 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

Over the last decade, Fluorescence Correlation Spectroscopy (FCS) has been utilized to investigate the dynamics of complex cellular processes. However, this powerful tool has significant drawbacks when observing slower moving molecules such as fluorescently labeled components diffusing in cell membranes. In order to average over a sufficient number of independent diffusion events, the optimal measurement time for an FCS measurement has to be increased. This in turn increases the risk of introducing artifacts (e.g., drift, or sample movement) or photobleaching.

Scanning FCS (sFCS) was developed to counteract these issues. The confocal volume is moved with respect to the sample, thus reducing the residence times of the molecules. In this scenario, photobleaching is decreased while increasing the statistical accuracy. An added advantage of the scanning process is the ability to determine the observation volume without prior calibration.

As we use the confocal time-resolved fluorescence microscope MicroTime 200 STED equipped with a FLIMbee galvo scanner, we have access to the fluorescence lifetime information. In our case, multi species STED measurements are performed with labels featuring similar emission wavelengths and a single STED laser. The different labels can be discriminated by applying a unique pattern matching analysis method [1].

André Weber¹, Yury Prokazov², Evgeny Turbin², Werner Zuschratter¹

¹LIN Magdeburg, Brenneckestr. 6, 39118 Magdeburg
²Photonscore GmbH, Brenneckestr. 20, Brenneckestr. 20, 39118 Magdeburg

Metal induced energy transfer (MIET) is a nanoscopy technique to measure the distances from the fluorescent molecules to the metal membrane. Here we present MIET acquisition with wide-field fluorescence lifetime imaging microscope (FLIM) equipped by a position sensitive single photon counting system. The system is based on a micro-channel plate photomultiplier tube equipped with a charge dividing anode for position readout that grants better than 20μm position precision across 25mm of detector’s sensitive area. A combination of high image quality with better than 50ps of temporal resolution enables fast and precise wide-field FLIM acquisition.
 

Elin Sandberg¹, Johan Tornmalm¹, Mihailo Rabasovic², Jerker Widengren¹

¹Royal Institute of Technology (KTH), Dept. Applied Physics, AlbaNova University Center, SE-106 91, Stockholm, Sweden
²Univ Belgrade, Inst Phys, Pregrevica 118, Belgrade 11080, Serbia

Fluorescent molecules can provide information via the “traditional” parameters of their emitted fluorescence light (wavelength, intensity, polarization and fluorescence lifetime), but also from their fluorescence blinking behavior. This blinking is highly environment sensitive, to local oxygen concentrations, pH, redox conditions and viscosities. Transient state (TRAST) imaging allows such blinking behavior to be monitored in a widely applicable manner, does not require single-molecule detection conditions, or any particular time resolution on the detection side [1-3].

In this work, we introduce laser-scanning TRAST imaging with near-infrared two-photon excitation (TPE). The excitation beam of a Titanium-Sapphire laser was circularly scanned in aqueous solution with the auto-fluorescent coenzyme nicotinamide adenine dinucleotide (NADH). The blinking properties of NADH was determined from the variation in fluorescence intensity upon systematically varying the scanning speed (and the excitation dwell time) in the sample, and was then compared to corresponding TRAST measurements with stationary one-photon excitation in the UV. From the measurements different redox-environments could be identified.

Furthermore, the auto-fluorescence from C2C12 cells was imaged by TPE scanning TRAST, opening for the use of NADH blinking as additional label-free reporters of cellular environment, together with e.g. the NADH lifetime.

Saptaswa Sen, Joachim Piguet, Johan Tornmalm, Elin Sandberg, Jerker Widengren

AlbaNova University Center, KTH-Royal Institute of Technology, Department of Applied Physics, 10691 SE, Stockholm, Sweden

Cell membrane viscosity is a crucial parameter that determines the rate of diffusion-controlled reactions in many biological processes. Changes in cell membrane viscosity can be found in many diseases, including cancer, diabetes, and Alzheimer’s disease, and such changes may thus also serve as biomarkers.

Fluorescence techniques currently offer high sensitivity, fast readouts, and the use of low concentrations of probes, thereby minimizing perturbation on the cellular membranes. Unravelling the mechanisms of biomolecules, cells and organisms rely on the development of fluorescent-based tools. Creative probe design makes molecular fluorescence an extremely useful tool for in vitro biophysical and biochemical analyses as well as for in vivo cellular imaging.

The purpose of our research is to characterize and further investigate the physical properties of natural and artificial biological membranes with novel environment-sensitive fluorescent probes. On the basis of different fluorescence readout modalities and probes occupying different locations in the membranes, microviscosity of cell membranes can be sensed. Building further on previous work [1], combining complementary approaches e.g. fluorescence correlation spectroscopy (FCS) and transient state imaging (TRAST), we demonstrate that the mobility of those novel fluorescent probes can be a useful parameter to resolve differences in membrane microviscosity in live cells.

