Julie Biteen

University of Michigan, Departments of Chemistry and Biophysics, Ann Arbor, USA, jsbiteen@umich.edu

Because of the small size of bacterial cells, the mysteries of their subcellular structure, dynamics and cooperativity are well-suited to single-molecule and super-resolution investigations. Our lab has been developing new methods to locate, track, and analyze single molecules to answer fundamental, unanswered questions in live bacterial cells. I will discuss how we are measuring and understanding the dynamical interactions essential for DNA mismatch recognition and DNA replication in living Bacillus subtilis cells, as well as our ongoing work to extend our targets from single cells to pathogens and microbial communities. Overall, our results provide fundamental insight of relevance to human health and disease.

Ephrem Sitiwin^1,2, Michele Madigan^1,2, Robert Conway², Svetlana Cherepanoff^2,4, Alexander Macmillan³

¹School of Optometry and Vision Science, The University of New South Wales, Rupert Myers Building (North Wing), Barker St, Kensington NSW 2033
²Save Sight Institute, South Block, Sydney Eye Hospital, 8 Macquarie Street,Sydney NSW 2000
³Biomedical Imaging Facility, Room LG12, Lower Ground, Lowy Cancer Research Centre (C25), Kensington UNSW Sydney NSW 2052
⁴SydPath, St Vincent's Pathology, St Vincent's Hospital, 390 Victoria Street, Darlinghurst NSW 2010

The progression of choroidal melanoma (CM) is complex, involving genetic/immune-related factors. Melanin/pigmentation genes and forms (eumelanin/dark and pheomelanin/light) can also impact on CM progression. An optimised 2-photon FLIM, with phasor analysis, was used to identify melanin fluorescence_lifetime (FL) profiles in formalin-fixed, label-free paraffin sections of human melanoma/naevus, with surrounding heterogeneously-pigmented melanocytes. Sections of ‘light_CM’(n=3) with ‘mixed_surrounding_melanocytes’ and ‘dark_naevi’(n=3) with ‘dark_surrounding_melanocytes’ were imaged with FLIM(sampled-regions=3). FLs were measured at every image pixel, Fourier transformed and presented in a ‘fit-free’ phasor_plot. These plots were segmented by 7-phasor_clusters of linearly-increasing FLs mapped to lesion/cell-localised melanin. The fraction of FLIM image-pixels linked to each melanin-mapped cluster was obtained for all sampled regions to form melanin FL profiles. The measured sampled regions displayed distinct intracellular-melanin (eumelanin:pheomelanin) profiles with varying dominant melanin-mapped clusters. The dominant ‘highest-pixel-fraction’ cluster measured in ‘light_CM’ mapped to long FLs, implying a low eu:pheo ratio. The ‘mixed_pigmented_melanocytes_around_CM’ showed a mixed eu:pheo content based on the dominant mid-valued FL cluster. The ‘dark_naevi_and_surrounding_melanocytes’ were mapped to mostly short FLs (high eu:pheo ratio). Our FLIM-phasor method provides a fast ‘model-free’ way to unmix melanin FLs in melanocytes, naevi and CM, and provides a basis for exploring the role of melanin forms in eye melanoma pathogenesis.

Rhys Dowler, Paja Reisch, Ben Kraemer, Mariano Gonzales Pisfil, Marcelle Koenig, Sandra Orthaus-Mueller, Marcus Sackrow, Matthias Patting, Tino Roehlicke, Hans-Juergen Rahn, Michael Wahl, Felix Koberling, Rainer Erdmann

PicoQuant, Rudower Chaussee 29, 12489 Berlin, Germany

Increasing the speed of Fluorescence Lifetime Imaging (FLIM) is essential to cementing its importance as a tool in the Life Sciences. This technique is already well established, but imaging dynamic processes requires shorter acquisition times. Our novel rapidFLIM approach dramatically reduces the acquisition time through a combination of fast scanning, hybrid photomultiplier detectors which are capable of handling very high count rates, and TCSPC modules with ultra short dead times. With the new FLIMbee fast scanning add-on for the MicroTime 200, this technique can be used with our microscopy platform as well as being offered as an upgrade kit for conventional Laser Scanning Microscopes (LSMs).

With this hardware combination, excellent photon statistics can be achieved in significantly shorter time spans, allowing fast processes to be measured with the high resolution achieveable in confocal microscopy. Depending on the image size, rapidFLIM allows imaging at a rate of several frames per second, enabling dynamic processes, such as protein interactions, FRET dynamics, or chemical reactions to be imaged in a time-resolved manner. With these high frame rates, FLIM can also be used on highly mobile species such as cell organelles and for other live cell imaging applications.

Recently, we have further pushed the limits of this method by systematically reducing the effects of decay distortions at very high count rates, allowing quantitative data analysis to be performed even at count rates >> 10 Mcps. This technique has been applied to quantitatively analyze FRET measurements using fluorescent proteins.

Fast, accurate scanning has also enabled scanning fluorescence correlation spec­troscopy (scanningFCS) measurements to be performed, with a view to ob­tai­ning better correlation curves from shorter measurement times. We present first results from diffusion in lipid bilayers, showing a significant improvement in the quality of data produced in comparison to conventional single point FCS using the same acquisition times.

Belinda Wright¹, Tara Bartolec², Tony Cesare², Elizabeth Hinde¹

¹EMBL Australia node in Single Molecule Science, School of Medical Sciences, University of New South Wales.
²Genomic Integrity Group, Children’s Medical Research Institute, Westmead, Sydney.

Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging¹. Here we employ fluorescence lifetime imaging microscopy (FLIM) for FRET detection of nucleosome arrangement in live cells and monitor the structural rearrangements of chromatin during DNA repair. With this technology we demonstrate that genomic double strand breaks (DSBs) induce both local and global condensation events in the chromatin network and the detected chromatin dynamics appear to facilitate DNA repair factor recruitment specifically to the lesion site

Alexandra Z. Andreou, Ulf Harms, Brighton Samatanga, Dagmar Klostermeier

University of Muenster, Institute for Physical Chemistry, Muenster, Germanydagmar.klostermeier@uni-muenster.de

DEAD-box helicases catalyze ATP-dependent RNA unwinding. Their enzymatic function is provided by a common helicase core, formed by two flexibly linked RecA-like domains. RNA unwinding is linked to an ATP-driven conformational cycle of this core: Closure of the cleft between the RecA domains upon binding of RNA and ATP leads to local destabilization of the bound RNA duplex and allows dissociation of one of the RNA strands. Phosphate release then triggers reopening of the core and release of the second strand, enabling subsequent catalytic cycles. In single-molecule FRET experiments on freely diffusing and surface-immobilized helicases, we show that modulation of the conformational cycle of the helicase core is used to regulate helicase activity. The RNA unwinding activity the eukaryotic translation initiation factor eIF4A is stimulated by the factors eIF4B and eIF4G that differentially accelerate its conformational transitions. In the B. subtilis DEAD-box helicase YxiN, binding of RNA to an RNA-binding domain (RBD) following the core induces a substantial movement of the RBD and allosterically stimulates ATP hydrolysis and RNA unwinding activities of the helicase core. Regulation of core activities through modulation of the conformational dynamics of the helicase core may constitute a widely-used regulatory mechanism of DEAD-box protein activity.

Joseph GALLAGHER², Monika DOLEGA¹, Irène WANG¹, Jacques DEROUARD¹, Joerg ENDERLEIN³, Giovanni CAPPELLO¹, Antoine DELON¹

¹Université Grenoble Alpes, Laboratoire Interdisciplinaire, de Physique, Grenoble, France
²Université Grenoble Alpes, Laboratoire Interdisciplinaire, de Physique, Alpao SAS, Grenoble, France
³Third Institute of Physics - Biophysics, Georg August University Goettingen

Fluorescence Correlation Spectroscopy (FCS) yields measurement parameters (number of molecules, diffusion time) that characterize the concentration and kinetics of fluorescent molecules within a supposedly known observation volume. Absolute derivation of concentrations and diffusion constants therefore requires preliminary calibrations of the confocal Point Spread Function with phantom solutions under perfectly controlled environmental conditions. However, any source of optical aberrations occurring when measuring in depth or even through cellular assemblies strongly biases the estimated parameters. Using a deformable mirror in open loop stabilizes these parameters. Interestingly enough the molecular brightness (as uniquely provided by FCS) scales as the Strehl ratio squared and thus, is a quite efficient metric for adaptive optics optimization.

We report recent FCS measurements performed in control solution, while excitation and detection optical paths propagate through micro-beads and cellular assembly layers. We also performed measurements inside cellular aggregates.

We present a theoretical approach to improve content and quantitativeness of measurements associated with in depth imaging, which is a challenging issue for optical microscopy. In addition we suggest an experimental approach, alternative to adaptive optics.

Kristina Bruun, Carsten Hille

Physical Chemistry, Institute of Chemistry, University of Potsdam, 14476 Potsdam, Germany

Drug delivery systems are used for the targeted transport of pharmaceuticals and their controlled release at a destined target site. One of the most commonly used drug carriers are liposomes. These systems can protect the drugs from degradation, increase the drug solubility, deliver them selectively to the target site, decrease toxic effects and improve therapeutic outcome. In addition, the fusion process of such liposome carriers can be used as a model system for studying membrane fusion at a molecular level. Thus, an improved understanding of the spatiotemporal interactions of liposomes with biological systems is required for systematic advances in drug delivery especially in cancer research. Fluorescence spectroscopy is notably suitable for investigating these aspects because of its high sensitivity, non-invasive application and the availability of highly specific fluorescent dyes.

The aim of our work is to improve the understanding of the carrier / drug uptake mechanism by applying fluorescence lifetime imaging (FLIM) and dual-colour fluorescence cross-correlation spectroscopy (FCCS), both combined with two-photon excitation (2P). For that, we are using giant unilamellar vesicles (GUVs), which act as simple model system for cell membrane, labelled with the amphiphilic fluorescent dye 3,3'-dioctadecyloxacarbocyanine (DiO). On the other hand, large unilamellar vesicles (LUVs) with a diameter of 100 nm containing an encapsulated, spectrally different fluorescent drug imitate sulforhodamine 101 (SRh101), act as drug carrier system. The preparation of such vesicles composed of different lipids allows then for studying the effects of electrostatic attraction on the liposome fusion process as well as the transfer of the drug imitate from the fusogenic LUVs to the stable GUV.

