26^th International Workshop on
“Single Molecule Spectroscopy and Super-resolution Microscopy”

Philip Tinnefeld

Department of Chemistry, LMU Munich , Butenandtstr. 5 - 13, 81377 Munich, phtipc@cup.uni-muenchen.de

Graphene can act as a broadband energy transfer acceptor in single-molecule biophysics experiments and can provide exquisite z-resolution in superresolution microscopy. In this presentation, we present our efforts in preparing graphene-on-glass-coverslips and characterizing them. We then discuss how graphene quenching reports on the distance of dyes to graphene by fluorescence intensity of better, by fluorescence lifetime measurements. Using DNA origami nano-positioners, we place sensing units at the most sensitive height and report on new biosensing formats for nucleic acid. Finally, we combine graphene energy transfer with DNA PAINT and p-MINFLUX for superresolution and tracking with nanometer resolution and time resolution in the millisecond range.

Oleksii Nevskyi¹, Jan Christoph Thiele¹, Dominic A. Helmerich², Nazar Oleksiievets¹, Roman Tsukanov¹, Eugenia Butkevich¹, Markus Sauer², Jörg Enderlein¹

¹III. Institute of Physics – Biophysics, Georg‐August‐University Göttingen, Friedrich‐Hund‐Platz 1, 37077 Göttingen, Germany
²2Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by a conventional microscopy. Also nowadays, single molecule localization microscopy (SMLM) techniques have become one of the most successful and widely applied methods of super-resolution fluorescence microscopy. At the moment, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy.[1] In contrast, all SMLM techniques which utilize wide-field cameras completely lack the lifetime dimension. Here we demonstrate a combination of fluorescence-lifetime confocal laser-scanning microscopy (CLSM) with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging, which can be used for environment sensing applications and for multiplexing on samples with different labels that differ only by fluorescence lifetime but not by their spectral properties.[2] The technique is straightforward to be implemented on a commercial confocal scanning microscope setup with TCSPC capability and fast laser scanning unit. The method combines all the advantages of CLSM with those of SMLM: axial sectioning, shot-noise limited single-photon detection, pixel-free continuous position data, and fluorescence lifetime information acquired by CLSM with the exceptional spatial resolution and single-molecule identification of SMLM. Moreover, method is conceptually free of chromatic aberrations.

N.T.M. van der Voort¹, J. Budde¹, P. Lauterjung^1,2, C. Herrmann², A. Barth¹, C.A.M. Seidel¹

¹Institut für Physikalische Chemie MPC, HHU, Düsseldorf, Germany
²Physical Chemistry I, Ruhr-University Bochum, Germany

Introduction Biomolecules organize in 3D assemblies to drive the fundamental processes of life. To access this scale, we present a correlative FRET-nanoscopy approach where we make the transition from 2D to 3D at a molecular scale. Using localization on two-channel STED images (colocalization, cSTED), we resolve xy-projections with high precision (<4 nm, seamless resolution), which is increased to 4 Å precision using particle alignment and averaging. By applying appropriate corrections and using sub-ensemble averaging, we obtain accurate FRET-based distances¹ under STED conditions with high precision (<5 Å), independent of the molecular orientation. Using Pythagoras’ theorem, we make the transition into 3D molecular resolution on short (<12 nm) scales. Results We apply FRET-nanoscopy on a rectangular origami platform labelled with two FRET pairs separated by 75 nm, well below the diffraction limit. We show that FRET nanoscopy enables a quantitative FRET analysis of the two FRET pairs. Application We provide direct evidence for the extended state of the protein hGBP1 in vitro, which has been suggested to undergo a conformational change and self-assemble into a complex polymer structure upon activation². Inaccessible to either STED or FRET alone, we measure a donor acceptor distance of 28nm.

Jan Christoph Thiele¹, Dominic A. Helmerich², Marvin Jungblut², Roman Tsukanov¹, Markus Sauer², Oleksii Nevskyi¹, Jörg Enderlein¹

¹Third Institute of Physics – Biophysics, Georg August University Göttingen, Germany
²Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Germany

Over the last decade, single molecule localization microscopy (SMLM) has become well established for super-resolved imaging. However, until recently, SMLM has not been able to capture fluorescence lifetime information. With advances in instrumentation, including fast confocal laser scanning microscopy and single-photon sensitive cameras, first Fluorescence-Lifetime SMLM (FL-SMLM) implementations have been demonstrated. As it is independent of the intensity, the lifetime is a versatile, additional information which can be exploited for local-environment sensing, quantitative FRET or for multiplexed imaging.

Here, we present confocal lifetime-resolved STORM in combination with metal induced energy transfer (MIET). This energy transfer to a thin metal film on the substrate causes a distance-depended lifetime quenching. MIET enables a precise lifetime-based axial localisation which augments the lateral super-resolution of STORM to 3D. By utilising spectral splitting, we demonstrate dual-colour 3D STORM on fixed cells and simultaneously resolve microtubules and clathrin-coated pits. Unlike common PSF-engineering approaches, our method provides close to isotropic 3D super-resolution and does not require calibration or channel-registration.

Jonas Zähringer¹, Fiona Cole¹, Florian Steiner¹, Luciano A. Masullo², Fernando Stefani², Philip Tinnefeld¹

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

Super-resolution microscopy techniques have revolutionized the use of optical microscopes to study biological systems [1], resolving positions and dynamics well below the diffraction limit. The latest development, MINFLUX, [2] takes the resolution limit to the size of a label, but requires very sophisticated experimental setups.

We recently extended MINFLUX with pulsed interleaved excitation (p-MINFLUX) [3], thus simplifying the experimental setup and additionally acquiring the fluorescence lifetime information. This enables nanometer-resolution fluorescence lifetime imaging (FLIM) which can be used for a multitude of applications.

In this talk, we show applications of super-resolved colocalization of dyes using the fluorescence lifetime to distinguish individual molecules. At very short distances, i.e. within FRET range, the nanometer precision of p-MINFLUX coupled with fluorescence lifetime can be applied to detect energy transfer pathways. Using nanometer precise tracking, we visualize the energy transfer in donor-acceptor dye pairs (smFRET). Moreover, energy transfer to an energy accepting surface, e.g. graphene, enables super-resolution in three dimensions [4]. An isotropic optical resolution of ~ 1-2 nm is demonstrated on DNA origami reference systems.

Nazar Oleksiievets¹, Yelena Sargsyan², Jan Christoph Thiele¹, Nikolaos Mougios^3,4, Shama Sograte-Idrissi^3,4, Oleksii Nevskyi¹, Ingo Gregor¹, Felipe Opazo^3,4,5, Sven Thoms^2,6, Jörg Enderlein^1,7, Roman Tsukanov¹

¹III. Institute of Physics -- Biophysics, Georg August University, 37077 Göttingen, Germany.
²Department of Child and Adolescent Health, University Medical Center Göttingen, 37073 Göttingen, Germany.
³Institute of Neuro-and Sensory Physiology, University Medical Center Göttingen, 37073 Göttingen, Germany.
⁴Center for Biostructural Imaging of Neurodegeneration (BIN), University of Göttingen Medical Center, 37075 Göttingen, Germany.
⁵NanoTag Biotechnologies GmbH, Rudolf-Wissel-Straße 28a, 37079, Göttingen, Germany.
⁶Biochemistry and Molecular Medicine, Medical School, Bielefeld University, Bielefeld, Germany.
⁷Cluster of Excellence: Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cell (MBExC), Georg August University, Göttingen, Germany.

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging applications [1]. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT (together FL-PAINT) and use the lifetime information as a multiplexing parameter for targets identification. We employ short DNA strands (imagers) that carry fluorophores with similar emission spectrum but different fluorescence lifetimes to label different targets. In contrast to Exchange-PAINT [2], fluorescence lifetime PAINT (FL-PAINT) images multiple targets simultaneously, therefore shortening the total acquisition time, and requires no fluid exchange, thus both leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM (equipped with commercially available lifetime camera) [3] and time-resolved Confocal Laser Scanning Microscopy (CLSM), and we implement 3D multiplexed imaging of cells using CLSM. FL-PAINT can be readily combined with other DNA-PAINT based techniques of multiplexed imaging, and therefore FL-PAINT has a great potential for highly multiplexed bio-imaging.

Eitan Lerner

Dept. of Biological Chemistry, the Alexander Silberman Institute for Life Sciences, the Hebrew University of Jerusalem, Israel, Eitan.Lerner@mail.huji.ac.il

Intrinsically disordered proteins (IDPs) are recognized today as ubiquitous protein components of the cell, extending the structure-function relationship towards multiple structures promoting multiple functions, with their ability to bind multiple biomolecular partners and sometimes even stabilize different partially- or fully-folded structures. One such famous IDP is α-Synuclein (α-Syn) that can fold in helical structures when interacting with membranes to support dopamine-containing vesicles, but can change conformation towards beta strand-based structures that promote its self-association towards forming oligomers and amyloid fibrils somehow associated with the intracellular pathophysiology of Parkinson's disease (PD).

Previous single-molecule FRET (smFRET) studies reported that the α-Syn monomer in solution undergoes overall conformational dynamics within few microseconds or even in hundreds of nanoseconds, rendering its structures too transient to be considered as ones that promote its future binding to its binding partners. We tested these findings using a fluorescence-based proximity sensor sensitive to proximities shorter than smFRET does, namely single-molecule protein-induced fluorescence enhancement (smPIFE)^1-3. Doing so, for freely-diffusing single α-Syn monomers in the confocal modality we show distinct fluorescence lifetime sub-populations, reporting on different α-Syn species where the site-specifically attached dye experiences different local environments⁴. These results suggest that while the overall structure of the α-Syn monomer in solution is very flexible and too unstable, it still contains regions that gain local structures that stay stable for milliseconds. FRET-restrained discrete MD simulations verified against data from several experimental results further provide an insight onto the different structural sub-populations of the α-Syn monomer in solution, where some of the resolved structural sub-populations exhibit high homology with high resolution structures of α-Syn when bound to the outer leaflet of a membrane or when it is a subunit of the α-Syn amyloid fibril4.

Using smPIFE, we also show how α-Syn binds membranes in a manner different than what has previously been reported, and by that explain how it is shielding its aggregation-prone segment, when bound to the membrane (unpublished). Measurements in the presence of physiologically-normal and -toxic levels of manganese provide potential explanations for the well-documented acceleration in PD-related events following high manganese exposures, regarding α-Syn as one of the PD-related targets (unpublished).

This work exhibits the power of using smPIFE to probe local structures and their dynamics in IDPs, as a method complementary to smFRET^5,6 and of combining single-molecule spectroscopy experiments with other experiments and modelling in an integrative approach to recover the structural features of proteins otherwise referred to as unstructured or disordered⁴.

This talk will present certain procedures utilized by our lab to promote open science in our single-molecule spectroscopy studies and in the community⁵.

Monique Honsa¹, Tim Schröder¹, Philipp Nickels², Tim Liedl², Philip Tinnefeld¹

¹Department Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13 Haus E, 81377 München, Germany
²Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, 80539 München, Germany

Forces in biology at single-molecule level are usually studied by atomic force microscopy as well as optical and magnetic tweezers. Not only do these methods lack high throughout, but they are also limited by their physical connection to the macroscopic world.

Nickels et al. [1] constructed a nanoscopic force clamp made of DNA, which can exert piconewton forces onto biomolecules in a highly parallelized fashion. So far, only constant forces could be generated within this force clamp.

We develop the idea of the molecular force clamp further by introducing a toehold exchange mechanism to alter the applied force reversibly during an experiment. This approach uses single-molecule FRET as readout mechanism and will path the way for a more versatile tool to study the impact of forces on biomolecules.