Akshita Sharma, Narain Karedla, Sebastian Isbaner, Arindam Ghosh, Ingo Gregor, Jörg Enderlein

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

Understanding membrane diffusion provides a deeper insight into transport mechanisms and the functioning of proteins in several cellular processes. In this work, we determine the diffusion of labeled lipids in lipid bilayers in a leaflet-dependent manner. We use the recently established single-molecule Metal Induced Energy Transfer (smMIET)[1] technique together with Line-Scan Fluorescence Lifetime Correlation Spectroscopy (LS-FLCS). In smMIET, the excited-state lifetime of a fluorescent molecule varies monotonically with its distance from a metal surface. This is due to the strong distance-dependent energy transfer from the dye to the surface plasmons of the metal. Here, we use Indium-Tin Oxide (ITO) as a substrate. Due to the steep variation of the fluorescence lifetime with distance from the ITO substrate, we observe a bi-exponential decay of fluorescence from the labeled lipids diffusing in the excitation focus. We estimated the thickness of the bilayer, which is on the order of 5 nm. Using LS-FLCS, we separate the temporal- and spatial- intensity autocorrelation functions of the lipids diffusing in the two leaflets. We fit the spatio-temporal autocorrelation functions using a 2D diffusion-sticking model. We found significant sticking of the bottom leaflet to the substrate. The sticking of the top leaflet is indicative of the strong interleaflet coupling in DOPC bilayers. We also observe two distinct sets of population for the top and bottom leaflet of the bilayer by Single Particle Tracking[2]. This method will have huge potential in understanding transport mechanisms in membranes.

Jan Sýkora², Jana Brejchová¹, Lenka Roubalová¹, Jiří Janáček¹, Martin Hof², Petr Svoboda¹

¹Institute of Physiology of the Czech Academy of Sciences, Vídeňská 1083, 14220, Prague 4, Czech Republic
²Department of Biophysical Chemistry, J. Heyrovsky Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 2155/3, 18223, Prague 8, Czech Republic

The mobility of G protein-coupled receptors (GPCR) within the plasma membrane is believed to play a significant role in their function. For example, the diffusion of mu-opioid receptor (MOR) was reported to be agonist dependent (1). We therefore decided to investigate the mobility of delta opioid receptors (DOR) in living HEK cells by means of the imaging fluorescence correlation spectroscopy (imFCS) (2). This recently introduced technique has certain advantages over the classical single point FCS. First of all, the extent of photobleaching is reduced and the method enables reconstruction of the spatially resolved 2D diffusion maps. The gained data are then discussed in the framework of the results obtained with other techniques providing information on the receptor mobility, e.g. fluorescence recovery after photobleaching (FRAP), and raster image correlation spectroscopy (RICS) (3).

The support of Grant Agency of the Czech Republic via 17-05903S is greatly acknowledged.

Jan Christoph Thiele¹, Sebastian Isbaner¹, Katharina Dabow², Roman Tsukanov¹, Jörg Enderlein¹

¹III. Institute of Physics – Biophysics, Georg August University Göttingen, Germany
²Institute of Physical Chemistry, Georg August University Göttingen, Germany

Polymer brushes are coatings with surface-tethered polymers and widely used to tailor surface properties including wetting behaviour, friction, and interactions with biomolecules. Brushes change the hydrodynamic properties of a surface in a complex fashion. Experiments on capillaries showed an unexpected large flow reduction by the brush, while recent simulations predict a backflow within the brush layer.^[1]
We investigate the interaction of a polymer brush with shear flow and characterise the nanoscale movement of the polymer chains. For this, we fabricate microfluidic channels containing a fluorescently labelled polymer brush on a gold surface. To follow its movement, we utilise Metal-Induced Energy Transfer (MIET):^[2] MIET causes a modulation of fluorescence lifetime and brightness in proximity to the gold surface. This enables us to obtain the surface distance (height) of a dye at the chain end by measuring its fluorescence lifetime. By applying flow, we observe a decrease of the average height from 15 nm to 7 nm. Additionally, we correlate the fluorophore’s brightness fluctuations over time as in fluorescence correlation spectroscopy (FCS) to quantify the timescale of the vertical motion in the brush. The high spatial and temporal resolution of MIET enables us to quantify the polymer movement on the molecular scale.

Milan Vala, A. García Marín, Yong He, Roberto Fernández, Marek Piliarik

Institute of Photonics and Electronics, Czech Academy of Sciences, Prague, Czech Republic

Interferometric detection of scattering (iSCAT) is a powerful optical imaging technique that enables observation of nanoscale objects with sub-nanometer localization precision and microsecond temporal resolution. In recent years, this technique was used in various bioimaging applications such as tracking of movements of single gold nanoparticle-labeled proteins ¹, detection of medium to large-sized unlabeled proteins ² or direct visualization of sub-wavelength biomolecular processes such as lipid bilayer formation ³. As the iSCAT signal originates from interference between the scattered light and a reference wave, its magnitude depends on relative intensities as well as phases of these two waves. With the increasing complexity of the system under study, influenced by multiple scattering objects within the probed volume and heterogeneity of the size and shapes of scattering labels, iSCAT signal can become ambiguous and challenging to interpret in a quantitative manner.

In this contribution, we employ iSCAT imaging in ultrafast tracking of the dynamics of model nanoparticle-protein constructs. We combine optical image with detailed reconstruction of the particular geometry of scatterers by means of atomic force microscopy (AFM) correlated with the iSCAT signal.