Herein, we present results of the charged-dependent fusion between anionic GUVs and cationic LUVs using time-resolved fluorescence spectroscopy and dual-colour FCCS. By using this technique, the exchange kinetics and structural changes of the liposome carriers during the fusion process are investigated. Finally, by applying FCCS and FLIM the interaction of these drug-loaded liposome carriers with living cell membranes will be investigated.

Anjali Gupta, Shuangru Huang, Lim Shi Ying, Nirmalya Bag, Thorsten Wohland

Department of Biological Sciences, NUS Centre for Bio-Imaging Sciences, National University of Singapore

The plasma membrane organization is hugely debated, especially about the existence of domains consisting of cholesterol, sphingolipids and proteins. Many fundamental signalling pathway events take place at the plasma membrane and membrane organization can have important implications in the outcome of signalling. In this study, we investigate the dynamics and organization of five different closely related cell lines using up to four different lipid probes. For the outer membrane leaflet, we use DiI-C₁₈, a marker of free diffusion and GFP-GPI (glycosylphosphatidylinositol anchored protein) as a marker of transient domain-confined diffusion. For the inner membrane leaflet, we use PH-PLCδ-RFP, a PIP₂ binding protein, and PMT-GFP, a GFP tagged plasma membrane targeting domain that is shown to exhibit cytoskeleton sensitive diffusion. We applied Imaging Fluorescence Correlation Spectroscopy (imaging FCS) on a Total Internal Reflection Microscope (TIRFM) which provided diffusion coefficients (D), the Arrhenius activation energy for diffusion (E_Arr), and the FCS diffusion law intercept (τ₀), which report on membrane fluidity, molecular packing, and diffusion mode of the probes (free, domain, or hop diffusion), respectively. Our results showed that each of the probes is characterized by a unique combination of parameters (D, E_Arr, τ₀) which varies with cell-type and temperature. Hence, organization and mode of diffusion of probe molecules is strongly environment dependent. This raises the question of the comparability of measurements, which are conducted with the same probe at different temperatures and within different cell lines.

David Li, Haochang Chen

608 Hamnett Wing Building, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, Scotland, UK

Rapid advances in semiconductor manufacturing have allowed single-photon avalanche diodes (SPAD) to be minaturised in a large 2D [1] or linear array [2] integrated with signal processing units [3]. Ten years ago, researchers hardly believed that silicon SPADs could offer satisfied quantum efficiency (QE) and dark noise performances for impactful applications in biomedical imaging. Now SPADs can easily offer a QE over 50% [4] or excellent timing accurcy within 100ps [5]. Moreover, innovative time-to-digital conversion (TDC) techniques with high resolution, excellent linearity, but neglegible dead time (< 500ps) [6], promising real-time TCSPC applications [7-10].

Enrico Gratton, Leonel Malacrida, Per Niklas Hedde

Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, 3210 Natural Science 2, Irvine CA 92967, egratton22@gmail.com

The coordination of cell functions requires that molecules move in the cell interior to find their partners. In the cell, the mechanisms for molecular motion are poorly understood.  While in an isotropic fluid, diffusion is the default mechanism of motion, in the cell interior diffusion is hindered by barriers and by transient binding.  Also molecules can move by active transport.  One universal transport processes which is still debated is the shuttling of molecules between the cell membrane and other locations where the molecules will deliver a signal.  Although we have made progresses in understanding directed motion, less is known about paths for diffusion and in general the connectivity of the cell interior. In this presentation I will discuss the development of tools that could help us in measuring the path that molecules follow in the cell to reach their target. Among the tools needed for measuring diffusion on the entire cell, or at least in a plane of the cell is a microscope capable of acquiring images fast and in 3D over large field of view. Although the SPIM microscope has some of the characteristic needed, the specific implementations proposed so far suffer from problems when imaging cells in cultures.

Stephan Uphoff

Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, United Kingdom

The accurate detection and repair of DNA damage is crucial for genome stability in all organisms. Despite extensive characterization of DNA repair pathways using genetics and biochemistry, it remains unclear how repair proteins perform their function within the cellular environment. Here, I will present our developments of single-molecule tracking and super-resolution microscopy techniques to investigate DNA repair in live cells. Using these methods, we obtained unprecedented insight into the search for DNA damage sites [1], the kinetics of repair [2], and regulation of DNA damage responses [3]. Mechanistic interpretation of microscopy observations crucially depends on specific biological perturbations, but this has been challenging in live-cell experiments. We have developed a perturbation method for rapid and complete removal of the bacterial chromosome, which allowed us to distinguish between non-specific DNA binding and free diffusion of proteins that are searching for DNA lesions. Furthermore, we used microfluidics to monitor dynamics and heterogeneity in the DNA damage response in individual cells. We found that activation of the damage response was highly stochastic and this caused cell-to-cell variation in mutation rates. Beyond applications of these methods in molecular microbiology, I will show results from single-molecule imaging of chromosome maintenance processes in human cells [4].

Till Zickmantel, Young-Hwa Song, Christian Hübner

Institut für Physik, Universität zu Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany

Human Dipeptidyl Peptidase III (hDPPIII) is a ubiquitous peptidase with broad specificity that cleaves dipeptides from the N-terminus of neuropeptides and a range of other substrates. Since substrate binding and catalytic activity are accompanied by a large conformational change, hDPPIII is an attractive system for FRET experiments. The protein captures the ligand in a cleft between two rigid lobes and closes around its target. Single molecule FRET data reveal the existence of three discrete conformations along this reaction coordinate. The relative occupancy can be shifted by varied ligand concentration. We examined the effect of point mutations in the active site and an associated amino acid interaction triad that may stabilize the individual conformations. With a Python based analysis pipeline and examination of internal dynamics in solution smFRET data, we relate the changes in conformational occupancy to a molecular latch model that may govern the opening and closing of hDPPIII.

Bianxiao Cui

Stanford University, Department of Chemistry, USA

Cell-substrate integration is crucial for many biomedical applications such as medical prosthetics and implantable electrodes for brain recording. Nanotopography and nano materials are being explored to improve the cell-substrate interactions. Recently, we and other groups show that vertical nanopillars protruding from a flat surface support cell survival and can be used as subcellular sensors to probe biological processes in live cells. Vertical nanopillars deform the plasma membrane inwards and induce membrane curvature when the cell engulfs them, leading to a reduction of the membrane-substrate gap distance. We found that the high membrane curvature induced by vertical nanopillars significantly affects the distribution of curvature-sensitive proteins and stimulates several cellular processes in live cells. Our studies show a strong interplay between biological cells and nano-featured surfaces, which is an essential consideration for future development of interfacing devices.

Klaus Yserentant, Siegfried Hänselmann, Felix Braun, Wioleta Chmielewicz, Dirk-Peter Herten

Bioquant & Institute of Physical Chemistry, Heidelberg University, Im Neuenheimer Feld 229, 69120 Heidelberg, Germany

A crucial prerequisite for quantitative fluorescence microscopy is the precise knowledge about the degree of labeling (DOL), that is the ratio of fluorescently-labeled to non-labeled targets. Although most fluorescent labeling schemes achieve sub-stoichiometric labeling efficiencies and/or attach more than one fluorophore per target binding site, relative DOL measures and not the absolute fluorophore to target ratio are usually being reported.

To overcome this limitation, we have developed a staining efficiency probe that allows for measuring the absolute DOL for genetically encoded protein tags in situ on a single-molecule level. We demonstrate the use of our probe by determining absolute DOLs for two self-labeling protein-tags, SNAP-tag and HaloTag, across a wide range of labeling conditions and in different cell lines. We found that for both tags, the absolute DOL is substantially below one for all conditions tested. We are currently investigating to which extent the achievable DOL is influenced by the fluorophore substrate and by cell line-specific properties.

Our approach is readily expendable to other protein-tags and can also be used to determine the maturation efficiencies of fluorescent proteins allowing for quantitative applications with a wide range of tags and fluorophores in both live and fixed cells.

Kristjan Leiger, Juha-Matti Linnanto, Margus Rätsep, Arvi Freiberg

Institute of Physics, University of Tartu, Estonia

Single-particle (-molecule, -protein, -organelle or -cell) spectroscopy, by getting rid of unwanted ensemble averaging, has become a valuable tool in biophysical research. Yet in these studies rather high excitation intensity is necessarily used that may significantly modify the properties of the studied particles. Here, the spectral responses of peripheral LH2 antenna complexes from photosynthetic purple bacteria on intense continuous-wave optical irradiation at ambient temperature were investigated. Both individual complexes and bulk solutions were studied. As a result, we revealed important permanent modifications of the absorption, fluorescence, and circular dichroism spectra related to singlet excitons in the bacteriochlorophyll a containing B850 domain of LH2. Analyses of the experimental spectra involving numerical simulations imply that majority of these changes are due to photo-oxidation of various numbers of the B850 chromophores and not because of damaging the structure of the surrounding protein scaffold. The limited photo-protective role of carotenoids present in the LH2 structure is also discussed.

Philip Tinnefeld

TU Braunschweig - BRICS, Institut für Physikalische und Theoretische Chemie, BRICS - Braunschweig Institute for Systems Biology, LENA - Laboratory for Emerging Nanometrology, Rebenring 56, 38106 Braunschweig

In recent years, DNA nanotechnology has matured to enable robust production of complex nanostructures and hybrid materials. We have combined DNA nanotechnology with sensitive optical detection to create functional single-molecule devices that enable new applications in single-molecule biophysics. Here, we present a new molecular force spectroscopy employing DNA origami force clamps that work autonomously without any physical connection to the macroscopic world. We used the conformer switching of a Holliday junction as a benchmark and studied the TATA-binding protein–induced bending of a DNA duplex under tension. The observed suppression of bending above 10 piconewtons provides further evidence of mechanosensitivity in gene regulation.

W. E. Moerner

Stanford University, Department of Chemistry, Stanford, CA 94305, wmoerner@stanford.edu

It is worth remembering that the first optical detection of single molecules arose out of an industrial research lab in the late 1980’s, while exploring the fundamentals of molecular frequency domain optical storage at low temperatures. This work led to the observations of blinking and optical switching, key concepts that provide the foundations of super-resolution imaging with single molecules. Super-resolution microscopy has opened up a new frontier in which biological structures and behavior can be observed in fixed and live cells with resolutions down to 20-40 nm and below, and many examples abound. Current methods development research addresses ways to image in thick cells and to extract more information from each single molecule such as 3D position and orientation, as well as to assure not only precision, but also accuracy. Further, new labels are needed which provide more photons before photobleaching. At the same time, it is worth noting that in spite of all the current focus on super-resolution, even in the “conventional” low concentration, single-molecule tracking regime where the motions of individual biomolecules are recorded rather than the shapes of extended structures, much can be learned about dynamic biological processes when ensemble averaging is removed.