Julian Folz, Suren Felekyan, Ralf Kühnemuth, Claus Seidel

Heinrich-Heine-University, Düsseldorf

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

This phenomenon we applied to a wide range of biomolecules with a varying size from 40 kDa to 1.4 MDa showing different dynamics and kinetics on timescales from µs to hours. To study this we combine established methods like Multiparameter Fluorescence Detection (MFD) of freely diffusing molecules and Total Internal Reflection (TIRF) microscopy using immobilized molecules, together with new approaches pushing the collection efficiency to detection signals to hundreds of kHz. The outcome are observables describing (a) different structural states and dynamics of the human Guanalyte Binding Protein (hGBP1) and its behavior in the farnesylated state, (b) the live exchange of different molecular states of a freely diffusing 4 way junction on a µs timescale in dependence of the salt concentration and (c) the structural behavior of the TcToxin on a ms-timescale giving insights into fast/straight forward and complex/distributed transitions crucial for its biological functionality.

Fabio D. Steffen², Felix Erichson¹, Roland K.O. Sigel², Richard Börner¹

¹Laserinstitut Hochschule Mittweida, University of Applied Sciences Mittweida, Mittweida, Germany
²Department of Chemistry, University of Zurich, Zurich, Switzerland

As RNA molecules are often too large for NMR and too flexible for x-ray crystallography, RNA structure prediction with the help of computational approaches helps to improve our understanding of RNA structure and function. To develop these RNA structure models, Förster resonance energy transfer (FRET) as a molecular distance measure comes into play. Although only single distance coordinates are recorded at the time, these have a time resolution in the range of ns and a spatial resolution in the Å range [1]. Our goal is to integrate this distance information into hybrid structural models as a distance constrain to predict unknown RNA structures. At the same time, we follow the reverse approach and monitor the evolution of existing RNA structures in time via MD simulations and predict FRET of dynamic structure models. The latter is essential to develop dynamic RNA models and allows to compare and validate MD with different levels of coarse-graining simulations with experimental FRET distributions. We have developed a complete hybrid model that covers both, the experimental and the computational part. Our python-based tool FRETraj allows to label RNA and DNA structures in PyMOL with the fluorescent dyes and to predict FRET. We use an accessible-contact volume (ACV) model to describe how the fluorophores move with respect to the RNA and therefore influence the FRET value [2]. This dye model also takes experimentally observed interactions of the fluorophore with the RNA surface into account. This allows the prediction of very accurate FRET values. Our method is scalable. We use it to calculate multiple ACVs along a simulated MD trajectory, i.e., a structure ensemble with multi-ACV, and thus predict the FRET distribution of dynamic structures taking experimental parameters such as the photon noise into account. Conversely, we are able to integrate FRET into de novo models as a distance constrain to reduce the ensemble of structures predicted [3]. We calibrated our method using carbocyanine labelled DNA double strands and tested it on different RNA structural motifs, such as the classical tetraloop receptor [4].

Ivan Maslov^1,2,3, Oleksandr Volkov^4,5, Polina Khorn¹, Philipp Orekhov¹, Anastasiia Gusach¹, Pavel Kuzmichev¹, Andrey Gerasimov^1,6, Aleksandra Luginina¹, Quinten Coucke³, Andrey Bogorodskiy¹, Valentin Gordeliy^1,4,5, Simon Wanninger⁷, Anders Barth⁷, Alexey Mishin¹, Johan Hofkens^3,8, Vadim Cherezov^1,9, Thomas Gensch¹⁰, Jelle Hendrix^2,3, Valentin Borshchevskiy^1,4,5

¹Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia
²Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre, Biomedical Research Institute, Agoralaan C (BIOMED), Hasselt University, Diepenbeek, Belgium
³Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Leuven, Belgium
⁴Institute of Biological Information Processing (IBI-7: Structural Biochemistry), Forschungszentrum Jülich, Jülich, Germany
⁵JuStruct: Jülich Center for Structural Biology, Forschungszentrum Jülich, Jülich, Germany
⁶Vyatka State University, Kirov, Russia
⁷Physical Chemistry, Department of Chemistry, Center for Nano Science (CENS), Center for Integrated Protein Science (CIPSM) and Nanosystems Initiative München (NIM), Ludwig-Maximilians-Universität Munich, Munich, Germany
⁸Max Plank Institute for Polymer Research, Mainz, Germany
⁹Bridge Institute, Departments of Chemistry and Biological Sciences, University of Southern California, Los Angeles, CA, USA
¹⁰Institute of Biological Information Processing (IBI-1: Molecular and Cellular Physiology), Forschungszentrum Jülich, Jülich, Germany

G-protein coupled receptors (GPCRs) orchestrate critical processes in human body and are targets for 30% out of all FDA-approved drugs. The multi-state conformational behavior of GPCRs delineates their complex pharmacology and, therefore, challenges modern drug design. The adenosine receptor A2A is a G-protein coupled receptor (GPCR) that regulates the cardiovascular tonus and promotes healing of inflammation-induced injuries. A2A is a promising target for drugs against insomnia, chronic pain, depression, Parkinson’s disease, and cancer. Here we applied single-molecule FRET (smFRET) to investigate the conformational dynamics of the A2A in freely diffusing lipid nanodiscs without immobilization.

We combined fluorescence intensity, lifetime, and anisotropy information to measure smFRET between two dyes attached to genetically introduced cysteines in the A2A. We observed that FRET efficiency in the double-labeled A2A increases upon agonist binding. Sub-millisecond dynamics were revealed by several complimentary burst-wise fluorescence analysis approaches: E vs TauD plot, FRET-2CDE, BVA, and fFCS.

We propose a dynamic model of A2A activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2A, explaining the receptor’s constitutive activity. For the agonist-bound A2A, we detected faster (390±80 µs) ligand efficacy-dependent dynamics.

Nynke Dekker

Department of Bionanoscience, Kavli Institute of Nanoscience, TU Delft, , Van der Maasweg 9, 2629 HZ Delft, The Netherlands, N.H.Dekker@tudelft.nl

Many transactions on DNA are carried out by molecular machines that operate at the nanometer-scale. How they do so effectively is a question of long-standing interest. We are particularly interested in studying the dynamics of these molecular machines using single-molecule techniques. I will briefly highlight how the field of single-molecule biophysics has advanced such techniques over the past decades, allowing the dynamics of diverse motor proteins to be accurately followed.

I will next describe how single-molecule techniques are being used to tackle new challenges, including the probing of complex molecular machines built up from many different components. The replisome that copies DNA is such a complex machine. While the overall outline of replisome assembly is understood, little is known about the dynamics of the individual proteins on the DNA and how these contribute to the formation of proper replisomes. I will show that using integrated optical trapping and confocal microscopy, one can dissect how protein binding, diffusion, sequence recognition, and protein-protein interactions play important roles in the first steps of replisome assembly, and discuss the implications thereof.

Christian Franke^1,2, Prasath Paramasivam², Martin Stöter², Andreas Höijer³, Stefano Bartesaghi⁴, Alan Sabirsh³, Lennart Lindfors³, Marianna Yanez Arteta³, Anders Dahlén⁵, Annette Bak⁶, Shalini Andersson⁵, Yannis Kalaidzidis², Marc Bickle², Marino Zerial²

¹Institute of Applied Optics and Biophysics, Friedrich-Schiller-University, Jena, Max-Wien Platz 4, 07743 Jena, Germany
²Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307. Dresden, Germany.
³Advanced Drug Delivery, Pharmaceutical Science R&D, AstraZeneca, Gothenburg, Sweden.
⁴Bioscience Metabolism, Research and Early Development Cardiovascular, Renal and, Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
⁵Oligonucleotide Discovery, Discovery Sciences R&D, AstraZeneca, Gothenburg, Sweden.
⁶Advanced Drug Delivery, Pharmaceutical Science R&D, AstraZeneca, Boston, USA

Delivery of exogenous mRNA using lipid nanoparticles (LNP) is a promising strategy for therapeutics. However, a bottleneck remains the poor understanding of the parameters that correlate with endosomal escape vs. cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those employed in mRNA-based vaccines, in primary human adipocytes, fibroblasts and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery and different LNP vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By multi-colour SMLM we could resolve for the first time single LNP-mRNA within sub-endosomal compartments. Furthermore, we illuminate the nano-structure of arrested endosomes and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations towards higher efficacy and lower cytotoxicity [1].

Lennart Grabenhorst, Martina Pfeiffer, Philip Tinnefeld, Viktorija Glembockyte

Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 München

Many enzymes in nature are multivalent, meaning that they can bind more than one substrate unit simultaneously. Often, these binding sites are communicating to each other and occupation of one binding site modulates the binding properties of the other binding sites. Understanding how this communication works may enable the rational design of tunable biosensors¹ for customizable target concentration windows, and with that, better diagnostic tools. Because of its modular nature and excellent nanopositioning capabilities, DNA origami² is a very suitable backbone material for shedding light on such interactions.

In this contribution, we present our progress on employing a well-studied hinge-like dynamic DNA origami structure^3,4, for the systematic investigation of the influence of parameters such as the number of binding sites, the spacing between them and the relative stabilities of the „bound“ conformational state of the structure on the macroscopic ligand binding behavior of the biosensor. Using DNA-DNA interactions as a model system for ligand binding and single-molecule FRET as a readout, we show that the overall dose-response of a structure comprising several of these interactions can be affected without changing the single binding interaction itself.   

Gail McConnell

Department of Physics, University of Strathclyde, 107 Rottenrow, Glasgow G4 0NG, United Kingdom, g.mcconnell@strath.ac.uk

For more than a century, the design of microscope objectives has been guided by the angular acuity of the human eye. At 4x magnification, this requires a numerical aperture no greater than 0.1 or 0.2, which can be achieved cheaply and easily by simple optical designs. With the advent of confocal and multiphoton microscopy, however, it became apparent that the poor axial resolution of more than 30 microns with low magnification objectives was intolerable for these 3D methods.

To overcome this, we have developed a new and complex objective with a magnification of 4x and a numerical aperture of just less than 0.5 which we call the Mesolens [1]. We originally specified this lens for mammalian embryology, and have shown that it can image every cell of a 6mm-long embryo 3mm thick with sub-cellular resolution if the tissue is cleared appropriately. A by-product of the high numerical aperture is that the optical throughput is approximately 20x greater than a conventional 4x objective. The pupil size of the lens is so great that it cannot be used with a conventional microscope frame, so we have built the imaging system around the lens, and use either wide-field camera or laser point-scanning detection to create images.

I will present an overview of the Mesolens technology, some examples of new and emerging applications that use this new instrument, and I will explain how we would like to develop this technology further.

C. Eggeling^{1, 2, 3}, M. Booth ⁴

¹ Friedrich‐Schiller‐University Jena, Institute of Applied Optics and Biophysics, 07743 Jena, Germany
² Leibniz Institute of Photonic Technology e.V., Department Biophysical Imaging, 07745 Jena, Germany
³ University of Oxford, MRC Human Immunology Unit and Wolfson Imaging Center, University of Oxford, Oxford OX3 9DS, United Kingdom
⁴ Department of Engineering Science, University of Oxford, Oxford OX1 3PJ, United Kingdom
e-mail: christian.eggeling@uni-jena.de

Molecular interactions are key in cellular signaling. They are usually ruled by the organization and mobility of the involved molecules. We present a fluorescence microscopy study to determine such information and potentially extract interaction dynamics in the cellular cytosol. For example, we employ the combination of super-resolution STED microscopy with fluorescence correlation spectroscopy (STED-FCS). Here, cytosolic observations are hampered by optical aberrations, which can be corrected for using adaptive optics. We highlight how the latter can be implemented in STED-FCS, and how this can be used to reveal novel aspects of molecular interaction dynamics in the cellular cytosol, specifically on peroxisomal import proteins.

Maria Loidolt-Krüger, Mariano Gonzalez Pisfil, Sumeet Rohilla, Marcelle König, Benedikt Krämer, Matthias Patting, Felix Koberling, and Rainer Erdmann

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

Stimulated emission depletion (STED) in confocal fluorescence microscopy enables visualization of biological structures within cells far below the optical diffraction limit. To meet the demand in the field for simultaneous investigations of multiple species within a cell, a couple of different STED techniques have been proposed, each with their own challenges. By systemically exploiting spectral differences in the absorption of fluorescent labels, we present here a novel, beneficial approach to multispecies STED nanoscopy carried out with the time-resolved confocal fluorescence microscope MicroTime 200 [1].