Johann Georg Danzl^1,2, Sven Sidenstein², Carola Gregor², Nicolai Urban², Peter Ilgen², Stefan Jakobs², Stefan Hell²

¹Institute of Science and Technology Austria, Am Campus 1, 3400 Klosterneuburg, Austria, present address.
²Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.

Far-field superresolution microscopy or nanoscopy techniques “super-resolve” features residing closer than the diffraction-limit by transiently preparing fluorophores in distinguishable (typically on- and off-) states and reading them out sequentially. In coordinate-targeted superresolution modalities, such as stimulated emission depletion (STED) microscopy, this state difference is created by patterns of light, driving for instance all molecules to the off-state except for those residing at intensity minima. For high resolution, strong spatial confinement of the on-state is required. However, this also subjects fluorophores at intensity maxima to excess light intensities and photobleaching. In addition, as spatial confinement of the on-state is increased, state contrast between designated on- and off-regions has to be improved, too.

To address these issues, we introduced the concept of using multiple off-state transitions for coordinate-targeted nanoscopy [1]. Applied to STED microscopy, transfer of fluorophores to a second, inert off-state allowed protecting fluorophores in high intensity regions. Using reversible photoswitching as second off-transition led to a realization that we dubbed "protected STED". Our approach improved repeated imaging capability and augmented resolution and contrast through a synergistic effect of multiple off-transitions on molecular state contrast. This allowed decoding e.g. the structure of living brain tissue with 3D diffraction-unlimited resolution.

Sebastian Isbaner¹, Roman Tsukanov¹, Narain Karedla^1,2, Izabela Kaminska³, Ingo Gregor¹, Philip Tinnefeld³, Jörg Enderlein^1,2

¹III. Institute of Physics, University of Göttingen, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany
²DFG Research Center "Nanoscale Microscopy and Molecular Physiology of the Brain" (CNMPB), Göttingen, Germany
³Institute of Physical and Theoretical Chemistry, TU Braunschweig, Braunschweig, Germany

Superresolution microscopy has found tremendous applications in resolving structures on the order of a few nanometers, far below the optical diffraction limit. In particular, single-molecule localization methods are used routinely to resolve intricate biomolecular structures by making use of sparse turn-on events of fluorescent molecules. However, most of these techniques are limited to the sample plane and offer no or limited resolution along the axial direction. Here, we present a new method to localize several emitters along the optical axis with nanometer precision. The core principle behind this is the distance dependent fluorescence quenching of an emitter close to a metal surface, which we term Metal-Induced Energy Transfer (MIET). The fluorescence lifetime can be measured and converted into the molecule's distance from the surface using a theoretical model [1]. We apply this method to colocalize multiple emitters on a designed DNA origami structure labeled with dyes at known heights using step-wise bleaching. We achieve an axial localization precision of about 5 nm, within a distance range of 100 nm. The combination with the existing single-molecule localization methods would allow an isotropic nanometer localization accuracy.

Bartosz Turkowyd¹, Alexander Balinovic¹, David Virant¹, Haruko G. Gölz Carnero¹, Fabienne Caldana¹, Marc Endesfelder², Dominique Bourgeois³, Ulrike Endesfelder¹

¹Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology & LOEWE Center for Synthetic Microbiology (SYNMIKRO), Karl-von-Frisch-Str. 16, 35043 Marburg, Germany
²Institut für Assyriologie und Hethitologie, Ludwig-Maximilians-Universität München, GeschwisterScholl-Platz 1, 80539 München, Germany
³Institut de Biologie Structurale, CNRS, Université Grenoble Alpes, CEA, IBS, 38044 Grenoble, France

Live-cell single particle tracking using photoactivated localization microscopy (sptPALM) [1] allows following the dynamics of single proteins at a nanometer precision in vivo and at high protein abundance. Typically, sptPALM imaging is realized by photoactivatable and photoconvertible fluorescent proteins (paFPs and pcFPs) which are brought into a fluorescent state by illumination with near-UV light. However, introducing UV light easily causes strong photoinduced damage to living cells [2-4].

Lately, Dendra2 has shown to efficiently photoconvert by concurrent illumination with blue and infrared light, termed primed conversion (PC) [5]. This discovery suggests a new photoconversion mechanism with reduced phototoxicity by replacing the UV-light by longer wavelengths.

In our recent work [6, 7], we show that primed conversion is a general photoconversion mechanism which is immanent for Dendra and mMaple FPs and can be enabled for all Anthozoan green-to-red pcFP families (Eos pcFPs, KikGR, pcDronpa pcFPs) by controlling the conformation of residue arginine 66 through mutagenesis of residue 69. Next, to characterizing the underlying PC mechanism, we demonstrate that PC indeed reduces phototoxicity when imaging living cells and present a novel live-cell multi-color imaging approach based on this new photoconversion approach. 

Ron Tenne, Yonatan Israel, Dan Oron, Yaron Silberberg

Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot 76100, Israel

In the past two decades, several successful schemes to overcome the diffraction limit in microscopy were developed. Many of these utilize the concept of precise localization of a single emitter in a time series of sparse frames. Such methods require sparse scenes containing no more than a single emitter per diffraction limited spot per frame; slowing down the acquisition process. 

In order to alleviate the sparsity requirement in localization microscopy one needs a source of extra information. Determining the number of emitters contributing to a scene through photon correlation measurements can provide such valuable additional information. However performing such a measurement requires a fast, single-photon sensitive imaging detector. Our novel imaging device, the single-photon fiber bundle camera (SPFICAM), is an ultrafast low pixel-number camera. Each fiber from a fiber bundle acts as an effective pixel guiding photons to a single-photon avalanche photo detector. From the same SPFICAM dataset one can estimate the number of emitters and acquire images.

By incorporating the number-of-emitters information in the localization algorithm we perform precise super-resolved localization of a dense scene of quantum dots. A combination of this scheme together with standard super-resolution localization-based methods may shorten the long acquisition times that limit these methods.

Matz Liebel¹, Lukasz Piatkowski¹, Nicolò Accanto¹, Gaëtan Calbris¹, Sotirios Christodoulou^1,2, Iwan Moreels², Costanza Toninelli³, Niek F. van Hulst^1,4

¹ICFO – the Institute of Photonic Sciences, Barcelona Institute of Science & Technology, Barcelona
²Nanochemistry Department, IIT - Istituto Italiano di Tecnologia, Genova, Italy
³CNR-INO, Istituto Nazionale di Ottica, LENS, Sesto Fiorentino, Florence, Italy
⁴ICREA – Institució Catalana de Recerca i Estudis Avançats, Barcelona., e-mail: Niek.vanHulst@ICFO.eu, Web:www.ICFO.eu

Detecting single molecules in life science applications, one traces individual molecules both in space and in time, providing high-resolution images of dynamic processes beyond the ensemble. Yet capturing fast dynamics is fundamentally limited by the lifetime of the detected fluorescence. A host of important dynamic processes occurs on fs-ps timescale, such as electronic relaxation and dephasing, energy transfer, charge transfer, vibrational relaxation, quenching, internal conversion, photo-dissociation-ionization and photo-isomerization. For the ensemble, fs-ps resolution is obtained by transient absorption and 2D electronic/vibrational spectroscopy [1].

Here we present the first ultrafast transient absorption of a single molecule. Specifically, we trace the femtosecond evolution of excited electronic state spectra of single molecules over hundreds of nanometers of bandwidth at room temperature. The non-linear ultrafast response of the single molecule is probed using a broadband laser in an effective 3-pulse scheme with fluorescence detection. Two-dimensional electronic spectroscopy of single molecules is experimentally in reach [2].

Nanosecond spontaneous fluorescence decay is slow, and occurs from the lowest excited state thus missing on any ultrafast relaxation dynamics in the excited state. Stimulated emission has been used as alternative detection method [3] and is widely applied to enhance spatial resolution in STED microscopy [4]. Here we demonstrate direct detection of stimulated emission from individual quantum emitters at ambient conditions, with synchronized stimulated emission and luminescence contrast for imaging. We use time-resolved stimulated emission to disentangle ultrafast charge dynamics in the excited state, which occurs on sub-picosecond timescale. [5].

Finally, perspectives and challenges ahead of addressing “singles” in real nano-space on femtosecond timescale will be addressed.

Tim Schröder¹, Max B. Scheible², Philip Tinnefeld¹

¹Rebenring 56, Braunschweig, Germany 38106
²GATTAquant GmbH, Karlstr. 82, Braunschweig, Germany 38106

Fluorescence polymer beads are broadly used for characterizing fluorescence and super-resolution microscopes. But their broad distribution in size and brightness are a big drawback [1]. DNA origami nanotechnology can overcome this problem by placing a defined number of dyes at predefined positions on a template DNA structure. We alter the distance between two ATTO647N dyes at the single base pair level on the same DNA helix in order to find the minimum distance between two dyes without observing dye-dye interactions in a DNA origami structure. At close distances H-type dimer formation quenches the fluorescence intensity which drops by one order of magnitude. Shortened fluorescence lifetimes and a change in the absorption spectrum are also observed. Fluctuations of fluorescence intensity and fluorescence lifetime on a millisecond timescale reveal the dynamic nature of DNA. The dyes regain their known fluorescence properties at larger distances. The results are used in collaboration with GATTAquant to build a nanoscaled DNA bead with outstanding homogeneity and brightness [2].

Arindam Ghosh, Roman Tsukanov, Alexey Chizhik, Ingo Gregor, Narain Karedla, Jörg Enderlein

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

Förster Resonance Energy Transfer (FRET) is a widely used fluorescence technique to probe the dynamics of biomolecules at single molecule level [1]. However, due to the nature of dipole-dipole coupling FRET is limited to a short distance range of ca. 10 nm, and it requires labeling the sample with two fluorescent dyes. In this work, we report a novel method to study the fast conformational dynamics of single molecules using Metal-Induced Energy Transfer (DynaMIET). The method relaxes the labeling requirement to a single dye and is based on the distance dependent energy transfer from a molecule’s excited state to the metal surface plasmons [2]. This leads to temporal fluctuations of the intensity due to the dynamics of the single molecule orthogonal to the metal surface within a distance range of ca. 100 nm. We present proof of concept DynaMIET results by probing the chain opening and closing dynamics of individual DNA-hairpin molecules immobilized on a thin gold film and compare the obtained rates with existing FRET values [3]. This method can be potentially used to quantify sub-microsecond to millisecond relaxation time constants of biomolecules and polymer chains on length scales inaccessible to a conventional single-pair FRET experiment.