In our approach, we use three excitation wavelengths in nanosecond Pulsed Interleaved Excitation (PIE) mode to probe quasi simultaneously multiple fluorescent labels that have absorption maxima as close as 13 nm [2]. The acquired image is then decomposed into its single species contributions by application of a linear unmixing algorithm based on reference patterns.

For multispecies images containing single species regions, we introduce the image correlation map (ICM). Here, the single species regions easily can be identified in order to generate the necessary single species reference patterns. This avoids the otherwise cumbersome and artifact prone preparation and recording of additional reference samples. The power of the proposed imaging scheme persists in species separation quality at high speed shown for up to three species with established reference samples and dyes commonly used for cellular STED imaging.

Michael Scheckenbach, Tom Schubert, Carsten Forthmann, Viktorija Glembockyte, Philip Tinnefeld

Department of Chemistry and Center for NanoScience, Ludwig−Maximilians−Universität München, Butenandtstraße 5−13, 81377 München, Germany

In the last years, DNA nanotechnology and DNA origami technique have enabled the synthesis of reference structures for single-molecule microscopy and for super-resolution imaging techniques like DNA PAINT.^{[1, 2]} Fundamentally, such molecular devices are susceptible to rapid degradation and loss of their functionality due to the high proportion of surface atoms and fast damage rates the nanoscale.^[3] Hence, autonomous or non-autonomous, i.e. externally triggered, self-repair mechanisms are desirable. In this contribution, we exploit the self-assembly nature and reconfigurability of DNA origami nanostructures to establish dynamic self-repair mechanisms.^[4] By dynamically exchanging building units with intact analogues from solution, we are able to maintain the brightness of reference structures even after complete damage and to stabilize super-resolution nanorulers used for DNA PAINT over days. We illustrate the two possible self-repair mechanisms using this approach: the damage unspecific self-regeneration, which exchanges intact and defective building units, and the damage specific self-healing, i.e. the exchange of only defective building units. Furthermore, we will also discuss our recent work on extending these self-regeneration strategies to improve the photostability of DNA-PAINT docking sites enabling superresolution imaging spanning over hours even under harsh imaging conditions.

Jennifer Lippincott-Schwartz

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, U.S., lippincottschwartzj@hhmi.org

Powerful new ways to image the internal structures and complex dynamics of cells are revolutionizing cell biology and bio-medical research. In this talk, I will focus on how emerging fluorescent technologies are increasing spatio-temporal resolution dramatically, permitting simultaneous multispectral imaging of multiple cellular components. In addition, results will be discussed from whole cell milling using Focused Ion Beam Electron Microscopy (FIB-SEM), which reconstructs the entire cell volume at 4 voxel resolution. Using these tools, it is now possible to begin constructing an “organelle interactome”, describing the interrelationships of different cellular organelles as they carry out critical functions. The same tools are also revealing new properties of organelles and their trafficking pathways, and how disruptions of their normal functions due to genetic mutations may contribute to important diseases.

Gregor J. Gentsch¹, Christian Franke^1,2

¹Institute of Applied Optics and Biophysics, Friedrich-Schiller University,Helmholtzweg 4, 07743 Jena, Germany
²Abbe Center of Photonics, Albert-Einstein-Straße 6, 07745 Jena, Germany

Multi-color Single-Molecule Localization Microscopy (SMLM) has emerged as a powerful tool for the quantitative study of cell-biological questions. However, the limited number of available spectroscopically distinct and high-performance SMLM dyes poses a key bottleneck for many applications, while the range of best performing dyes is further restricted to the far-red spectrum (e.g. Alexa647).

Here we propose a novel computational method enabling the multiplexing of monochromatic SMLM of several unique biological structures. Our approach is based on the analysis of the local nano-environment in super-resolved image data, i.e. the sub-pixel precise extraction of 26 nano-textural Haralick-features [1]. We show, that the combination of these nano-texture features, by employing a machine learning based grouping, results in an organelle-specific texture fingerprint. Finally, we show the application of these fingerprint enables the (multi-colour) distinction of several cellular structures, including microtubules, endosomes and the nuclear pore complex, labeled with spectrally identical markers.

We reckon, that our approach not only opens up new avenues for multi-colour SMLM imaging, but also has potential in the quantitative assessment of nanometric labeling efficiency and quality (e.g. antibody screening), as well as the detection of nanometric changes in organelle morphology and texture, e.g. in in-vivo biological studies.[2]

Anežka Vacková^1,2, Ruth Winter¹, Michael Hausmann¹

¹Kirchhoff-Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg
²Institute of Physics of Charles University, Ke Karlovu 5, 12000 Praha 2

Exposure of a cell to a radiation source causes different types of damage in its DNA, one of the most severe is DNA double-strand-break (DSB). Cell response to such induced damage has to be fast, efficient and specific for a given site, and deeper understanding of this repair mechanism could lead to more exact and individualized applications in cancer and radiotherapy research.

In this work we investigate by Single-Molecule-Localization-Microscopy^[1] cells after ionizing irradiation, we address the influence of gold nanoparticles (NPs) of different sizes incorporated into them. As was described by previous research^[2], radiation enhancing effects take place in the presence of gold NPs. Radiation releases electrons into a nanoscale volume and the generated radicals intensify the pure radiation effect.

We used γH2AX phosphorylation as a measure for the effect of radiation and radicals in DNA damage.  We correlated the appearance of γH2AX foci in presence of different gold NPs after 2 Gy X-Ray irradiation. Particles of the following sizes were applied: 1, 5, 10 and 25 nm, which were incorporated into human dermal fibroblasts.

Size dependent enhancing effect was manifested in 6 hours after irradiation; in contrast, at earlier repair times the enhancing effect of NPs did not appear.  

Anoushka Handa¹, Edita Bulovaite², Leila Muresan³, Katie Morris⁴, Gregory Chant¹, Kevin O' Holleran³, Mathew Horrocks⁴, Seth Grant², Steven Lee¹

¹Yusuf Hamied Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW
²Centre for Clinical Brain Sciences, The University of Edinburgh, Chancellor's Building, 49 Little France Crescent, Edinburgh, EH16 4SB
³Cambridge Advanced Imaging Centre, Anatomy School, Downing Street, Cambridge CB2 3DY
⁴School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh, EH9 3FJ

The spatial organisation of the neuronal synapse occurs both below the optical diffraction limit and in an intricate 3D pattern. Advanced 3D super-resolution imaging techniques are particularly well suited to study this. The 3D double helix-point spread function (DH-PSF) is one such technique which has an increased depth of field (~4 μm) and is capable of isotropic resolutions of ~25 nm. This makes it highly compatible to investigate sub-synaptic diversity in a physiologically relevant environment. Here we demonstrate postsynaptic density 95 (PSD95) genetically expressed to mEos2 imaged in brain tissue using the 3D DH-PSF. PSD95 is known to form nanoclusters which make up the basic structural unit of an excitatory synapse. We have been able to image the hippocampus and perform cluster analysis on our results to better understand the role of these nanoclusters and also have visualised diffuse PSD95 protein. We have developed quantitative imaging analysis algorithms to compare the role of inter and intra synaptic diversity in the hippocampus. A deeper understanding of how PSD95 nanoclusters form and how mutations occur in these synapses in the hippocampus contributes knowledge to understanding how this can lead to schizophrenia, learning disabilities and autism.

Yu P. Zhang¹, Derya Emin¹, Evgenia Lobanova¹, Antonina Kouli², Caroline Williams-Grey², David Klenerman¹

¹Yusuf Hamied Department of Chemistry, University of Cambridge
²John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge

Alpha-synuclein and Abeta aggregates have significant pathologic relevance to the development of Parkinson’s disease (PD), the second most common neurodegenerative disease after Alzheimer’s. The characterisation of these two proteins in human biofluids offers insights into their pathological roles in disease progression as well as diagnostic potentials. In this work, we combined the aptamer assisted Single molecule pulldown (SimPull) imaging and Stochastic Optical Reconstruction Microscopy (STORM) to characterise Alpha-synuclein and Abeta aggregates in human serums in detail.

Our results demonstrate that the size and abundancy levels of both proteins in serum samples from PD and healthy controls (HC) patients differ. PD serum was found to have a higher level of alpha-synuclein with larger length in comparison to the HC. Meanwhile for abeta, PD serum has less total protein without clear size differences compared to the HC group. This differences could be used as a biomarker to distinguish HC from diseased patients in the future.

Andres Manuel Vera¹, Albert Galera², Michał Wojciechowski³, Bartosz Różycki³, Douglas V Laurents⁴, Mariano Carrión-Vázquez⁵, Marek Cieplak³, Philip Tinnefeld¹

¹Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13 Haus E, 81377 München, Germany
²Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90014 Oulu, Finland
³Institute of Physics, Polish Academy of Sciences, Al. Lotników, 32/46, 02-668 Warsaw, Poland
⁴Instituto de Química Física """Rocasolano""", CSIC, C/ Serrano 119, 28006 Madrid, Spain
⁵Instituto Cajal, CSIC, Avda. Doctor Arce 37, 28002 Madrid, Spain

Cellulosomes are extraordinary complex protein nanomachines with unmatched efficiency in degrading lignocellulose, the most abundant and hydrolysis-resistant biomaterial on earth. Over the last years, single-molecule techniques have unveiled surprising hidden features of these complexes, including remarkable mechanical sturdiness of their building blocks^[1] (cohesins) and record-breaking stability of their pivotal assembling interaction (the cohesin-dockerin interaction)^[2]. In this contribution, we use smFRET and molecular dynamics simulations to uncover two alternative binding modes of the cohesin-dockerin interaction. Using our single-molecule approach, we showed that the isomerization state of a single proline residue determine the population ratio between these binding modes, its assembly kinetics and the overall stability of the cohesin-dockerin interaction. Furthermore, these processes could be enzymatically modulated by a prolyl-isomerase^[3]. We propose that these results can answer the long-standing open question of how cellulosomes can dynamically change their cellulase composition despite the extremely high stability of the cohesin-dockerin interaction.

Prithu Roy, Aleksandr Barulin, Jean-Benoît Claude, Jérôme Wenger

Aix Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, 13013 Marseille, France

Proteins are building blocks of life; they perform vital cellular functions ranging from DNA replication to environment sensing. This development in the application of proteins has led to the necessity to study proteins in their physiological environment[1]. Thus the label-free study of proteins at the single-molecule level in their natural conditions (pH and concentration) is needed. The label-free technique overcomes the issues of labeling techniques like alteration in conformation and dynamics of target molecules due to fluorophores. However, a label-free technique that uses auto-fluorescence of the protein, struggles with intrinsically low UV auto-fluorescence signals and has scope for development [2-3]. In our work, we show a nano-aperture+ horn antenna combination, a novel design to reduce the interaction volume (nano-aperture), and enhancement by collecting emission at a larger angle (horn antenna). Using this horn antenna and auto-fluorescence we studied the cooperativity of Streptavidin-biotin interaction resolved to the single-molecule level. We also demonstrated the first real-time detection of UV auto-fluorescence signals from fixed single molecules.

Kay-E. Gottschalk, Fabian Port, Carolin Grandy

Institut für Experimentelle Physik, Universität Ulm, Albert Einstein Allee 11, 89077 Ulm

Cells adapt their actin cytoskeletons architecture to structural and mechanical cues of the environment. Focal adhesions function as anchoring points of actin to the extracellular matrix [1]. How manipulating cell shape or exerting force on individual focal adhesions influences the actin cytoskeletons z dimension is unstudied. Metal induced energy transfer(MIET)  is ideally suited to resolve actin structures in nm resolution in the crucial dimension normal to the imaging plane [2,3]. Therefore, we combined MIET with both cellular micropatterning [4,5] and high-resolution AFM measurements to study the influence of shape or force on actin structure. We show that these combinations reveal with unprecedented resolution how focal adhesions adapt to external stimuli.