Maabur Sow¹, Barak Gilboa², Laia Gines³, Olivier Williams⁴, Achillefs Kapanidis⁵

¹maabur.sow@physics.ox.ac.uk
²barak.gilboa@physics.ox.ac.uk
³GinesL@cardiff.ac.uk
⁴WilliamsO@cardiff.ac.uk
⁵kapanidis@physics.ox.ac.uk

The fluorescent nitrogen-vacancy defect (NV) in diamond exhibits exceptional photophysical properties such as remarkable photostability. This makes nanodiamonds very promising probes for bio-imaging [1,2]. Photophysical properties of the NV centre have been well studied in bulk diamond and in nanocrystals but less in solution because of the technical challenges it poses, especially for small nanodiamonds (< 15 nm) showing high diffusion coefficient and low brightness [2,3]. Here we report the use of wide-field epifluorescence imaging (including single-particle tracking) to characterise the size and the photophysics of nanodiamonds. Unlike methods using an averaged measurement of the sample such as dynamic light scattering, working at a single-particle level allowed us to analyse up to a thousand of nanodiamonds in one experiment. Distributions of nanoparticles in terms of brightness and photostability can be generated for immobilised nanoparticles on surfaces or diffusing in solution. Using intensity ratios (between green and red emission) we are able to investigate the transition of NV- to NV0 (rates and lifetimes) and the proportion of fluorescent nanocrystals. This characterisation approach will be useful to the material scientists designing smaller and brighter nanodiamonds which is one of the key challenges for more successful applications in bio-imaging.

Achillefs Kapanidis

Clarendon Laboratory, Department of Physics, University of Oxford, UK, a.kapanidis1@physics.ox.ac.uk

Transcription, the process responsible for the conversion of DNA information into RNA, is the first and most regulated step in gene expression in all organisms. Transcription is orchestrated by the multi-functional protein RNA polymerase. Despite the importance of transcription, the mechanisms and pathways of many transcription steps still remain obscure due to the presence of transient intermediates and heterogeneity, which challenge ensemble biochemistry and conventional structural-biology.

Here, I will focus on our recent discovery and characterization of new mechanistic steps and pathways of transcription by the bacterial RNA polymerase (RNAP) using single-molecule fluorescence. Specifically, I will discuss in vitro and in vivo single-molecule FRET studies of DNA conformational changes on immobilized initial transcribing complexes. Our work uncovered extensive pausing and backtracking during initial transcription, and identified protein, DNA and RNA elements that control and modulate the pausing, and can have major regulatory roles. I will also describe studies of RNA polymerase conformational changes that are crucial for opening the DNA and stabilising the transcription complex, while serving as targets for antibiotics. Our work paves the way for further analysis of major conformational changes occurring from initiation to early elongation.    

Flurin Sturzenegger, Franziska Zosel, Daniel Nettels, Erik Holmstrom, Benjamin Schuler

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

A transition path is the very short part of a molecular trajectory where the free energy barrier between two states is crossed. It is a single-molecule property and contains all the mechanistic information about the process. Measuring transition path times is challenging but was achieved recently for protein folding reactions using single-molecule FRET [1]. Here we study transition path times of the coupled binding and folding reaction of two intrinsically disordered proteins, ACTR and NCBD. We immobilize donor-labeled ACTR on a surface and observe binding and unbinding events of NCBD molecules, which are labeled with an acceptor dye, and analyze them on a photon-by-photon level with the maximum likelihood method developed by Gopich & Szabo [2]. The average transition path time was found to be 80 μs, at least an order of magnitude slower than for the unimolecular protein folding reactions studied so far [1]. The high photon count rates of the trajectories also allow us to characterize the distribution of transition path times, from which information about the barrier-shape can be deduced.

Mikayel Aznauryan, Victoria Birkedal

Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus, Denmark

G-quadruplexes are secondary nucleic acid strucrures that fold in the presence of physiological ions (K+ and Na+) on the basis of guanine-rich nucleic acid sequences. It is nowadays emerging that they play important regulatory roles in cell biology [1-2], are potential targets for anti-cancer therapies [3] and can also be utilized as biocompatible structures in DNA nanotechnology [4].

Here we employed single-molecule Förster Resonence Energy Transfer (FRET) microscopy to investigate the folding and underlying conformational dynamics of human telomeric G-quadruplex DNA. Our results yield a comprehensive thermodynamic and kinetic description of the folding of G-quadruplexes that we find to proceed through a complex multi-route pathway, involving several conformational states [5]. Our recent experiments in the presence of synthetic crowders as well as mammalian cell lysates probed the effect of macromolecular crowding on the folding of G-quadruplexes. Moreover, we compared our experimental data to the simplest model for the excluded volume effect on biomolecular processes - the scaled particle theory - and find a remarkable agreement between them. Altogether, these studies give a detailed picture of the folding dynamics of G-quadruplexes under broad experimental conditions thus uncovering common mechanistic features of their folding under cell-mimicking milieu.

Erik Holmstrom¹, Zhaowei Liu¹, 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

Like proteins, nucleic acids can also fold into intricate 3D structures with specific biological functions. In order to do so they must avoid any potential non-functional conformational traps that can complicate the folding process. Nucleic acid chaperones are a class of positively charged proteins, often intrinsically unstructured, that function to alleviate this notorious folding problem by facilitating the formation of natively folded RNAs and DNAs. Despite recent advances in our understanding of the functional mechanism associated with these protein, not much is known about the molecular structure of the nucleoprotein complexes that they form. Therefore, we have started to use a variety of time-resolved single-molecule FRET techniques to study the conformational dimensions of a model nucleic acid chaperone. Using a collection of intra- and inter- molecular FRET pairs it appears that this nucleic acid chaperone remains entirely unstructured while chaperoning the folding of a model nucleic acid. The experimental observations are nicely consistent with coarse grained molecular dynamics simulations that treat these biomolecules as non-specific charged polymers. These findings help provide a refined structural and functional view of intrinsically disordered nucleic acid chaperones.

Madhavi Krishnan

Department of Chemistry, University of Zurich , Winterthurerstrasse 190, CH-8057 Zürich, madhavi.krishnan@uzh.ch

The desire to “freely suspend the constituents of matter” in order to study their behavior can be traced back over 200 years to the diaries of Lichtenberg. From radio-frequency ion traps to optical tweezing of colloidal particles, existing methods to trap matter in free space or solution rely on the use of external fields that often strongly perturb the integrity of a macromolecule in solution. We recently introduced the ‘electrostatic fluidic trap’, an external field-free principle that supports stable, non-destructive confinement of single macromolecules in room temperature fluids, representing a paradigm shift in a nearly century-old field [1]. The spatio-temporal dynamics of a single electrostatically trapped object reveals fundamental information on its properties, e.g., size and electrical charge [2]. We have demonstrated the ability to measure the electrical charge of a single macromolecule in solution with a precision much better than a single elementary charge [3]. Since the electrical charge of a macromolecule in solution is in turn a strong function of its 3D conformation, our approach enables for the first time precise, general measurements of the relationship between 3D structure and electrical charge of a single macromolecule, in real time. I will present our most recent advances in this emerging area of molecular measurement which now includes spectrally resolved multi-color detection of single molecules trapped in parallel.

Iman Esmaeil Zadeh, Johannes Los, Ronan Gourgues, Violette Steinmetz, Sergiy Dobrovolskiy, Valery Zwiller, Sander Dorenbos

Single Quantum, Van der Waalsweg 8, 2628CH, Delft, The Netherlands

Single-photon detection with high efficiency, high time resolution, and high photon detection-rates is required for a wide range of optical measurements. Superconducting nanowire single-photon detectors offer exceptional performance in the near-infrared. To unleash the true potential of these detectors in bio-imaging applications, sub 10 ps time resolution and  count-rates in the order of tens of MHz are required. Here we show for the first time a superconducting nanowire single photon detector with a system detection efficiency >97% and a count-rate of >200MHz. We measure an unprecedented timing jitter of <3.7 ps (FWHM) at the count-rate of 50MHz. This combination of ultrahigh time resolution and count-rate and high efficiency opens great new possibilities for the field of femtosecond imaging/spectroscopy, time-resolved infrared spectroscopy, high-resolution imaging, non-invasive reflectometry and diffuse optical tomography
 

Dirk-Peter Herten¹, Felix Braun¹, Wioleta Chmielewicz¹, Siegfried Hänselmann¹, Klaus Yserentant¹, Kristin Grußmayer²

¹Institute for Physical Chemistry, Heidelberg University, Germany
²École Polytechnique Fédérale de Lausanne, Switzerland

With the diffraction barrier broken, optical microscopy can resolve various protein complexes in cells on a length scale of approx. 20 nm. At the same time single-molecule sensitivity allows kinetic measurements of the interaction of individual proteins on the milliseconds time scale. Both achievements offer solutions to biological questions which could not have been asked about 20 years ago. However, the ultimate dream of a microscopist is a method that also delivers the absolute number or at least a fair estimate of the protein copies along with an image of the cellular structure. Although several approaches have been developed to count copies in protein assemblies – mostly based on single-molecule spectroscopy - so far there is no method available that delivers quantitative images cellular structures, ideally with a time resolution sufficient to also follow their formation and decay in live-cell experiments. We envision that the principles of photon-antibunching can be used to achieve the ambitious goal of quantitative microscopy.[1-6] Along this line we not only tried to improve the method which is still based on point detection but also started developing strategies for stoichiometric labelling and for quantifying the labelling efficiencies which will be discussed with along with their shortcomings.

Steven Magennis

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

It is clear that a crowded environment influences the structure, dynamics, and interactions of biological molecules, but the complexity of this phenomenon demands the development of new experimental and theoretical approaches. We have used two complementary single-molecule FRET techniques to study DNA hairpins either freely diffusing in solution or immobilized on a surface under crowding conditions. We found that DNA base pairing and unpairing, which are fundamental to both the biological role of DNA and its technological applications, are strongly modulated by a crowded environment. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concentrations of the crowding agent polyethylene glycol (PEG). These experiments highlight the impact of a crowded environment on a fundamental biological process, DNA base pairing, and illustrate the benefits of single-molecule approaches to probing the structure and dynamics of complex biomolecular systems.