Petra Schwille

Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany, schwille@biochem.mpg.de

Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy for the biosciences, extending resolution down to the level of individual molecules. However, the dynamic information that these methods can yield on a biomolecular system has so far not been sufficiently exploited. Also, the quantitative interpretation of SMLM data is usually not straightforward and requires a thorough understanding of the underlying physical principles. In my talk, I will present our recent work on localization-based fluorescence correlation spectroscopy which allows to not only resolve the dynamics of surface-binding reactions in equilibrium, but also to determine absolute molecular copy numbers in DNA-PAINT images. I will further present a new solution to a long-lasting problem in single particle tracking (SPT) experiments: the limited observation times of fluorescently-labeled particles due to fast photobleaching of fluorophores within typically a few seconds. Our recent labeling approach exploiting DNA-mediated fluorophore exchange allows us to circumvent the limited photon budget of fluorophores and to perform SPT of target molecules for tens of minutes.

Viktorija Glembockyte¹, Kateryna Trofymchuk¹, Martina Pfeiffer¹, Lennart Grabenhorst¹, Florian Steiner¹, Cindy Close¹, Florian Selbach¹, Renukka Yaadav¹, Jonas Zähringer¹, Guillermo Acuna², Birka Lalkens³, Philip Tinnefeld¹

¹Department of Chemistry and Center for NanoScience, Ludwig Maximilian University of Munich, Germany
²Department of Physics, University of Fribourg, Switzerland
³Institute of Physical and Theoretical Chemistry, TU Braunschweig and Integrated Center of Systems Biology (BRICS), Germany

DNA nanotechnology have enabled facile design and synthesis of complex and functional nanostructures.¹ For example, the unprecedented addressability of DNA origami can be used to arrange plasmonic nanostructures and emissive molecules to create antennas for light on the nanoscale capable of enhancing fluorescence signals up to several hundred fold. In this contribution, I will share our latest progress on the development of dimer DNA nanoantennas with plasmonic hotspots cleared for the placement of biomolecular assays.² We incorporate a single molecule diagnostic assay specific to DNA of antibiotic resistant bacteria in the hotspot of a dimer nanoantenna composed of two 100-nm silver nanoparticles and demonstrate fluorescence enhancements reaching few hundred fold both in buffer as well as heat-deactivated human serum. The high signal amplification provided by the DNA nanoantennas enabled us to detect single DNA molecules on a portable, battery-driven and low cost smartphone device. I will also discuss our efforts to expand the scope of DNA nanoantennas beyond the detection of nucleic acids. By incorporating a nanoswitch containing a fluorophore and quencher pair as well as antibody recognition elements directly into DNA origami nanostructures we were also able to detect single antibodies on a smartphone camera.

Jonas Schütte^1,2,3, Martin Wolff^1,2,3, Linda Groeneweg⁴, Fabian Beutel^1,2,3, Helge Gehring^1,2,3, Matthias Häußler^1,2,3, Carsten Schuck^1,2,3, Noelia Alonso Gonzalez⁴, Wolfram Pernice^1,2,3

¹Institute of Physics, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany
²CeNTech – Center for Nanotechnology, Heisenbergstraße 11, 48149 Münster, Germany
³Center for Soft Nanoscience, Busso-Peus-Straße 10, 48149 Münster, Germany
⁴Institute of Immunology, University of Münster, Röntgenstraße 21, 48149 Münster, Germany

Microscopy techniques as fluorescent lifetime imaging (FLIM), stimulated emission depletion (STED) and Two-photon excitation each work at the limit of current detector technologies regarding timing resolution, efficiency as well as wavelength sensitivity.

Superconducting Single-Photon Detectors (SNSPD) have shown to excel in every key parameter for those microscopy techniques with high detection efficiency, GHz detection rates, timing accuracies down to few ps, and a spectral sensitivity window from UV to MIR with sub-Hz dark count rates, only limited by the optical interface [1].

By introducing wideband and efficient 3D printed optical interfaces to the input port of SNSPDs [2], we achieve high overall system detection efficiencies over 1000 nm of wavelength from 532-1650 nm. We observe a close relation of the transmission of our optical inputs and the resulting detection efficiency, revealing that our SNSPDs possess high internal quantum efficiencies.

In an interdisciplinary pilot project this coupling approach was used to image immune cells using a confocal laser scanning microscope in the wavelength regime of 650-800 nm with a better signal-to-noise ratio than state-of-the-art single photon avalanche diodes optimized for this wavelength range. This serves as a proof-of principle experiment for an advantage for new emerging microscopy schemes using broadband SNSPDs.

Tim Schröder, Johann Bohlen, Sarah Ochmann, Patrick Schüler, Stefan Krause, Don C. Lamb, Philip Tinnefeld

Department Chemie and Center for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 München, Germany

Fluorescence correlation spectroscopy (FCS) is a versatile tool to study fast conformational changes of proteins in single molecule Förster Resonance Energy Transfer (smFRET) experiments. The equilibrium constant is one of most important observables to describe the kinetics quantitatively. However, the correlation amplitude does not provide an unambiguous solution of the equilibrium constant if two unknown intensity levels are involved. As an easy and versatile approach, we introduce microtime gated intensity correlation (MiGIC) of the donor signal to extract the equilibrium constant of two intensity level trajectories. Therefore, we exploit the change in intensity contrast between the two intensity levels at different microtime gates. We demonstrate with simulations and a DNA origami-based model system the applicability of our approach in surface and solution experiments. We further demonstrate that MiGIC can distinguish between photo physics, e.g. triplet or radical blinking, and dynamic intensity changes due to a dark quencher like graphene.[1] Finally, we unravel the mechanism of a FRET-based membrane charge sensor with MiGIC. Compared to Fluorescence Lifetime Correlation Spectroscopy (FLCS) no prior knowledge of fluorescence lifetimes nor fluorescence intensity levels are required, making MiGIC an easy and applicable tool to study conformational dynamics in smFRET experiments.

Eli Slenders¹, Eleonora Perego¹, Alessandro Rossetta^1,2,3, Mattia Donato¹, Giorgio Tortarolo¹, Mauro Buttafava⁴, Enrico Conca⁴, Sabrina Zappone¹, Agnieszka M. Pierzynska-Mach³, Sami Koho¹, Federica Villa⁴, Alberto Tosi⁴, Giuseppe Vicidomini¹

¹Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Genoa, Italy
²DIBRIS, Università degli Studi di Genova, Genoa, Italy
³Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy
⁴Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milan, Italy

Fluorescence fluctuation spectroscopy (FFS) is a family of techniques for measuring the dynamics of (bio)molecules. FFS methods rely on the measurement of temporal and/or spatial fluctuations in the fluorescence intensity generated by fluorophores passing through the detection volume of a confocal microscope [1]. However, by integrating the fluorescence over the sensitive area of the detector, information on the spatial distribution of the photons in the image plane, and thus the diffusion modality, is lost. Typical FFS setups also integrate the signal in microsecond time bins. Consequently, information on the photon arrival times, i.e., the fluorescence decay, is lost. We solve these limitations by integrating a 5x5 pixel single-photon-avalanche-diode (SPAD) array detector in a confocal microscope [2-4] and reading out the detector with the BrightEyes time-tagging module (TTM). Having for each photon a 4D data set with the pixel coordinates, absolute time and start-stop time, our setup provides detailed information about the dynamics, while measuring the fluorescence lifetime [5]. We validate our implementation on freely diffusing particles and show results of GFP in live cells. We predict that the combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for confocal-based FFS.

Mark Nüesch, Miloš Ivanović, Jean-Benoît Claude, Daniel Nettels, Robert Best, Jerome Wenger, Benjamin Schuler

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

Single-molecule Förster resonance energy transfer (FRET) is a versatile technique for probing the structure and dynamics of biomolecules over a broad range of timescales and even in heterogeneous ensembles. However, the low fluorescence brightness per molecule has limited access to very rapid dynamics. Here we demonstrate that nanophotonic fluorescence enhancement in zero-mode waveguides enables measurements of previously inaccessible low-nanosecond dynamics and reduces data acquisition times by more than an order of magnitude. For the example of a disordered peptide, we show that a detailed interpretation of the underlying distance distributions and dynamics is enabled by all-atom molecular dynamics simulations, which agree remarkably well with the experiments. We expect this combined approach to be widely applicable to the investigation of ultrafast biomolecular dynamics.

Xiongfei Luo¹, Bernd Strehmel², Zhijun Chen¹, Shujun Li¹

¹Northeast Forestry University, Hexing Road 26, Xiangfang District, Harbin, China
²Niederrhein University of Applied Sciences, Institute for Coatings and Surface Chemistry, Department of Chemistry, Adlerstr.1, D-47798 Krefeld, Germany

Sustainable Carbon Nanodots synthesized from natural materials based on seaweed, kerria lacca, citric acid, and cellulose were investigated by time-resolved fluorescence spectroscopy. Global analysis was applied to analyze the wavelength-dependent emission caused by the particles exhibiting a size of 4-10 nm with no issue of toxicological response in a flow-cytometric assay. Excitation spectra also indicated a dependence wavelength indicating the availability of different structures contributing to fluorescence. Global analysis showed the contribution of 2-3 global decay times to the overall emission. Carbon dots (CDs) derived comprising porphyrin complement these experiments.

In addition, experiments were designed to align carbon dots in several matrices; that is zeolite and hydrogel-based materials. This resulted in emissive materials exhibiting a decay time in the micro-and millisecond time frame. This depicts a new approach to design long emissive materials.

These new emitters can attract the interest of several applications based on imaging or photocatalysis. Time-resolved measurements facilitated to receive a deeper insight into photophysical processes occurring in these materials. Experiments were also pursued with solid-state samples complementing the pattern of these new fascinating materials.

Alessandro Rossetta^1,2,3, Eli Slenders¹, Mattia Donato¹, Eleonora Perego¹, Francesco Diotalevi⁴, Sami Koho¹, Giorgio Tortarolo¹, Marco Crepaldi⁴, Giuseppe Vicidomini¹

¹Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Genoa, Italy
²DIBRIS, Università degli Studi di Genova, Genoa, Italy
³Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy
⁴Electronic Design Laboratory, Istituto Italiano di Tecnologia, Genoa, Italy

Single-photon (SP) array detectors are revolutionizing fluorescence laser-scanning microscopy (LSM) by granting access to a new set of single-photon information typically lost in conventional LSM [1,2] and triggering a new imaging/spectroscopy paradigm -- the so-called single-photon-LSM (SP-LSM). Because the huge perspectives of this paradigm, and the great momentum on single-photon-detection technology, there is a constant improvement in  SP array detectors tech specs [3,4], which demands for a similar improvement in the data-acquisition (DAQ) system counterpart.  In particular, there is a growing need for DAQs capable of coping with the mega-sized temporal information delivered by SPAD array detectors. In order to fill this gap and provide a benchmarking data acquisition architecture for SP-LSM applications, we developed an open-source multi-channel time-tagging DAQ solution based on a field-programmable-gate-array (FPGA).  This BrightEyes time-tagging module (TTM) can temporally tag single-photon events with ~30 ps precision and simultaneously process other synchronization events with ~4 ns precision. After testbench validation, we connected the module to a fluorescence LSM equipped with SPAD  array detector, thus enabling fluorescence lifetime image scanning microscopy (FLISM) and fluorescence lifetime fluctuation spectroscopy (FLFS) experiments. Being an open-access project, capable of plug-and-play deployment, the  BrightEyes-TTM can be upgraded, modified, and customized by the microscopy-makers. 

Luke D. Lavis

Janelia Research Campus, Howard Hughes Medical Institute,19700 Helix Drive, Ashburn, Virginia 20147, USA, lavisl@hhmi.org

Specific labeling of biomolecules with bright, photostable fluorophores is the keystone of fluorescence microscopy. An expanding method to label cellular components utilizes genetically encoded self‑labeling tags, which enable the attachment of chemical fluorophores to specific proteins inside living cells. This strategy combines the genetic specificity of fluorescent proteins with the favorable photophysics of synthetic dyes. However, intracellular labeling using these techniques requires small, cell-permeable fluorophores, thereby limiting utility to a small number of classic, unoptimized dyes. We discovered a simple structural modification to standard fluorophores that improves brightness and photostability while preserving other spectral properties and cell permeability. Inspired by computational experiments, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with a four-membered azetidine ring. This net addition of two carbon atoms doubles the quantum efficiency and improves the photon yield in living cells. The novel substitution is generalizable to fluorophores from different structural classes, yielding a palette of synthetically tractable chemical dyes with improved quantum efficiency and enabling multicolor single-molecule imaging experiments. These brighter versions of classic fluorophores can be further modified to fine-tune spectral and chemical properties for advanced imaging experiments in increasingly complex biological samples.

Michael Isselstein¹, Lei Zhang¹, Viktorija Glembockyte², Oliver Brix¹, Gonzalo Cosa³, Philip Tinnefeld², Thorben Cordes¹

¹ Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany
² Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, Haus E 81377 München, Germany
³ Department of Chemistry and Quebec Centre for Applied Materials (QCAM), McGill University, 801 Sherbrooke Street W., H3A 0B8 Montreal, Quebec, Canada
email: cordes@bio.lmu.de

Self-healing dyes have emerged as a new promising class of fluorescent labels. They consist of two units, a fluorescent dye and a photostabilizer. The latter heals whenever the fluorescent dye is in danger of taking a reaction pathway toward photobleaching. We describe the underlying concepts and summarize the developmental history and state-of-the-art, including latest applications in high-resolution microscopy, live-cell, and single-molecule imaging. We further discuss remaining limitations, which are (i) lower photostabilization of most self-healing dyes when compared to solution additives, (ii) limited mechanistic understanding on the influence of the biochemical environment and molecular oxygen on self-healing, and (iii) the lack of cheap and facile bioconjugation strategies. Finally, we provide ideas on how to further advance self-healing dyes, show new data on redox blinking caused by double-stranded DNA, and highlight forthcoming work on intramolecular photostabilization of fluorescent proteins.

Dominik Wöll, Silvia Centeno Benigno, Oleksii Nevskyi, Ashvini Purohit, Eric Siemes, Pia Lenßen, Laura Hoppe Alvarez

Institute of Physical Chemistry, Landoltweg 2, 52074 Aachen

The elucidation of the structure and functionalization of materials in the sub-micron range is a key to their further development and application.^[1] Microgels are a class of such soft materials with high potential for multiple fields.^[2] Several groups have learnt to functionalize and structure microgels in sophisticated ways, but the evaluation of a successful functionalization or the envisioned properties are often limited by the ways of analysis and visualization. The development of modern super-resolved fluorescence microscopy methods^[3] opened up new ways of nanoscopic visual­ization that had not been possible previously due to the diffraction limit of light prohibiting spatial resolution beyond approx. 300 nm.

In my contribution, our results on super-resolution fluorescence microscopy in microgels and some other soft matter systems will be shown. The power of these methods to address and answer scientific questions in materials science such as local polarity,^[4] positions of polymer cross-linkers^[5] and deformaion of soft particles^[6] will be discussed.

Francesco Reina¹, John M.A. Wigg², Mariia Dmitrieva³, Joël Lefebvre⁴, Jens Rittscher³, B. Christoffer Lagerholm⁵, Christian Eggeling^1,2,5,6

¹Leibniz-Institüt für Photonische Technologien e.V., Jena, Germany
²Institute of Applied Optics and Biophysics, Friedrich Schiller Universität, Jena, Germany
³Department of Engineering Science, University of Oxford, Oxford, UK
⁴Département d’informatique, Université du Québec à Montréal, 201 av. du Président-Kennedy, Montréal (Québec) H2X 3Y7, Canada
⁵Wolfson Imaging Centre, MRC Weatherall institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom
⁶MRC Human Immunology Unit, MRC Weatherall institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom

The study of lipid diffusion on model and plasma membranes has benefitted greatly from the application of Single Particle Tracking (SPT) and Fluorescence Correlation Spectroscopy (SPT). Recent improvements in SPT detection, which push the sampling rates to the kHz range, make the case for the comparison between these techniques, which have historically produced diverging results in this field. High temporal resolutions, such as the ones we employ, necessitate a more accurate treatment of the dynamic localization uncertainty in SPT detection. We develop a method for analysis of SPT trajectories which is capable of accurately estimating diffusion rates, and other physical characteristics of the environment in which the particles diffuse, provided an appropriate model of diffusion is introduced. We applied this method to experimental datasets of lipid diffusion on live plasma membrane, recorded at 2kHz through Interferometric Scattering Microscopy. The method was implemented in a Python library (TRAIT2D) we recently released, for integration in pre-existing analysis pipelines. Finally, we introduce a metric that allows the comparison of the present results with relevant experiments in literature, both from SPT and FCS experiments.

Esra Ahunbay, Fabio D. Steffen, Susann Zelger-Paulus, Roland K. O. Sigel

Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

Dynamic RNAs undergo dramatic structural rearrangements, strongly related to their function. Single-molecule FRET can display stages of RNA processing and track transitions between these different folds.¹ Visualizing RNA dynamics via smFRET requires site-specific positioning of fluorescent dyes on the molecule of interest.^2,3 When labeling large catalytically active RNAs, it is particularly important yet challenging to find the balance between relevant labeling positions and preservation of functionality. Previous labeling methods usually meet only one of the two requirements at the expense of the other.⁴

We present a dual-color RNA end-labeling strategy that is fast, efficient, site-specific and preserves the structural and functional integrity of the biomolecule. We establish an optimized protocol for bioconjugation with a FRET pair of fluorophores via chemical activation of the RNA termini, the 5'-phosphate and the 3'-ribose. The strength of the strategy is illustrated on a highly dynamic self-splicing wild-type group II intron, Sc.ai5γ (≈ 300 kDa). This ribozyme was covalently labeled while retaining structure and function.⁵ In summary, we demonstrate a universal strategy for covalent attachment of fluorescent dyes that is independent of RNA size, sequence and structure.

Swarupa Chatterjee(a,b), Dr. Christian Blum (a), Prof. dr. Mireille M.A.E. Classens (a)

^aDepartment of Nanobiophysics (NBP), University of Twente, The Netherlands
^bWetsus, European Centre of Excellence for Sustainable Water Technology, Leeuwarden, The Netherlands

Filtration membranes have been widely used for waterborne virus removal to obtain safe drinking water. Polyethylenimine (PEI) functionalized microfiltration polyethersulfone (PES) membranes retain and result in the inactivation of viruses by disassembly [1]. However, monitoring virus retention with inactivation is time-consuming, requires expertise in microbiology and specialized equipment. Recently, a fast and easy method has been introduced to quantify low virus concentrations (count) based on single-particle tracking(SPT) of fluorescently labeled cowpea chlorotic mottle viruses (model) [2]. This technique is used to quantify the virus retention of a PEI coated PES membrane which removes ~99% (log(2)) of the applied viruses. In the filtrate, a low number of bright particles of size ~28nm (SPT) is detected, indicating the presence of intact viruses. To study virus disassembly, a complex photophysical interplay of multiple fluorophores with a high degree of labeling (DOL) on one virus (quenched and redshifted fluorescence)[2] is exploited. Differentiation between the intact virus and the disassembled, free capsid proteins are made based on the above technique. Fluorescence emission (unquenched and blueshifted), lifetime (multiexponential to single exponential decay), and correlation spectroscopy (diffusion coefficient change:~17 µm2/s to ~100 µm2/s) additionally evidence the disassembly of viruses by the membrane (PEI) and in bulk experiments. By imaging the membrane it is shown viruses that remain behind are disassembled thus inactivated.

Tao Chen, Arindam Ghosh, Akshita Sharma, Jörg Enderlein

Drittes Physikalisches Institut - Biophysik, Georg August University, Göttingen

Single-molecule fluorescence microscopy (SMFM) has become an indispensable tool for almost all fields of research, from fundamental physics to the life sciences. The advent of SMFM (e.g., STED and SMLM) has pushed the limits of spatial resolution down to the molecular length scale. However, simple technical realization to achieve resolution in a similar order along the optical axis of a microscope still remained challenging. Recently, metal/graphene-induced energy transfer (MIET/GIET) was introduced as a simpler alternative that enables axial localization of fluorescent emitters with nanometer resolution [1-2], which can be substantially utilized in the direction of membrane biophysics. Here, we demonstrate that by using MIET/GIET to reveal the fluctuations of the membrane, the leaflet-specific diffusion, and the dynamics of the phospholipid bilayer membrane.  

Sam Daly¹, Anoushka Handa¹, Kevin O'Holleran², Steven F. Lee¹

¹Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK
²Cambridge Advanced Imaging Centre, University of Cambridge, Downing Site, Cambridge, CB2 3DY, UK

Single molecule localisation microscopy (SMLM) has enabled the study of biological systems below the diffraction limit of light. The development of 3D-SMLM techniques, such as astigmatism, biplane and the double-helix point spread function (DHPSF) have allowed biological systems to be imaged in their native environment.¹ However, these techniques are limited by their depth-of-fields of 0.5 to 4 μm, maximum resolvable labelling densities and optical complexity to implement. Recently, single molecule light field microscopy (SMLFM) was introduced as a novel 3D-SMLM technique that places a refractive microlens array in the back focal plane of a widefield microscope platform to produce a 2D array of 2D perspective views of the sample plane.² SMLFM can routinely achieve a 6-8 μm depth-of-field with up to 20 nm isotropic resolutions. We demonstrate the first microscope platform permitting direct quantitative comparisons between SMLFM and DHPSF. Furthermore, we characterise its performance by imaging a fixed T cell membrane over a 6-8 μm depth-of-field.

Begüm Demirkurt¹, Fred Brouwer¹, Daniel Bonn²

¹Van’t Hoff Institute for Molecular Sciences, Faculty of Science, University of Amsterdam
²Van der Waals – Zeeman Institute, Faculty of Science, University of Amsterdam

  Fluorescent molecules from the special group called molecular rotors have been used as sensors to measure local viscosity and, to visualize mechanical contact between two surfaces at sub–micron length scale^1,2. Bond rotation in a molecular rotor is related to non-radiative decay, and the rate of rotation is sensitive to the local rigidity. As a result, fluorescence quantum yields are low in non-viscous media and increase with molecular-scale confinement. In this study, a dicyanodihydrofuran-based molecular rotor (DCDHF) was used to produce a fluorescent contact sensitive mono-layer on glass and, we focus attention on single–molecule fluorescence properties of molecular rotor in confined spaces, which can be used for super-resolution imaging of mechanical contact.

  Controlled photobleaching yields a photostable and switchable molecular layer with long–lasting fluorescence blinking at the glass–air and glass–contact interfaces. Interestingly, the fluorescence blinking of this single DCDHF molecules was measured to possess an entirely different kinetic behaviour at the contact interface; where the power – law distribution of fluorescently on and off molecules at glass – air interface, was observed to switch to the exponential distribution, with remarkable lengthening of on and off times at contact interface. The synchronization of kinetic information with various experimental imaging conditions, allowed super–resolution imaging of the mechanical contact, with ~70 nm lateral resolution.

Paz Drori, Yair Razvag, Eitan Lerner

The Hebrew University of Jerusalem, Givat Ram.

We developed an optical setup that allows rapid detection of particles with diameters
larger than 50 nm based on coincident detection of their size and specific interactions
with antibodies. The solution couples a confocal microscope and microfluidics flow
of a mixture of nano-particles along with high concentration of fluorescent dyes and
low concentrations of fluorescently-labeled antibodies. The fluorescent dyes produce
a constant signal, however when a particle with a diameter >50 nm flows through the
laser focus, it excludes a fraction of the dyes proportional to the particle volume,
which leads to a dip in the signal. Additionally, the fluorescence of the dye-labeled
antibody conjugated to the nano-particle is detected in a different spectral regime with
a separate detector. If a particle that flows through the beam had antibodies attached
to it, it will produce a signal dip in one detector and a signal burst in another detector,
at the same time. By performing microfluidics hydrodynamic focusing, we were able
to enhance the sensitivity and reach the limit with a sample containing 10⁵
particles/mL. The setup allows for rapid detection of particles (>100 particles in <10
minutes) by combining laser confocal detection with microfluidic flow devices.