Felis Braun¹, Andreas Haderspeck¹, Dominik Brox¹, Marcel Best², Richard Wombacher², Dirk-Peter Herten¹

¹Institute for Physical Chemistry, Heidelberg University, Im Neuenheimer Feld 229, 69120 Heidelberg, Germany
²Institute for Pharmacy and Molecular Biotechnology, Heidelberg Universtity, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

Switching a fluorophore between fluorescent and non-fluorescent states is a key process for super-resolution techniques based on single-molecule localization. Most of these approaches use light-driven transitions for switching. The resulting highly-resolved image reconstruction however comes often at the cost of high irradiation intensities, requires special imaging buffers and is limited to a small number of suitable fluorophores. Our research focuses on alternative switching mechanisms using reversible chemical reactions. Previously, we reported switching between bright and dark states by reversible coordination of Cu²⁺-ions to a ligand.^[1] The stochastic nature of this process can be used in localization microscopy to decouple blinking from excitation, making additional laser irradiation obsolete and reducing phototoxicity.^[2] More recently we could exploit the Cu²⁺-complexation for more persisting reversible fluorescence switching to establish a novel mode of multiplexing.^[3] To date we have developed a modular system for Cu²⁺-switchable probes comprising different ligands, a large variety of fluorophores and diverse linkers for conjugation. This offers a large library covering the whole visible spectrum for all kinds of targeting approaches. Beyond the Cu²⁺-dependent fluorescence switching we now intend to enlarge the scope of chemical reactions e.g. by complexation of Ca²⁺-ions^[4] or pH-dependent spirolacton formation in rhodamines.

Arindam Ghosh¹, Sebastian Isbaner¹, Manoel Veiga², Ingo Gregor¹, Jörg Enderlein¹, Narain Karedla¹

¹III. Institute of Physics, Georg August University Göttingen,Göttingen,37077,Germany
²PicoQuant GmbH; 12489 Berlin, Germanz

Many complex luminescent emitters such as fluorescent proteins exhibit multi-exponential decay patterns of their excited state [1]. Single molecule spectroscopy revealed that this is true even for single emitters, indicating that they rapidly fluctuate between different emitting states. Here, we apply Fluorescence Lifetime Correlation Spectroscopy (FLCS) [2][3] to resolve the photophysical state dynamics of the prototypical Enhanced Green Fluorescent Protein (EGFP) [4]. We quantify the microsecond transition rates between its two fluorescent states, which otherwise show highly overlapping emission spectra. We relate these transitions to a rotational isomerization of an amino acid next to the protein's fluorescent center. With this study, we demonstrate the power of FLCS for studying the rapid transition dynamics of a broad range of light-emitting systems with complex multi-state photophysics, which cannot be easily done by other methods.

Lennart Grabenhorst, Sarah E. Ochmann, Carolin Vietz, Birka Lalkens, Philip Tinnefeld

Institute for Physical and Theoretical Chemistry, Braunschweig University of Technology, Rebenring 56, 38106 Braunschweig, Germany

Recent epidemics like the zika epidemic in south america in 2015 illustrated the need for new diagnostic tools that are both highly specific and affordable as well as easy to use as epidemics often hit hardest in areas with low access to medical facilities.

In this work, we employ nucleic acid hybridization probes to detect short single stranded DNA sequences specific to the zika virus in vitro. The resulting fluorescence signal is amplified in a plasmonic hotspot formed by silver nanoparticles held in place using DNA nanostructures created with the origami technique. We show that the probes are highly sensitive with respect to the target DNA sequence and that plasmonic interactions amplify the observed fluorescence intensity by severalfold, potentially enabling detection of the signal even with cheaper sensors like smartphone cameras.

The simple fabrication process as well as the remarkably high single molecule fluorescence intensity might provide the framework for future point-of-care instruments that enable the physician to differentiate between numerous different infections even before symptoms start to show.

Daniel Gudnason^1,2, Mikael Madsen^1,2, Abhichart Krissanaprasit^1,2, Kurt V. Gothelf^1,2, Victoria Birkedal^1,2

¹Center for DNA Nanotechnology, Interdisciplinary Nanoscience Center, iNANO, Aarhus University, Gustav Wieds vej 14, 8000, Aarhus C, Denmark
²Department of Chemistry, Aarhus University, Langelandsgade 140, 8000, Aarhus C, Denmark.

The photophysical properties of conjugated polymers are used in photonic devices such as organic light emitting diodes (OLEDs), solar cells and biosensors. These properties are sensitive to the polymers physical conformation, which is an important factor for device performance and stability [1]. Here, DNA specific interactions are used to conformationally control a DNA functionalized Poly(Phenylene-Vinylene) polymer (APPV-DNA) [2].

First, we investigate different ways to achieve conformational control of the polymer in solutions.  Polymer properties are probed using absorption and emission spectroscopy as well as atomic force microscopy (AFM). Our results show that the aggregation state of APPV-DNA can be tuned by DNA hybridization and addition of mono- or multivalent salts to the buffer, yielding polymer solutions that exhibit different photophysical properties. We also aim at controlling polymer conformation on surfaces and use DNA origami platforms to spatially organize polymers on the nanoscale [2]. We show through AFM and single molecule fluorescence microscopy, that different aggregation states of the polymer can be prepared on DNA origami tiles. Achieving a nanoscale spatial control of polymer conformation is interesting for a number of applications and can be used to investigate and potentially improve their energy transferring capabilities.

M. Hamkalo^1,2, D. Borycki¹, M. Nowakowski¹, P. Stremplewski¹, M. Wojtkowski^1,2

¹Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
²Institute of Physics, Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus University, Grudziadzka 5, 87-100 Torun, Poland

In case of all conventional microscopic techniques light propagation through scattering layers is inevitably connected with the image distortions. So far problems like wavefronts deformations or speckle patterns formations were tackled by closed-loop wavefront shaping, transmission matrix analysis or numerical post-processing, which is usually sample-dependent. We present a novel technique of spatio-temporal optical coherence (STOC) manipulation, which can suppress the image distortions with use of a proper phase modulation of the incident light. We have applied this method in the optical coherence microscopy (OCM) system in which the light propagating in the object arm was modulated by the spatial light modulator (SLM). Obtained images of static samples (below the scattering layer) show that application of STOC hinders speckles formation and improves imaging quality. Moreover, we are presenting the influence of different kind of phase modulations on the image quality improvement.

Maximiliann Huisman, Carlas Smith, Mathias Hammer, David Grunwald

UMASS Medical School, 368 Plantation Street, Worcester, MA 01605

Single-molecule detection in fluorescence nanoscopy continues to provide ever-more powerful tools to investigate biological phenomena, ranging from the investigation of super-resolved structures to high-speed tracking in live cells. Although technological advances have increased the amount of signal that can be obtained, smart image-processing strategies are still required to combat issues such as unspecific background and limited signal. Efforts to replace empirically set parameters and thresholds values with analytical models have further increased detection efficiency and are bringing us closer to being able to ‘place an error-bar on images’.

Statistical detection frameworks such as probability-based hypothesis testing can remove the need for expert knowledge and subjective parameter tweaking, increasing reliability as well as detection efficiency – especially when photons are scarce. For this strategy to work, we need a model of what a positive signal looks like under the imaging conditions it is examined in. An accurate implementation of such a model therefore requires information about the particles of interest (e.g. size, emission wavelength, mobility), but also needs to factor in knowledge about the imaging system (e.g. camera efficiency and noise characteristics, optical aberrations). In low-light conditions, the latter component start to weigh more heavily as the detection limits of the microscope are being pushed.

Here, we present an approach to systematically extract the imaging system parameters needed for parameter-free, statistical single-molecule detection algorithms on most commonly used microscopes. This workflow comprises a benchmarking strategy of the optical quality across the field of view as well as a calibration routine for the detector. The latter is rendered possible by the use of prototype calibration device we call the test tube: this inexpensive yet versatile device generates a well-defined series of illumination conditions that are subsequently used to measure the light-efficiency of the optical system, as well as extracting the noise-characteristics of the detector. The ability to extract these parameters allows statistical detection framework we presented earlier[1] to be extended beyond EMCCD detectors, opening this method up to sCMOS cameras and confocal microscopes.

Darui Li ¹, Krutika Bavishi ², Simon B. Jensen ^1,*, Stine Eiersholt ¹, Emma N. Hooley ¹, Tom Vosch ¹, Birger Lindberg Møller ², Tomas Laursen ³ and Nikos S. Hatzakis ¹

¹ Department of Chemistry & Nanoscience Center, University of Copenhagen, DK-1871 Frederiksberg C
² Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C
³ Feedstocks Division, Joint BioEnergy Institute, Emeryville, CA 94608, USA
^*Simon Bo Jensen (lzn282@alumni.ku.dk)

Cytochrome P450 oxidoreductase (POR) is the primary electron donor in eukaryotic cytochrome P450 (CYP) containing systems and activates a plethora of structurally diverse downstream partners. The intrinsic dynamic motions of POR are anticipated to both underlie its function and its plasticity of selectively activating the downstream CYPs^1,2.

Here, the establishment of two platforms for direct observation of conformational sampling of POR using single molecule Förster resonance energy transfer (smFRET) is described. We used full length POR from the crop plant sorghum bicolor, site-specifically labeled with Cy3 (donor) and Cy5 (acceptor) fluorophores and reconstituted in nanodiscs. Our smFRET measurements allowed us to directly observe the existence, abundance and dependence on regulatory cues of POR conformational states. We identified three dominant conformational sub-populations as well as two transient intermediates. An increase in the ionic strength caused POR to populate more open conformations, while changing the reconstitution environment, i.e. phospholipid bilayers (nanodiscs) composed of different lipid head group charges vs. detergent micelles, significantly altered the occupancy of conformational states. Strikingly, detergent solubilized POR displayed a higher occupancy of the closed conformations compared to POR in bilayers.

The observation of multiple conformational states of POR and the transitional pathways connecting them, aids our understanding of POR structural dynamics and provides links to its biological function^3,4. The existence of conformational heterogeneity may mediate selective activation of multiple downstream partners and association in protein complexes in the ER membrane².

S. Orthaus-Mueller, M. Koenig, R. Dowler, B. Kraemer, F. Jolmes, M. Wahl, H.-J. Rahn, M. Patting, F. Koberling, R. Erdmann

PicoQuant, Rudower Chaussee 29, 12489 Berlin, Germany

Imaging the dynamics of life requires high spatial and temporal resolution. Therefore, expanding the combination of excellent time resolution from Fluorescence Lifetime Imaging (FLIM) based on Time-Correlated Single Photon Counting (TCSPC) with high spatial resolution towards ultrafast image acquisition times is most desirable. Speeding up FLIM enables to study processes and changes occurring on short time scales, such as signal transduction pathways in cells or fast moving sub-cellular structures.