Besim Fazliji, Susann Zelger-Paulus, Kevin Kraft, Roland K.O. Sigel

Department of Chemistry, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

RNA serves as a great example where form follows function. Similar to DNA, RNA consists of only four building blocks. Still, RNAs can perform various functions due to their folding diversity. For instance, the self-splicing activity of group II introns is inevitably connected to the formation of intricate tertiary structures.

Group II introns are a class of ribozymes found in bacteria and eukaryotic organelles and are considered predecessors of the nuclear spliceosome.^[1,2] Their autocatalytic activity independent of cofactors in vitro^[3] facilitates the application of various methods to study their reaction mechanism and architecture. Advancements in crystallography and cryo-EM have led to the discovery of the complex tertiary structures of group II introns.^[2,4] Nevertheless, knowledge on their conformational dynamics remains scarce. Thus, we are applying smFRET on a construct derived from group II intron Sc.ai5γ, an intron that resides in the mitochondrial genome of Saccharomyces cerevisiae. In vitro, high Mg²⁺ concentrations allow efficient self-splicing of group II introns.^[5] With this in mind, we trigger the active state by a Mg²⁺ titration and monitor the conformational changes by smFRET. We show the influence of Mg²⁺ on the folding ribozyme by analysis with our home-written MATLAB-based software.^[6]

Daniel Fersch¹, Pavel Malý¹, Jessica Rühe², Matthias Hensen¹, Frank Würthner^2,3, Tobias Brixner^1,3

¹Institut für Physikalische und Theoretische Chemie, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
²Institut für Organische Chemie, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
³Center for Nanosystems Chemistry (CNC), Universität Würzburg, Theodor-Boveri-Weg, 97074 Würzburg, Germany

Generally speaking, the photophysical properties of ensemble-averaged bulk samples can differ vastly from those of the respective single molecules. While setups for static single molecule imaging and even time-resolved spectroscopy on the picosecond timescale are nowadays commercially available, access to the femtosecond dynamics of single molecules remains a challenge.

In this work, we present a new setup using a spectrally tunable femtosecond laser source, and a scanning confocal fluorescence microscope with fully reflective excitation geometry and single molecule sensitivity. We use both a liquid-crystal-based pulse shaper as well as a phase-stable Mach–Zehnder interferometer to create two identical pulse copies with variable time delay. The molecular excitation spectrum can then be measured by detecting the sample fluorescence as a function of the inter-pulse delay and subsequent Fourier transformation. By pumping the molecule with an additional pulse, we can then probe the dynamics of the excited state as a function of the pump-probe delay, resulting in a fluorescence-detected pump–probe spectrum [1]. By applying this technique, we want to investigate the dynamics of individual molecules on the first hundreds of femtoseconds, such as spectral diffusion and intramolecular energy relaxation.

Veronika Frank¹, Benedikt Sohmen¹, Thorsten Hugel^1,2

¹Institute of Physical Chemistry, University of Freiburg, Albertstraße 21, 79104 Freiburg, Germany
²Signaling research centers BIOSS and CIBSS, University of Freiburg, Schänzlestraße 18, 79104 Freiburg, Germany

Fluorescence based methods have mainly been used to understanding protein dynamics on the millisecond to minutes timescale, in particular for large proteins. Here we extend this timescale by combining advanced fluorescence methods with molecular dynamics (MD) simulations to cover timescales from nanoseconds to milliseconds for the heat shock protein Hsp90. The ATPase Hsp90 is a homo dimer with a molecular weight of about 90 kDa per monomer. Its association to oncoproteins makes the understanding of this protein machinery significant for drug-targeting in medicine therapeutics [1].

Here we will first show how ATP hydrolysis in Hsp90 causes allosteric changes at a distant protein binding site on hierarchical timescales [2]. Then we show with nsFCS (nanosecond Fluorescence Correlation Spectroscopy) how fluctuations on the nanosecond timescale are affected or regulated by interactors. Finally, quenching by tryptophan via photoinduced electron transfer (PET) is used to further quantify and localize these nanosecond fluctuations.

Altogether, we show for the well-investigated heat shock protein Hsp90 how fast kinetics are regulated in a multi-domain protein.

Thomas Gensch

Thomas Gensch; IBI-1 (Molecular and Cellular Physiology); Forschungszentrum Juelich, Wilhelm-Jonen Str., 52428 Juelich, Germany

Autophagy is a homeostatic process, which enables cells to survive under conditions of stress and starvation and plays a role for basal turnover of intracellular proteins and maintenance of homeostasis under stress. Autophagosome formation critically depends on the Atg8 family of proteins.

Subcellular structures containing autophagy-related proteins of the Atg8 protein family have been investigated with conventional wide-field fluorescence and single molecule localization microscopy (SMLM). Fusion proteins of GABARAP and LC3B, respectively, with either EYFP or Dendra2 were overexpressed in HEK293 cells. The size distributions for all four fusion proteins were found to be similar, nor was an influence of the fluorescent protein (FP) on the shape distributions. The type of Atg8 protein, however, lead to dramatically different shape distributions. FP-GABARAP favours circular structures while FP-LC3B has elliptical or U-shape structures as majoritarian fraction. On the contrary, the comparison between fed and starved conditions revealed very similar shape distributions for both, GABARAP and LC3B.

Our experimental results point towards highly different localizations of the two Atg8 protein families, which appear to label structures representing distinct autophagy stages. The superiority of SMLM over conventional fluorescence microscopy is evident when comparing images of the same cell (better resolution revealing more structural details).

Carolin Grandy, Fabian Port, Kay-Eberhard Gottschalk

Institut für Experimentelle Physik, Universität Ulm, Germany

Cells adapt their actin cytoskeletons architecture to structural cues of the environment in all three dimensions. Nevertheless, how manipulating cell shape influences the actin cytoskeletons z dimension is unstudied, but crucial for an understanding of the mutual influence of cell shape, cell tension and actin architecture. Metal-induced energy transfer (MIET) is a super-resolution microscopy technique perfectly suited for studying the z-dimension of the actin cytoskeleton in response to different stimuli [1,2]. To study the effect of shape on the z-dimension of the actin cytoskeleton we combine MIET with protein micropatterning on gold [3]. This allows us not only to precisely manipulate the shape of the cell but also to regulate forces by changing the shape while studying specific actin structures with super-resolution in the third dimension.

Ingo Gregor, Eugenia Butkevich, Joerg Enderlein, Soheil Mojiri

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

One of the most widely used microscopy techniques in biology and medicine is fluorescence microscopy, offering high specificity in labeling as well as maximum sensitivity. For live cell imaging, the ideal fluorescence microscope should offer high spatial resolution, fast image acquisition, three-dimensional sectioning, and multi-color detection. However, most existing fluorescence microscopes have to compromise between these different requirements. Here, we present a multi-plane multi-color wide-field microscope that uses a dedicated beam-splitter for recording volumetric data in eight focal planes and for three emission colors with frame rates of hundreds of volumes per second. We demonstrate the efficiency and performance of our system by three-dimensional imaging of multiply labeled fixed and living cells.

Aleš Holoubek¹, Marie Olšinová², Aleš Benda², Kateřina Kuželová¹, Barbora Brodská¹

¹Institute of Hematology and Blood Transfusion, U Nemocnice 2094/1, 128 00 Prague 2, Czechia
²BIOCEV, Imaging Methods Core Facility, Průmyslová 595, 252 50 Vestec, Czechia

Function of proteins with important role in acute myeloid leukemia (AML) often depends on their oligomerization state. Nucleophosmin (NPM), a phosphoprotein occurring in nucleolus and nucleus in pentamers, is relocalized to the cytoplasm due to mutations characteristic for AML with normal karyotype. NPM participates in cell stress response via interaction with tumor suppressors, such as p53. The mutated NPM (NPMmut) causes delocalization of many of its interaction partners, including wild-type NPM (NPMwt) and p53, from nucleus to cytoplasm. We constructed a set of fluorescently labeled protein constructs serving as controls for fluorescence cross-correlation spectroscopy (FCCS). Mix of non-oligomerizing NPM variants (NPMcut) labeled with eGFP or mRFP1 served as a negative control for FCCS in the nucleoplasm. Both, oligomerizing and non-oligomerizing, NPM variants double-labeled with mRFP1 and eGFP were used as positive controls. Consequently, we introduced the AML-specific mutation to target the controls to cytoplasm and we compared FCCS in both cell compartments. Positive cross-correlation was detected for all positive controls, being more pronounced for oligomerizing variants. The results will be used for fluorescence monitoring of AML-related proteins interconnection in live cells.

Supported by MHCR (conceptual development of research organization No 00023736), MEYSCR (Large RI Project LM2018129 Czech-BioImaging) and ERDF (CZ.02.1.01/0.0/0.0/18_046/0016045).

Johan Hummert¹, Jonas Euchner¹, Caroline Berlage², Marcelle König³, Alexander Krull⁴, Dirk-Peter Herten¹

¹Institute of Cardiovascular Sciences , College of Medical and Dental Sciences, Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
²Einstein Center for Neurosciences, NeuroCure Cluster of Excellence, Charité-Universitätsmedizin Berlin, Berlin, Germany
³PicoQuant GmbH, Berlin, Germany
⁴School of Computer Science, Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

Fluorescence microscopy based molecular counting can provide unique insights into protein oligomerization and the composition of intracellular protein complexes. Photon antibunching can be used to directly measure the number of fluorescent labels without additional calibration [1]. However, robust and generalisable algorithms for density mapping directly from images containing photon coincidence information are lacking. We are working towards an analysis approach based on deep learning to extract density maps from confocal photon coincidence images. Fully convolutional neural networks are trained and validated with synthetic data from Monte Carlo simulations. We test for experimental applicability of the analysis by counting fluorophores on DNA origami with known fluorophore numbers, to benchmark the counting range of the analysis. In addition  to fast density mapping  the analysis can also provide a modest improvement in image resolution. We envision a similar approach by combining image scanning microscopy with single-photon sensitive array detectors [2], enabling molecular counting on less specialized microscope setups.

Nitish Jain^1,2, Tobias B. Gäbler^1,2, Josué R. León Torres^1,2, Patrick Hendra^1,2, Markus Gräfe^1,2

¹ Fraunhofer Institute of Applied Optics and Precision Engineering IOF, Albert-Einstein-Straße 7, D-07745 Jena, Germany
² Friedrich Schiller University Jena, Institute of Applied Physics, Abbe Center of Photonics, Albert-Einstein-Straße 15, D-07745 Jena, Germany

We demonstrate a comprehensive study of potential fluorophores for entangled 2-photon fluorescence microscopy. Using cw-pumped ppKTP crystals, we have obtained linear absorption rates with entangled photon pairs for commonly used fluorophores in life science, like rhodamine derivates or acridine orange. Our work aims to establish new prospects for low photon flux multiphoton absorption-based imaging techniques.

2-photon fluorescence microscopy is a non-linear live-cell imaging technique based on the simultaneous absorption of two photons to induce fluorescence. The use of a focused high-intensity infrared light beam to excite fluorophores provides deeper depth penetration, reduced photodamage, inherent z-sectioning, and lack of out-of-focus bleaching. However, the use of high peak power pico- and femtosecond pulsed lasers to achieve enough simultaneously absorbed infrared photons causes photodamage, photobleaching, and heating in the biological sample.

Our work addresses the shortcomings arising due to the use of high peak power pulsed lasers in classical 2-photon absorption (c2PA) by exploiting the concept of entangled 2-photon absorption (e2PA). The absorption rate for c2PA of two independent single photons depends quadratically on their flux. Whereas entangled photon pairs act in this case as a single quantum particle and, consequently, a linear absorption rate is obtained. Therefore, e2PA dominates at low photon fluxes with a linear absorption rate dependence on the photon pair rate.

We have clearly reported linear absorption rates for different concentrations of widely applicable commercial fluorophores, indicating e2PA, upon illumination with entangled photon-pairs at 810 nm. We further calculated the e2PA cross-section for different fluorescent dye concentrations.

Our experimental results, together with our modular and fiber-coupled setup design, may pave the way towards low-cost, high-efficiency, and sensitive 2-photon fluorescence microscopy.

Tomas Janovic, Tomas Brom, Martin Stojaspal, Pavel Veverka, Denisa Horakova, Ctirad Hofr

LifeB, Chromatin Molecular Complexes, CEITEC and Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno CZ-62500, Czech Republic.

Telomeric repeat binding factor TRF1, TRF2 together with TIN2 protein play essential roles as shelterin-core subunits. Their dynamics are important for regulating the assembly of shelterin complex. We applied FCCS – Fluorescence Cross-Correlation Spectroscopy as an in vitro single-molecule approach to quantitatively describe the exchange of TRF1 and TRF2 in the complex of TIN2. We found that TRF1 can effectively exchange TRF2 in the TIN2-TRF2 complex, which is essential regarding the function of shelterin during specific recognition of chromosome ends and telomerase activity regulation. We extended the FCCS study with the addition of TPP1, a TIN2 binding partner. We tested whether TPP1 presence could alter the TIN2-TRF2 interaction and enable TIN2 to interact simultaneously with TRF1 and TRF2, hence originating TRF1-TIN2-TRF2 shelterin-core complex. Our FCCS data shows that TPP1 indeed, upon binding to TIN2, induces an allosteric effect improving the binding capacity so the complex TPP1-TIN2 can accommodate both TRF1 and TRF2 simultaneously. The TPP1 plays a crucial role in the proper formation of the TRF1-TIN2-TRF2 shelterin-core complex.

Agnes Koerfer¹, Francesco Reina^1,2, Cristian Eggeling^1,2

¹Friedrich-Schiller-Universität Jena, Germany
²Institut für Photonische Technologien, Germany

Diffusion of molecules in living cell membranes is influenced by several factors, such as interactions with other molecules and the characteristics of their environment. By observing the diffusion of biomolecules, we can study their interaction and hence function during different cellular processes. Here, we present a novel, versatile and open accessible program based on Python for simulating molecular membrane diffusion, specifically for three dominant models: Brownian diffusion, compartmentalized diffusion (with transient confinement in larger areas), and trapped diffusion (with transient slowdowns due to interactions). A Voronoi transformation on a uniform random distribution of seed points is performed for compartmentalized and trapping diffusion simulations to construct, respectively, the underlying network of distinct boundaries and random distributed trapping sites. We simulate signals obtained for different fluorescence microscopy measurement modes such as single-particle tracking and various Fluorescence Correlation Spectroscopy based approaches. Our ultimate goal is to develop a simulation toolbox that enables a better understanding of diffusion dynamic data obtained in living cells.

Tino Roehlicke, Sebastian Kulisch, Maximilian Diedrich, Torsten Langer, Michael Wahl, and Rainer Erdmann

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

Time tagging and Time-Correlated Single Photon Counting (TCSPC) are powerful tools in many areas of applied physics. In optical quantum science, they are widely used for the characterization of non-classical light emitters and the detection of coincident photon arrival events. In light of the recent quantum technology initiatives, these timing devices play a central role as crucial technological building blocks. Here we present a new integrated TCSPC and time tagging system design that complies with the demands for speedy data acquisition and parallelization of the detection via multiple channels. This design provides a novel high speed interface for data transfer to one or more external FPGAs where custom algorithms for real-time data processing can be implemented. The interface can carry a total rate of up to 1.8 Gtags/s from up to 64 synchronized input channels, each with an extremely short dead-time of 650 ps and a digital timing resolution of 5 ps. Apart from showing design features and benchmark results of the instrument as such, we present results from fluorescence lifetime imaging and a development snapshot of other high throughput applications.

Daniel Marx, Oleksii Nevskyi, Arindam Ghosh, Jan Christoph Thiele, Roman Tsukanov, Jörg Enderlein

Fakultät für Physik, Friedrich-Hund-Platz 1, D-37077 Göttingen

Single-molecule optical DNA mapping offers a possibility to get a long range sequence information. This allows the fast characterizing of unknown biological samples or identifying the position of a DNA-fragment inside of a large DNA-code.

Nowadays, enzyme-free DNA optical mapping methods are in more demand.

The advantage of the techniques mentioned above is that no extensive sample preparation is required and you can receive continuous sequence information. Most popular enzyme-free methods are utilizing competitive binding or denaturation mapping. [1, 2] 

Earlier papers stated that the excited state lifetime of dyes like TOTO or YOYO-1 intercalated into DNA strands strongly depends on the GC-AT content. [3]

Here we present a new approach utilizing fluorescence lifetime imaging to measure the GC-AT content along the DNA strand directly. This could allow short measurement times and could suite high-throughput applications.

Therefore, for proof-of-principle experiment, λ- and T7-DNA were labelled with YOYO-1 and stretched using different stretching methods.  

The lifetimes along the DNA-strands were measured with a new FLIM-camera utilizing wide-field setup. Here we investigate the applicability of fluorescence lifetime measurements to receive a predictable, reproducible and genome specific response.

Fabian Port, Carolin Grandy, Kay-Eberhard Gottschalk

Institut für Experimentelle Physik, Universität Ulm

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

For a detailed analysis of the focal adhesion architecture coupled with force response, we necessitate a method to measure small distances while manipulating the cells with an AFM. To meet this challenge, we couple AFM techniques [3] with Metal Induced Energy Transfer (MIET) [4] to resolve positions at the nanoscale level. Here we show an initial analysis of the interplay between focal adhesion associated actin and force.

Yury Prokazov¹, Evgeny Turbin¹, André Weber², Werner Zuschratter²

¹Photonscore GmbH, Brenneckestr. 20, 39118 Magdeburg
²Leibniz Institute for Neurobiology, Brenneckestr. 6, 39118 Magdeburg

Photonscore LINCam is a position sensitive single photon counting system that enables time-domain fluorescence lifetime imaging (FLIM) acquisition. In combination with a pulsed laser source the LINCam upgrades virtually any widefield fluorescence microscope into a cutting edge FLIM system with picosecond time resolution. Here we present our latest developments of the LINCam camera system and some of the applications.

One of the well-known drawbacks of widefield microscopy imaging is low axial resolution compared to confocal microscopy. Several methods are known to overcome those limitations introducing the confocallity. In this poster we demonstrate FLIM images acquired with optical sectioning or axial superresolution methods using (i) a spinning disk confocal unit, (ii) light-sheet illumination or (iii) Metal Induced Energy Transfer (MIET) technique in combination with TIRF illumination. In addition, we highlight the results of single molecule imaging where a finite mixture of spectrally indistinguishable chromophores was resolved solely based on the lifetimes of individual molecules.

Debayan Purkait

Saha Institute of Nuclear Physics, Sector 1, AF Block, Bidhannagar, Kolkata, Pin Code - 700064, West Bengal, India.

RecD2 from Deinococcus radiodurans (DrRecD2) is the functional analog of the RecBCD complex that is found in Escherichia coli. DrRecD2 has important contributions towards the organism’s unusually high tolerance to gamma radiation (unto 15 kGy) and hydrogen peroxide. Previous atomic level structural studies have identified the DNA binding domains of the protein and proposed a possible mechanism of translocation but further experimental validation of the model is required. In this study we probed the DNA unwinding process by DrRecD2 at single-molecule resolution by single-molecule Forster resonance energy transfer (smFRET). Additionally, we have also identified the exact binding spot and the mechanism of binding of DrRecD2 by single-molecule protein-induced fluorescence enhancement (smPIFE). Our study reveals that the protein shows an ATP-independent ‘continuous’ unwinding and an ATP-dependent ‘discontinuous’ unwinding of the DNA. The ATP-dependent ‘discontinuous’ unwinding fits well in the previously hypothesised ‘Power-Stroke Model’. Also we show that DrRecD2 approaches the DNA from the 5’ terminal end of the overhang region either by slow ‘drifting’ or fast ‘collision’. These finding may help in better understanding of the mechanisms of the less studied SF1B family of helicases.

Niels Radmacher, Ingo Gregor, Jörg Enderlein

Drittes Physikalisches Institut - Biophysik, Georg August Universität Göttingen, Friedrich-Hund-Platz 1, 37077 Göttingen

In many fields of biology and medicine, it is crucial to capture high-resolution images of living cells. Especially in diagnostics, knowing the spatial arrangement and relative composition of proteins in cells can be an important clue for the health of an organism.
Image Scanning Microscopy (ISM) can double the resolution of a confocal microscope by replacing the single point detector with a CCD chip. Another excellent tool for bio-imaging is Fluorescence Lifetime Imaging Microscopy (FLIM), which allows for distinguishing different markers by their specific lifetime, in addition to their colour.
These two techniques can be combined into a Fluorescence Lifetime Image Scanning Microscope (FL-ISM) using an array of single-photon detectors such as SPADs (Single Photon Avalanche Diodes), thereby enabling super-resolution microscopy with fluorescence lifetime multiplexing for live-cell and tissue imaging.
In first results, we show that a suitable selection of fluorescent dyes can be used to create lifetime-multiplexed images for simultaneously imaging different structures while greatly reducing data acquisition time.

Damir Sakhapov, Ingo Gregor, Jörg Enderlein

Drittes Physikalisches Institut - Biophysik, Georg-August-Universität, Friedrich-Hund-Platz 1, D-37077 Göttingen, GERMANY

Fluorescence correlation spectroscopy (FCS) is a widely used technique to measure diffusion constants (aka hydrodynamic radii), to study conformational changes of biomolecules, or to measure photophysical rate constants. The latter depends on the excitation intensities seen by the studied dye molecules, a quantity that is notoriously difficult to determine in FCS experiments.

We use fluorescence antibunching measurements to determine absolute values of excitation intensity in the confocal volume, and we use this value for obtaining absolute values of photophysical rate constants from an FCS measurement. The core idea is to measure antibunching times at different excitation intensities, which can then be used to extract absolute numbers for the excitation rate of the studied molecules.

Here, the intersystem crossing rate constant of Atto655 is smaller than its phosphorescent rate so that in all practical FCS experiments, the photophysics of Atto655 is fully dominated by its phosphorescent rate and is quasi-independent on excitation intensity. That makes Atto655 an ideal tool for Photo-Electron Transfer FCS (PET-FCS) where intramolecular dynamics of a peptide are in the same time range as the dye’s photophysics. Inversely, rhodamine shows considerable intersystem crossing.  We use our method for determining the absolute values of their corresponding photophysical rate constants.

Clovis Ndege Simisi, Sobhan Sen

Spectroscopy Laboratory, School of Physical Sciences, Jawaharlal Nehru University, , New Delhi 110067, India

Ligand/drug binding to biomolecules is of paramount importance in biology, biotechnology, chemistry and   medicine. Fluorescence signal from ligand molecules of interest is routinely used to track binding of ligands to bio-macromolecules because of its ultimate sensitivity, which reveal important thermodynamics information of ligand binding to target molecules. However, it is more important to track the kinetics of such interaction for better understanding the ligand/drug efficacy. Moreover, most of relevant drug/ligand used for therapeutic purpose are either non-fluorescent or weakly fluorescent. Hence, often it becomes difficult to use such ligand molecules for fluorescence based titration. To circumvent such problem we propose here a simple fluorescence correlation spectroscopy based strategy that uses site-specific fluorescently (Cy3) end-labelled G-quadruplex DNA structures which can probe the interaction of (non-fluorescent) ligands with the DNA through monitoring the quenching of fluorescence of labelled-Cy3. This method has the capability of monitoring position dependent ligand binding kinetics to DNA. As a proof of concept, here we use well known ligands, TMPyP, BRACO-19, Hoechst 33342 and Hoechst 33258 (non-fluorescent at excitation wavelength used) to extract binding kinetic parameters. Our results show differential chemotype-specificity of binding kinetics of ligands to different G-quadruplex DNA structures.