In contrast to other conventional FLIM methods, our rapidFLIM approach allows combining high optical resolution of confocal laser scanning microscopes with acquisition speeds of more than 15 frames per second, while retaining the high temporal resolution of TCSPC and offering good lifetime contrast even for samples with multiple fluorescent species. Thus, rapidFLIM is essential to monitor even smallest changes in the fluorescence behavior of living or mobile samples.

The method exploits recent hardware developments such as the TimeHarp 260 Nano TCSPC card with virtually no dead time allowing to record huge photon numbers without loss. This, in combination with our extreme low dead time detector, enables to achieve significantly higher count rates (up to 40 MHz) within a specific time frame than with a classic setup and to speed up FLIM image acquisition by more than a factor of 100.

Even complex fluorescence decay data can be efficiently and quickly analyzed with our easy-to-use pattern matching method. Now, complete turn-key systems such as stand-alone units or upgrade kits enable to study fast and dynamic processes such as protein interactions involved in endosome trafficking.

Tanja Kaufmann¹, Sebastien Herbert¹, Lijuan Zhang³, Joachim Garbrecht¹, Josef Gotzmann², Kareem Elsayad³, Dea Slade¹

¹Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, Vienna, Austria
²BioOptics Facility, Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9, Vienna, Austria
³Advanced Microscopy Facility, Vienna Biocenter Core Facilities (VBCF), Dr. Bohr-Gasse 3, Vienna, Austria

DNA damage elicits a complex response in human cells comprised of DNA damage sensing, signalling and repair. Protein-protein interactions are essential to ensure timely and precise recruitment of repair factors to DNA damage sites. Conventionally, DNA damage is induced globally by irradiation or chemical treatment and the effect of the damage on the interaction of repair proteins is measured on a whole population of cells. However, this approach precludes full understanding of the complexity of DNA-damage dependent protein interaction dynamics. To tackle these limitations we integrated a controllable pulsed 355 nm UV laser add-on module on a commercially available confocal FLIM microscope from Picoquant. This experimental configuration allows us to induce localized DNA double strand breaks in the nucleus of single cells and quantify DNA-damage dependent changes in protein interactions by FLIM-FRET measurements. We have previously verified that Poly(ADP-ribose) glycohydrolase (PARG) is recruited to DNA damage sites in a proliferating cell nuclear antigen (PCNA)-dependent manner, identified a novel binding site for PCNA on PARG, confirmed FRET between PARG-EGFP and mRFP-PCNA and found that a single mutation in this binding site is sufficient to abolish the binding of PARG to PCNA as measured by FRET [1,2]. We will present first insights into dynamic changes in the PARG-PCNA interaction monitored at a single cell level in response to localized UV laser-induced DNA damage using our in-house developed setup.

Lars Kreutzburg, Christian Hübner

Institut für Physik, Universität zu Lübeck, Ratzeburger Allee 160, 23562 Lübeck

The interface of the blood circulatory system and tissue is characterized by a dynamic matrix
of membrane-bound glycoproteins, proteoglycans and adsorbed proteins of the blood
plasma. This endothelial surface layer (ESL) not only acts as a steric barrier to molecules from
the blood stream but also shows an electric net charge, caused by sulphate-chains that may
affect the dynamics of any particles in or near the ESL. In order to understand the process of
drug release and distribution, it is crucial to gain knowledge of the particle dynamics in the
proximity of the capillary wall. To address this issue, we apply fluorescence correlation
spectroscopy to investigate the transition from directed motion in model vessels to diffusive
motion inside a model ESL.

Yury Prokazov³, Sebastian Tannert⁴, Rhys Dowler⁴, Torsten Langer⁴, Felix Koberling⁴, Claus Seidel², Michael Beek¹, Bernd Müller⁵, Ottmar Jagutzki⁶, Rainer Erdmann⁴, Werner Zuschratter³

¹Best Systeme GmbH, Münchner Strasse 123a, 85774 Unterföhring
²HHU Düsseldorf, Universitäts Str. 1, Geb 26.32.02, 40225 Düsseldorf
³LIN Magdeburg, Brenneckestr. 6, 39118 Magdeburg
⁴PicoQuant, Rudower Chaussee 29, 12489 Berlin
⁵ProxiVision GmbH, Robert Bosch Str. 34, 64625 Bensheim
⁶RoentDek Handels GmbH, Im Vogelshaag 8, 65779 Kelkheim

Time-resolved fluorescence microscopy is a technology of significant interest for biological and biomedical applications. It is used to study cell morphology as well as to unravel cellular signalling pathways, protein folding, and interactions between biomolecules. Today the majority of systems for fluorescence lifetime imaging (FLIM) is based on confocal laser or sample scanning. Here, we present a different approach. Assembling a dedicated detector head with a segmented anode structure enables the construction of a time and spatial resolving single photon counting detection system. The photon to electron converting detector head contains a photocathode, a two stage microchannel plate stack, and a resistive layer for charge mirroring. The detection system is coupled onto a widefield fluorescence microscope. We implemented picosecond pulsed epi-fluorescence and total internal reflection excitation.

The detector records the time interval between an excitation pulse and a detected photon on a picosecond timescale, spatial information, as well as the macroscopic arrival time. A time resolution of less than 100 ps could be achieved. This fits well to most common organic dyes and all fluorescent proteins. It allows furthermore time-resolved FRET studies.

In this poster, we present recent improvements of the detection system to lower the dead time and to enhance the photon throughput. Since the system is free of scanning parts it is well suited for long term life cell imaging under low light level conditions.

This research has been performed by a joint German research cooperation funded by the ministry of eduction and research (BMBF, project code 13N12672, tCam4Life).

David Li, Haochang Chen

608 Hamnett Wing, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, Scotland, UK

Time-to-digital converters (TDCs) are high-precision stopwatches that are capable of measuring fast physical events in many time-resolved applications due to their excellent performances in timing resolution. They have been widely applied in ranging, biomedical (such as Time-Correlated Single Photon Counting, TSCPC, in fluorescence lifetime imaging microscopy (FLIM), positron emission tomography, time-of-flight mass spectrometry and Raman spectrum), nuclear physics and quantum communications. The TDC can be implemented in application-specific integrated circuits (ASIC) and field programmable gate arrays (FPGA). Comparing with ASIC-based TDC, FPGA TDCs provide great flexibility with a shorter developing cycle for fast prototyping and research experiments. FPGAs are re-programmable, easy to access (low cost), and promising for product developments. However, traditional FPGA-TDCs are suffered from the poor linearity. This work presents low nonlinearity, missing-code free TDCs that achieves a least significant bit (LSB; resolution) of 10.5ps implemented in a Xilinx Virtex 7 FPGA device with a novel bin-width calibration method.

Alex Macmillan¹, Elizabeth Hinde², Sharon Sagnella^3,4, Maria Kavallaris^3,4 & Renee Whan¹

¹Biomedical Imaging Facility, University of New South Wales, Australia 20522
²Centre for Vascular Research, University of New South Wales, Sydney, Australia 2052
³Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, NSW, Australia
⁴ ARC Centre of Excellence in Convergent Bio-Nano Science and Technology and Australian Centre for NanoMedicine, UNSW Sydney, NSW, 2052

Soheil mojiri, Ingo Gregor, Joerg Enderlein

III Institute of Physics, Georg August University, Göttingen, Germany

Super Resolution Optical Fluctuation (SOFI), is a post processing fast way based on temporal correlation calculation of blinking emitters for obtaining images beyond the diffraction limit of light with a classical wide-field microscope.  Beside superior property of background reduction, SOFI enables optical sectioning results in increased resolution in all three dimensions. For employing the 3D Sectioning capability of SOFI and fast dynamic observation through the live mechanisms it is highly  favorable to simultaneous imaging of 3D volume instead of plane scanning. To this end, we have already combined our wide-field microscope with a multi-plexed 3D imaging system by implementing an 2×4 image splitter prism and two perpendicular cameras. The simultaneous acquisition of multiple focal planes reduces the acquisition time and thus the photobleaching. Fluorescent beads are scanned and imaged to characterize the brightness response and microscope specifications.

Anita Toulmin¹, Michael J. Morten², Tara Sabir¹, Laura E. Baltierra-Jasso^1,2, Peter McGlynn³, Gunnar F. Schröder⁴, Brian O. Smith⁵, Steven W. Magennis²

¹The School of Chemistry, The University of Manchester, Manchester, UK
²WestCHEM, School of Chemistry, University of Glasgow, Glasgow, UK
³Department of Biology, University of York, York, UK
⁴Institute of Complex Systems (ICS-6), Forschungszentrum Jülich, Germany
⁵Institute of Molecular, Cell & Systems Biology, University of Glasgow, Glasgow, UK

Branched structures of nucleic acids are widely observed in nature as intermediates during DNA repair, recombination and replication, as well as being key components of DNA nanostructures. Using high-resolution single-molecule Förster resonance energy transfer (SM-FRET), we recently showed that a DNA three-way junction (3WJ) was not fully paired at the branchpoint, in spite of having a fully complementary sequence [1]. Here, we report the results of SM-FRET experiments alongside molecular dynamics simulations and the first use of 19F NMR on a fully complementary 3WJ, which shows that a 3WJ can adopt at least two major conformations, depending on the branchpoint sequence. This structural diversity has implications for processing 3WJs in vivo and their use in nanodevices.

Franziska Zosel¹, Dominik Haenni², Andrea Soranno³, Daniel Nettels¹, Benjamin Schuler^1,4

¹Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
²Center for Microscopy and Image Analysis, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
³Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, 63110, USA
⁴Department of Physics, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

Intrinsically disordered proteins (IDPs) are a class of molecules that can exert essential biological functions even in the absence of a well-defined three-dimensional structure. Understanding the conformational distributions and dynamics of these highly flexible proteins is thus essential for explaining their function. smFRET is a powerful tool for probing intramolecular distances and the rapid long-range distance dynamics in IDPs. We combine it with PET quenching to monitor local loop-closure kinetics at the same time and in the same molecule. Here we employed this combination to investigate the intrinsically disordered N-terminal domain of HIV-1 integrase. The results show that both long-range dynamics and loop closure kinetics on the sub-microsecond timescale can be obtained reliably from an analysis with a comprehensive model of the underlying photon statistics including both FRET and PET. A detailed molecular interpretation of the results is enabled by direct comparison with a recent extensive atomistic MD simulation of integrase[1]. The simulations are in good agreement with experiment and can explain the deviation from simple models of chain dynamics by the formation of persistent local secondary structure. The results illustrate the power of a close combination of single-molecule spectroscopy and simulations for advancing our understanding of intrinsically disordered proteins.