Lorenz Sparrenberg, Kristian Berwanger, Benjamin Greiner, Steffen Krüger, Michael Fuchs, Moritz Balg, Alexander Schuster, Harald Mathis

Digital Health/BioMOS, Fraunhofer Institute for Applied Information Technology (FIT), FhG e.V. Institutszentrum Birlinghoven (IZB), Schloss Birlinghoven 1, 53757 Sankt Augustin

Today, DNA sequencing is an indispensable tool for addressing biological questions. An important step before the actual sequencing is the characterization of the DNA libraries, which is a multi-step process. We demonstrate a simplification and acceleration of this process by analyzing fluorescence fluctuations. Using a confocal plate reader, samples are automatically measured in a microtiter plate. By cumulant analysis [1][2] we derive the mean mass concentration, fragment length and molarity of the mixture. As calibration we use DNA solutions of defined composition. We also implemented a first order correction of the detector artifacts deadtime and afterpulsing [3] and a simple on/off isomerization model extension to account for photokinetic effects on small time scales [1][4]. Finally, we compared our results to a prevalent standard characterization process, thus showing the suitability of the approach.

Andreas W. Stark¹, Gregor Gentsch¹, D. Weigel^1,3, Lars Schmidl², Christian Geis², R. Kowarschik^1,4, Christian Franke^1,4

¹ Institut of Applied Optics and Biophysics, Friedrich Schiller University Jena, 07743 Jena, Germany
² Section for translational neuro immunology, University Hospital, 07743 Jena, Germany
³ Analytic Jena GmbH, Konrad-Zuse-Straße 1, 07745 Jena, Germany
⁴ Abbe Center of Photonics, Albert-Einstein-Straße 6, 07745 Jena, Germany

Structured illumination has been successfully adapted for nanoscopy to allow an increased spatial resolution with low photon doses, such as SIM or SOFI-microscopy, with a wide range of biological applications, especially under live cell conditions. An alternative to highly regular pattern are so called speckles, i.e. statistical, coherent patterns, caused by the self-interference of a coherent beam when statistically scattered. The resulting structures of such a scattering process are, if adapted to microscopic scale, diffraction limited at best. Speckle-based fluorescence microscopy is therefore restricted in its resolution, if only one wavelength or speckle pattern is used. Here we present the concept of a novel technique of nanoscopic imaging using multiplexed, coherent, structured speckle illumination. By utilizing two or more overlapping speckle pattern by multiplication, the resulting structures can be smaller than diffraction limited areas [1]. We propose this concept and applications in different variations thereof to mimic e.g. 2-photon microscopy, STED, SOFI or classic SMLM, by multiplexed speckle-field illumination. If successfully applied, we reckon to achieve comparable spatio-temporal resolutions to classic SIM with significantly reduced technical complexity and cost. Finally, the precise mapping of the resulting excitation speckle-field could open up new avenues to achieve also axial super-resolution.

Frederik Steiert^1,2, Armin Lambacher³, Petra Schwille¹, Thomas Weidemann¹

¹Max Planck Institute of Biochemistry, Cellular and Molecular Biophysics, Am Klopferspitz 18, 82152 Martinsried, Germany
²Department of Physics, Technical University Munich, 85748 Garching, Germany
³Max Planck Institute of Biochemistry, Molecular Medicine, Am Klopferspitz 18, 82152 Martinsried, Germany

The molecular brightness of fluorescently labeled biomolecules represents an intuitive readout parameter for oligomerization in model systems and live cells. A prerequisite for faithful interpretation of brightness experiments is, however, that the molecular brightness of the labeled molecule is carefully calibrated. Here, by taking dark states into account [1], FCS measurements, multi-component PCH and single molecule imaging of multimers of mNeonGreen (mNG) consistently validated that about 20% of the domains remained non-fluorescent after purification. These reference measurements proved to be of crucial use to characterize the oligomerization of FRB–FKBP fusion proteins [2] tagged with mNG in living cells. Depending on the protein concentration, we could monitor a monomer-to-tetramer transition of FRB–FKBP constructs and quantify the abundance of intermediate states by combined FCS and PCH analysis. Thus, our study successfully reconciles observations made with independent methods and showcases the feasibility to determine the stoichiometry of dynamic binding reactions in living cells.

Bernd Strehmel

Niederrhein University of Applied sciences, Department of Chemistry and Institute for Coatings and Surface Chemistry, Adlerstr. 1, D-47798 Krefeld, Germany

Different cyanines absorbing in the NIR between 750-930 nm were applied to study their fluorescence efficiency.  Variation of the connecting methine chain and structure of the terminal indolium moiety provided a deeper insight between the structure of the cyanine NIR-sensitizer and the efficiency to generate initiating radicals and conjugate acid. The connecting polymethine chain  either an open or bridged chain pattern resulting in different emission efficiencies. Photophysical studies were pursued by fluorescence spectroscopy providing a deeper understanding regarding the lifetime of the excited state and contribution of nonradiative deactivation resulting in generation of additional heat in the polymerization process. Decay time measuremnts were pursued with a FluoTime 300 and a respective integrating sphere from PICOQUANT to determine fluorescence quantum yields. These cyanines emitting in the NIR comprise as terminal groups different indolium groups that enhance either non-radiative deactivation or promote emission. This will give respective guidelines for their uses in material sciences as prospective fluorescent probes or photocatalyst. 

Stanimir Tashev¹, Johan Hummert¹, Robert Neely², Dirk-Peter Herten¹

¹Institute of Cardiovascular Sciences , College of Medical and Dental Sciences, Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
²School of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

The advances of detection systems and the development of more stable fluorophores have enabled single molecule quantification by fluorescence microscopy. However, most techniques are not live cell compatible, or they lack the temporal resolution to detect the dynamics of protein complexes. We seek to expand counting on photon statistics (CoPS) towards quantification of the assembly and disassembly of protein complexes in live cells. As a calibration-free counting technique with sub second time-resolution[1], CoPS is ideally suited for this purpose. We have validated the technique by using DNA origami standards with a known emitter number distribution and are now proceeding to validate dynamic counting on origami with associating and dissociating imager strands. The binding kinetics of which can be modified by the systematic variation of their length and sequence to outline the limits of dynamic CoPS. After dynamic counting by CoPS is established, we aim to study the kinetics of the central supramolecular activating cluster’s components during the activation of T-cells.

Roman Tsukanov¹, Christeena Mathew², Nazar Oleksiievets¹, Jan Christoph Thiele¹, Oleksii Nevskyi¹, Ingo Gregor¹, Jörg Enderlein^1,3

¹III. Institute of Physics -- Biophysics, Georg August University, 37077 Göttingen, Germany.
²Complex Systems Chemistry Graduate School, University of Strasbourg, France.
³Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), Georg August University, 37077 Göttingen, Germany

Super-resolution microscopy (SRM) has revolutionized our understanding of biological processes. Single-molecule localization-based microscopy (SMLM) has become increasingly popular due to the simplicity of implementation. Fluorescence Lifetime Microscopy (FLIM) provides an additional information, as compared to conventional intensity-based imaging. The synergy of SMLM and FLIM is extremely beneficial, as it combines superior resolution with a lifetime information. SMLM-FLIM can be performed by two complementary experimental approaches: confocal-laser scanning microscopy (CLSM) [1] and wide-field microscopy (WFM). The latter can be realized using extremely promising technology: electron multiplying micro-channel plates (MCP), as for example the novel commercially available lifetime camera LINCam (Photonscore) [2]. Despite a great potential of both SMLM-FLIM approaches, no direct comparison has been reported up to a date. Here, we perform systematic comparison between a confocal and a wide-field SMLM-FLIM imaging with single-molecule sensitivity of commonly used fluorophores in three spectral regions: blue, orange and far-red. In addition, we examine the performances of the two approaches for the metal-induced energy transfer (MIET) application and demonstrate DNA-PAINT imaging of fixed cells in particularly challenging for the lifetime camera far-red spectral region. Finally, we provide a practical advice on the SMLM-FLIM experiment design.

Bela Vogler¹, Francesco Reina^1,2, Christian Eggeling^1,2,3

¹Institute of applied Optics and Biophysics (IaOB), Philosophenweg 7, 07743 Jena
²Leibniz Institute of Photonic Technologies (Leibniz-IPHT), Albert-Einstein-Straße 9, 07745 Jena
³MRC Human Immunology Unit, Radcliffe Department of Medicine, University of Oxford

Understanding and retracing intracellular particle motion promises great insight on mechanisms of life. As these processes are diffusion driven and can be modeled quite well with respect to mathematical models, the study of diffusion dynamics and paths could deepen our knowledge of biological systems of interest. To this end, the recently developed MINFLUX technique delivers high-framerate and high-precision 2D & 3D single particle tracking.

We are working on a multi layered analysis program capable of identifying the diffusion modes of each detected trajectory. By combining the analysis of several trajectories of particles moving inside a set field-of-view, a diffusion mode map could be generated, highlighting areas of heterogeneous diffusion. Thus, a detailed understanding of the diffusion patterns of complex biological systems can be achieved.

For this purpose, we will combine conventional data analysis routines with recently developed machine-learning strategies for pattern recognition.

Therefore, we aim to develop a fully machine-learning based diffusion pattern recognition model for Single Particle Tracking and distribute it as a cloud service.

Zunhao Wang, Julia Molle, Stefan Wundrack, Rainer Stosch

Physikalisch-Technische Bundesanstalt (PTB), Bundesallee 100, 38116 Braunschweig, Germany

For surface-enhanced Raman spectroscopy (SERS) measurements at the single molecule detection limit,  high enhancement of Raman scattering is crucial. This is achieved by placing the analyte molecule in between two gold nanoparticles making use of the gap-effect – also called plasmonic hot-spot. However, in this instance, the Raman enhancement is highly dependent on the gap distance of these two gold nanoparticles. Here, we demonstrate the feasibility of SERS experiments at the single molecule detection limit using gold dimers with a DNA-origami controlled small gap size for the detection of single ATTO 633 molecules. Our results clearly prove the presence of ATTO 633 at ultra-low concentration ranges in the SERS experiments. Nevertheless, AFM analysis reveals high DNA-nanostructure density aggregation, thus, we expect that more than one ATTO 633 molecule contributes to the collected SERS signal. In future work, DNA-nanostructure aggregation will be avoided by spatially separating the “gold dimer origami structures” to finally ensure that not more than one of them is measured.

Sofia Zaer¹, Joanna Zamel¹, Jiaxing Chen², Nikolay Dokholyan², Eitan Lerner¹

¹Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Faculty of Mathematics & Science, The Edmond J., Safra Campus, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel
²Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA

Elucidating structural information of proteins is highly valuable and crucial for the accurate understanding of protein function. In the case of intrinsically disordered proteins (IDPs), however, it is challenging to obtain structural information using traditional structural biology tools. One such well-known IDP is α-Synuclein (α-Syn) that can gain helical structures when binding with membranes to support dopamine-containing vesicles,  nonetheless, it can  also change conformation, forming beta strand-based structures that induce self-association and forming oligomers and amyloid fibrils that known to be associated with Parkinson's disease (PD). single-molecule FRET (smFRET) studies, previously reported that the α-Syn monomer in solution exhibiting a single averaged-out sub-population of multiple conformations interconverting in hundreds of nanoseconds. While past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to test this protein using single-molecule Protein induced Fluorescence enhancement (smPIFE)^1-3, where we track the fluorescence lifetime of Cy3-labeled α-Syn proteins one at a time. Interestingly, using this shorter-range spectroscopic ruler, Cy3-labeled α-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. This work demonstrates the power of utilizing smPIFE to probe local structures and their dynamics in IDPs as a method complementary to smFRET^4,5.