Max Nobis¹, David Herrmann¹, Sean C. Warren¹, Shereen Kadir², Wilfred Leung¹, Monica Killen¹, Astrid Magenau¹, David Stevenson², Morghan C. Lucas¹, Nadine Reischmann¹, Clarie Vennin¹, James R.W. Conway¹, Michael S. Samuel³, Marina Pajic¹, Ewan J. McGhee², Owen J. Sansom², Heidi C.E. Welch⁴, Jennifer P. Morton², Douglas Strathdee², Kurt I. Anderson⁵, Paul Timpson¹

¹The Garvan Institute of Medical Research, St Vincent's Clinical School, Faculty of Medicine, Sydney, NSW 2010, Australia
²Cancer Research UK Beatson Institute, Switchback Road, Bearsden, Glasgow, G611BD, UK
³Centre for Cancer Biology, SA Pathology and University of South Australia School of Medicine, University of Adelaide, Adelaide, South Australia 5000, Australia.
⁴Signalling Programme, Babraham Institute, Cambridge CB223AT, UK
⁵Francis Crick Institute, London NW11AT, UK

Small GTPases such as RhoA enable cells to migrate during development as well as metastasize during cancer progression. More specific, time-resolved monitoring of RhoA activity in tissues that have upregulated RhoA activity could therefore be done in an in vivo setting with the use of Förster resonance energy transfer (FRET) biosensors to track the effect of therapeutic intervention.

Here, we describe the generation and characterization of a RhoA-FRET mouse to examine RhoA activity in an in vivo setting in a variety of cell types in homeostasis as well as in mouse models of cancer. FLIM-FRET (fluorescence lifetime imaging microscopy) was performed in conjunction with a multiphoton set up in tissues and live mice using optical windows. RhoA activity was monitored live in mechanosensing of osteocytes in bones, during melanocyte migration in the embryonic skin, neutrophil migration, in crypt cells of the small intestine, in the mammary gland during gestation, as well as in breast cancer and progressive stages of pancreatic ductal adenocarcinoma. Elevated levels of RhoA activity were observed in the polyoma-middle-T-antigen (PyMT) driven breast cancer model as well as at the invasive borders and liver metastasis of the KPC (KRas^G12D/+ and p53^R172H/+) pancreatic cancer model. Finally, longitudinal imaging of the indirect inhibition of RhoA activity live in vivo was achieved by employing optical windows implanted on top of developed tumours.

In conclusion, the development and use of the RhoA-FRET biosensor mouse represents a strong resource in understanding tissue context specific signaling events during migration, mechanosensing and drug target validation in vivo.

Jan Pavlita, Christian Hübner

Institut für Physik, Universität zu Lübeck

Spectroscopic investigations rely on the optical performance of the employed dye. Thereby each technique utilizes some specific kinetic properties of the respective dye and works with considerably different irradiation powers (e. g. high-power and bleaching sensitive super resolution techniques like STED or low-power and brightness sensitive methods like smFRET). The determination of the photo physical properties is thus of great importance, and in particular single molecule methods like fluorescence correlation spectroscopy (FCS) offer excellent capabilities to do so. Therefore, we compare the kinetic parameters of different widely used dyes and discuss the observed intensity dependent behaviour under continuous-wave and pulsed excitation in terms of accordance to actual theoretical frameworks and simulated data.

Mariano Gonzalez Pisfil^1,2, Marcelle König¹, Benedikt Krämer¹, Paja Reisch¹, Felix Koberling¹, Matthias Patting¹, Andreas Herrmann², Rainer Erdmann¹

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

At present, fluorescence fluctuations of single molecules can be observed on time-scales ranging from sub-nanoseconds to seconds thanks to techniques such as Fluorescence Correlation Spectroscopy (FCS). In FCS, single molecule dynamics are studied inside a confocal volume. By measuring the fluorescence fluctuations caused by molecules entering or leaving the volume, parameters such as the diffusion coefficient and molecular concentration can be determined. Over the last decade, this method has allowed elucidating complex cellular processes.

Although being a very powerful tool, FCS has significant drawbacks for slower moving molecules like fluorophores diffusing in model or cell membranes. Photobleaching is one of the main issues which can be avoided by employing very low excitation powers. Furthermore, in order to average over a sufficient number of independent events the optimal measurement time for an FCS measurement is increased for slower moving species which in turn increases the chances of introducing artifacts (e.g., drift, or sample movement).

Scanning FCS (sFCS) was developed to counteract these issues. By using fast linear or circular scans the confocal volume is moved with respect to the sample. Thus the residence times of the fluorophores are reduced. In this scenario, photobleaching is decreased all while the statistical accuracy is increased. In sFCS an increased number of molecules can be observed in a given time period compared to conventional FCS due to the scanning behavior: diffusion species are observed on a line rather than just in one point. As an added advantage, the scanning process allows determining the volume without prior calibration.

As we use the confocal time-resolved fluorescence microscope MicroTime 200 STED equipped with a FLIMbee galvo scanner, we also have access to the fluorescence lifetime information. In our case, e.g. multi species STED imaging is performed with a single STED laser and labels featuring similar emission wavelengths. The labels can be discriminated by applying a unique pattern matching analysis method1. This approach can also be applied to discriminate two species in FCS and in STED FCS.

In contrast to STED FCS, STED scanning FCS will allow to achieve data from fluorophores diffusing in model and cell membranes with observation spots smaller that 50 nm overcoming not only averaging issues along long transit paths, but also reducing photobleaching, measurement times and improving statistics. By adjusting the STED laser power, the observation volume can be tuned in a gradual manner enabling, for example, the type of hindered diffusion to be determined in lipid membrane studies (STED spot variation FCS). And thanks to our pulsed illumination scheme via pulsed interleaved excitation (PIE) it is possible to check online in a straightforward way whether the STED laser has an influence on the investigated dynamics in STED-FCS.

Shun Qin, Sebastian Isbaner, Ingo Gregor, Jörg Enderlein

III. Physikalisches Institut, Georg-August-Universität Göttingen

Spinning Disk Confocal (SDC) microscope significantly speeds up imaging process, as it allows multi-laser beams scanning sample simultaneously by the introduction of a spinning disk unit. However, the optical resolution of SDC is just among the range of normal wide-field microscope. Image Scanning Microscopy (ISM) is a novel technique [1] to achieve super-resolution based on normal SDC [2]. Instead of scanning continuously, SDC-ISM images the sample from different rotation angle of spinning disk and makes sure that all responses of laser excitations are separated. Because all those response patterns are not overlapped, it is very easy to achieve super-resolution image with very simple reconstruction algorithm. It has been theoretically demonstrated that SDC-ISM can be able to achieve twice lateral resolution enhancement ideally compared to wide-field microscope [2]. The other important aspect of SDC-ISM is that it does not require very complicated extra components, but only a hardware control core and GUI application for data acquisition and analysis. Now the easy-to-use software developed in our group has been available for easily building your SDC-ISM system and calculating super-resolution image. A control core based on FPGA is designed for generating precise synchronization signal for SDC-ISM camera and laser triggering. All the GUI and acquisition program (a plugin) are based on the free software, Micro-Manager, a more and more popular open-source platform which supports many devices for optical imaging. Now our software has been able to supported 2D and 3D ISM raw image measurement and super-resolution image calculation within only one click. Software package for SDC-ISM will be released and it is for free to use for all potential users. All these features make it easy to build SDC-ISM system with relatively low cost. We believe that SDC-ISM will be a powerful technique for fast 3D super-resolution confocal microscopy with our solution.

Kim Colin Reiter, Prof. Dr. C. Hübner

Institute of Physics, University of Lübeck, Ratzeburger Allee 23562 Lübeck

Fluorescence resonance energy transfer (FRET) is one of the popular methods for the quantification of inter- and intramolecular distances on a nanometer scale. In general there are two principally different variants of measurements: Single molecule FRET (smFRET) and time resolved FRET (trFRET), respectively. We compared both measurement techniques in order to verify if the time resolved variant can be regarded as an alternative or complementation to the smFRET measurements. For the experiments, double stranded deoxyribonucleic acid (dsDNA) was used. Both methods show comparable results for donor-acceptor-distances within the Förster radius. Moreover, the time resolved method allows for detection of fast dynamics, which cannot be resolved by smFRET. However, for larger donor-acceptor-distances or multiple populations with identical donor-acceptor-pairs the transfer efficiency cannot be determined with sufficient accuracy. Therefore, the time resolved technique can not be regarded as an alternative to the single molecule measurement technique but as a promising supplement for resolving faster dynamics.

Sumeet Rohilla¹, Benedikt Krämer¹, Paja Reisch¹, Felix Koberling¹, Matthias Patting¹, Ingo Gregor², Rainer Erdmann¹, Andreas Hocke³

¹PicoQuant, Rudower Chaussee 29, Berlin, Germany
²Georg-August-University, Friedrich-Hund-Platz 1, Göttingen, Germany
³Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Germany

The separation of overlapping fluorescence emission of biological samples has been improved in the last years by using spectral confocal microscopy in combination with linear unmixing. However, the separation of multiple labels in biological samples remains still challenging, especially when strong tissue autofluorescence (AF) covers specific labeled structures. Therefore, the combination of spectrally resolved detection and fluorescence lifetime measurements allows detecting simultaneously spectral and lifetime parameters, which could significantly improve the separation quality.

To demonstrate this, we use highly autofluorescent human lung tissue, where the fluorescence signals of the specific staining was even weaker than tissue AF itself. In order to obtain an improved discrimination between multiple labels and tissue AF, a new hard- and software setting for spectrally resolved imaging and fluorescence lifetime measurement was established. We use a spectral FLIM (sFLIM) detection system featuring eight separate TCSPC timing channels and acquire sFLIM data for three different cell-type specific labels including AF with 485 nm and 561 nm pulsed interleaved excitation (PIE). The recorded data is analyzed by applying a unique pattern matching technique1, which results in an improved discrimination for multiple labels in human lung tissue.

Leonardo Berlim Schneider^1,3, Arandi Ginane Bezerra Jr.², Wido Herwig Schreiner³, Wallance Moreira Pazin¹, Thiago Siqueira Ramin², Amando Siuiti Ito¹

¹Universidade de São Paulo
²Universidade Tecnológica Federal do Paraná
³Universidade Federal do Paraná

The handicraft using the Syngonanthus nitens stem, the golden grass, aims to mimic artefacts made of gold. These unique works are famous abroad and they represent an important source of income for the artisans of Jalapão, a micro-region of the Brazilian state of Tocantins. The main scientific interest of the golden grass found in the literature is around its preservation and about the sustainability of the families that explore this culture. Our group has recently described the mechanism responsible for their golden glow, showing that the flavonoids residing in the epidermis of the stem of this plant absorb light in the violet and blue UV band, reflecting the red-shifted part of the incident light from the solar spectrum. This red-shifted reflection and the smooth surface stem profile are responsible for the golden colour and brightness. Also, it was shown that the golden grass stem flavonoids are fluorescents. Flavonoids are composed of the family of polyphenols with a small number of atoms, of which fifteen are carbons. Essentially, the structure is composed of two aromatic rings, joined by a carbon bridge in the form of a heterocyclic ring. The different radicals, as well as their relative positions to the molecule, determine their biological action. In plants, flavonoids are secondary metabolites and receive attention because of their health benefits. In this way, the study of the optical properties of these compounds, until then not investigated, became the main objective of our group, which will be presented in this workshop.

Dharmendar Kumar Sharma¹, Shuzo Hirata¹, Lukasz Bujak¹, Vasudevanpillai Biju², Tatsuya Kameyama³, Marino Kishi³, Tsukasa Torimoto³, Martin Vacha¹

¹Department of Materials Science and Engineering, Tokyo Institute of Technology, Ookayama 2-12-1-S8-44, Meguro-ku, Tokyo 152-8552, Japan
²Research Institute for Electronic Science, Hokkaido University, N20W10, Kita Ward, Sapporo 001-0020, Japan
³Department of Crystalline Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan

I-III-VI semiconductor nanocrystals such as AgInS₂, CuInS₂ and their derivatives are investigated as potential non-toxic alternatives of the II-VI quantum dots [1, 2]. Though, photoluminescence quantum yield (PL QY) of (AgIn)_xZn_2(1-x)S₂ (ZAIS) solid solutions could reach upto ~80%, spectral width of ∼400 meV originating from defect related Donor-Acceptor pair recombinations, limits the application of these NCs in the fields where color purity is desirable. To understand the factors controlling homogeneous and inhomogeneous line broadening of ZAIS, we performed PL characterization from ensemble to single particle (SP) level under ambient and varied conditions. Ensemble measurements show that alloying with Zn generates new radiative centers and improves PL QY; however beyond a particular limit it places the radiative recombination centers close to each other, leading to undesired interactions among charge carriers and to non-radiative transitions [3]. Further SP spectroscopy shows that size distribution and the spectral diffusion are the key factors of PL line broadening of ZAIS NCs. Two color excitation above (3.30 eV) and below (2.33 eV) ZAIS bandgap pointed to the coexistence of two energy levels within a single particle activation and deactivation of one such pair is proposed as the main contributor to the spectral diffusion [4].

Krzysztof Sozański¹, Evangelos Sisamakis², Xuzhu Zhang¹, Robert Hołyst¹

¹Institute of Physical Chemistry PAS, Kasprzaka 44/52, 01-224 Warsaw, Poland
²PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany

Combination of stimulated emission depletion (STED) and fluorescence correlation spectroscopy (FCS) allows for diffusion studies at length-scales of tens of nm and time-scales down to μs. This is invaluable for biological and anomalous diffusion studies. Also, STED offers FCS measurements at probe concentrations higher by orders of magnitude than in the diffraction-limited case and access to higher reaction rate constants in experiments concerning reaction kinetics.

Until now, STED-FCS has been successfully applied to diffusion studies in 2D systems, such as membranes. However, severe deficiencies, including overestimation of the detected number of probes as well as underestimation of their diffusion coefficients (both parameters differing from expected by up to an order of magnitude) impeded STED-FCS studies in solutions. Here, we introduce a realistic 3D model of the detection volume for STED-FCS and use it to resolve the apparent inconsistencies. Our calculations and simulations are validated experimentally – we use the model to recover the concentrations and diffusion rates of a range of probes freely diffusing in solutions. We define the limitations of the method and provide simple guidelines for data analysis. This paves a way towards facile application of the increasingly popular, turnkey STED systems to diffusion studies in solutions.

Jan Sýkora², Jana Brejchová¹, Pavel Ostašov¹, 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 the 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.

Matias Emil Moses ^1,*, Sara Thodberg², Johannes Thomsen^1,*, Birger L Moller², Tomas Laursen³, Nikos S Hatzakis¹

¹ Department of Chemistry & Nanoscience center, University of Copenhagen, Denmark
² Plant Biochemistry Laboratory, Department of Plant and Environmental Science, University of Copenhagen, Denmark
³ Feedstocks Division, Joint BioEnergy Institute, Emeryville, CA 94608, USA
^*Presenting author: Johannes Thomsen (johannes.thomsen@chem.ku.dk)

Intrinsic protein behaviour, both folding and function, is governed by dynamic exploration of the conformational space (conformational sampling). However, mechanistic insights into how conformational sampling is linked to enzyme activity and plasticity - an enzymes ability to activate structural diverse substrates - cannot be gained by conventional ensemble studies, but only becomes perceivable by single molecule characterisation(1). Cytochrome P450 Oxidoreductase (POR) governs a wealth of essential metabolic processes by relaying electrons from NADPH to a spectrum of cytochrome P450 enzymes, exhibiting a remarkably high degree of plasticity.

We have established a high throughput single molecule assay for characterisation of POR functional dynamics (static and dynamic disorder), which allows us to quantify the extend of conformational sampling and corelate this to the extend of plasticity. We use total internal reflection fluorescence (TIRF) microscopy to detect enzymatic turnover of pre fluorescent substrate (resazurin) by hundreds of individual pathogenic human POR mutants reconstituted in native like membrane systems (nanodiscs)(2-4). Our data suggest that pathogenic point mutations in POR affect its conformational sampling which consequently alter its selective activation of downstream partners, the effect of which propagte all the way to clinical phenotype. Our results thus provide the first clues that conformational sampling may govern the selective activation of structural diverse substrates and may pave the way for controlling plasticity.

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

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

Common super-resolution techniques suffer from low axial resolution in 3D. We present a new technique, single-molecule Metal-Induced Energy Transfer (smMIET), for measuring distance of a fluorescent molecule from a metal surface. The core principle behind this method is the energy transfer from the excited molecule to surface plasmons of a metal film, which enhances the decay rate of the excited state [1]. As a proof of principle, we measured the excited state lifetimes of individual fluorophores attached to rigid three-dimensional DNA origami structures [2] at designed heights and then converted these lifetime values into measured heights by using our theoretical model [3]. We show several nanometers accuracy in colocalization of two dyes located on surface-immobilized DNA origami structures, in axial direction. Combining with existing superresolution methods, one can potentially achieve isotropic nanometer localization precision [4]. This will facilitate intra- and intermolecular distance measurements in biomolecules and their complexes. Moreover, we show the potential use of smMIET in biomolecular dynamics investigation. DNA hairpin, well-characterized with smFRET [5], was used as a model molecule to develop the dynamic smMIET experiment and data analysis. We show consistent measurements of DNA hairpin dynamics in full agreement with smFRET data.

Roman Tuma^1,2, Tomas Fessl^1,2, Peter Oatley¹, William J. Allen³, Robin A. Corey³, Richard B. Sessions³, Steve A. Baldwin¹, Sheena E. Radford¹, Ian Collinson³

¹Astbury centre for Structural Molecular Biology, University of Leeds, LS2 9JT UK
²Faculty of Science, University of Southern Bohemia, Ceske Budejovice, Czech Republic
³School of Biochemistry, University of Bristol, BS8 1TD UK

​Most membrane proteins in bacteria are transported across and inserted by the Sec machinery. In prokaryotes, many proteins are transported post-translationally via translocation through the SecYEG at the expense of ATP hydrolysis by SecA. Using combination of biochemical, molecular dynamics and single molecule FRET techniques we have shed light on the coupling between ATP hydrolysis and directional movement [1]. In the model, ATP binding by SecA causes opening of the SecY-channel, by allosteric effect at site 5 nm away while substrates at the channel feedback to the active site and regulate nucleotide exchange. Such two-way communication results in a 'Brownian ratchet' mechanism in which ATP binding and hydrolysis bias the direction of substrate diffusion and drive it across the membrane.

Using recurrence analysis of single molecules and confocal FRET setup we improved time resolution to millisecond time scale and investigated early steps in the transport initiation. In addition, total internal reflection microscopy and FRET was used to observe duration of individual translocation events which in turn yielded an estimate of the translocation rate. The initiation is rate limiting, is driven by ATP hydrolysis and depends on activation by signal peptide and is followed by processive translocation. 

Daniel Zalami¹, Oliver Grimm², Felix H. Schacher², Jürgen Köhler¹, Uwe Gerken¹

¹Experimental Physics IV and Bayreuth Institute of Macromolecular Research (BIMF), University of Bayreuth
²Institute for Organic Chemistry and Macromolecular Chemistry (IOMC) and Jena Centre of Soft Matter (JCSM), Friedrich-Schiller University Jena

We apply single-particle orbit tracking (SPOT) [1] for investigating the three-dimensional void size distribution of nanoporous polystyrene-block-polyisoprene-block-poly(N-isopropyl acrylamide) (PS₄₃-b-PI₄₀-b-PNIPAAm₁₇) triblock terpolymer membranes under liquid filled conditions. In order to do so, we monitor the diffusion trajectory of single dye-labeled particles with a spatial precision of about 10 nm [2], thereby mapping out the void structure of the membrane. Our results are in agreement with scanning electron microscopy (SEM) data obtained from the same type of membrane.

Donata Janickaite¹, Mathew Stracy², Achillefs Kapanidis², David Sherratt², Pawel Zawadzki¹

¹Division of Molecular Biophysics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland
²University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Using single-molecule fluorescence imaging, we provide a comprehensive characterisation of the lesion search, recognition, and verification process in living cells. We reveal that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB. Then UvrA recruits UvrB from solution to the lesion. UvrA monomer possess two independent ATP binding sites (denoted as ‘proximal’ and ‘distal’), therefore functional UvrA dimers have four ATP binding sites. The role of the individual binding sites in the repair process is still not clear. We demonstrate that coordinated and subsequent ATP binding and hydrolysis in both sites is required for efficient repair process. We show that initial UvrB-independent damage recognition by UvrA requires ATPase activity in the distal site only. Subsequent UvrB recruitment requires ATP hydrolysis in the proximal site. Finally, UvrA is dissociated from the lesion complex, allowing UvrB to orchestrate the downstream NER reactions.