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

Katrin Heinze

Julius-Maximilians-University Würzburg

G protein-coupled receptors (GPCRs) are the most commonly targeted transmembrane proteins by approved drugs. GPCRs undergo conformational changes upon binding with ligands, which allow them to carry out their biological functions within living cells. Several factors affect or modulate the functionality of GPCRs, including molecular diffusion, kinetics, and ligand-dependent oligomerization; however, the tools and time-resolved methods to capture the underlying dynamics are still limited. Particularly crucial is the required wide temporal range when studying GPCR conformational changes which is flanked by other challenges related to data sparsity and data noise. Here, a combined approach utilizing fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), and fluorescence lifetime imaging microscopy (FLIM) provided crucial information regarding GPCR conformational fluctuations and oligomerization. Despite the limited signal-to-noise levels in live cell experiments, this combined approach has allowed us to unravel previously elusive mobility constants and oligomeric states of GPCRs, which is crucial for refining our understanding of GPCR activation and signaling — a prerequisite for the development of more targeted therapies.

Sonja Schmid

Wageningen University, Stippeneng 4, 6708WE Wageningen,THE NETHERLANDS

Proteins are the molecular makers in our body. Researchers successfully identified a vast proteome, a dense web of metabolic interactions, and many thousands of static 3D structures. But the essential dynamic processes causing protein function are still challenging to detect – yet they are the key to the energetics controlling proteins. We therefore focus on acquiring time-resolved information to reveal the nanodynamics of biomolecular systems, such as a cancer-assisting chaperone, kinase proteins, and CRISPR-associated proteins.

In this talk, I will present our latest results from optical and electrical single-molecule experiments. FRET allows us to watch single proteins at work in real time [1,2,3], and with our DyeCycling approach [4], we can now overcome its notorious photobleaching limitation and detect previously hidden kinetic phenomena. In addition, we recently presented the NEOtrap 2.0, a label-free technique to monitor the time evolution of single unmodified proteins electrically, using nanopores [5,6]. Lastly, I will share how we revealed the hidden power of type-III CRISPR-Cas – one-by-one. Our overarching mission is to push beyond current detection limits, to learn how biomolecular function arises at the nanoscale.

Stanimir A. Tashev^1,2, Johan Hummert^1,2,6, Michael Scheckenbach³, J. Shepard Bryan IV⁴, Philip Tinnefeld³, Steve Presse⁴, Dirk-Peter Herten^1,2,5

¹Institute of Cardiovascular Sciences , University of Birmingham, Birmingham, UK
²Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and Nottingham, Midlands, UK
³Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, München, Germany
⁴Center for Biological Physics, Arizona State University, Phoenix, USA
⁵School of Chemistry, University of Birmingham, Birmingham, UK
⁶PicoQuant GmbH, Berlin, Germany

Advances in quantitative microscopy have made it possible to determine the stoichiometry of proteins inside complexes and receptor clusters. However, studies have mostly been done in fixed cells and model systems. The aim of this project is to bring protein counting methods to live cells, which with the inclusion of a temporal element this would also allow to study the kinetics of cluster formation. Two techniques are being developed one relying on the detection of fluorescence intensity state changes, and another based on Counting on Photon Statistics which is a photon antibunching technique^[1]. DNA origami with multiple binding sites for fluorophore-bound imager strands was used to mimic the binding and unbinding of proteins inside of clusters to establish these techniques. The kinetics of binding of this model system can be modified by systematic changes of the concentration and length of the imager strands, as well as the number of binding sites per origami^[2]. This will be used to determine the temporal resolution limits of the two techniques. Additionally, CoPS will need to be adapted for dynamic measurements. Together with Bayesian statistics and machine learning the kinetics of binding and the photophysical effects can be used to determine binding kinetics in live cells. We would then use inspect the recruitment of SLP76 to the cSMAC in T cells^[3]. With the development of SPAD array cameras we hope to merge these techniques in the future to increase throughput^[4].

Giovanni Ferrari¹, Alan M. Szalai¹, Lars Richter¹, Jakob Hartmann¹, Merve-Zeynep Kesici¹, Bosong Ji¹, Annika Jäger¹, Robert Hübsch¹, Andrés Vera Goméz¹, Ingrid Tessmer³, Izabela Kamińska^1,2, Philip Tinnefeld¹

¹Department Chemie and Center of Nanoscience (CeNS), Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 München, Germany
²Institute of Physical Chemistry of the Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
³Rudolf Virchow Center, University of Würzburg, Josef Schneider Str. 2, 97080 Würzburg, Germany

Single-molecule FRET (smFRET) is a powerful technique in dynamic structural biology, but its application has been limited by its requirement for double labelling, its small dynamic range, as well as photobleaching and complex acceptor photophysics. Furthermore, when investigating DNA bending, smFRET lacks accuracy as additional torsions and displacements of the DNA double helix can affect the donor-acceptor distance.

We introduce Graphene-Energy-Transfer with vertical Nucleic Acids (GETvNA) as a new tool to study DNA conformational changes and DNA-protein interactions with sub-nanometre spatial resolution. The method is based on our recent discovery that double-stranded DNA (dsDNA) with a single-stranded DNA overhang is immobilized perpendicularly on graphene, which also acts as an unbleachable broadband energy transfer acceptor for fluorescent dyes. We employ GETvNA to study both static and dynamic DNA  bending in the presence of bulges and enzymes such as Endonuclease IV, and show that it outperforms smFRET. Finally, we study the diffusion of AGT alkyltransferase along dsDNA, observing steps with single base spatial and milliseconds time resolutions on a standard confocal setup.

Hendrik Sielaff^1,2, Wilfried Engl^1,2, Aliz Kunstar-Thomas^1,2, Siyi Chen^1,2, Woei Shyuan Ng^1,2, Ziqing Winston Zhao^1,2,3

¹Department of Chemistry, Faculty of Science, National University of Singapore, Singapore 119543, Singapore
²Centre for BioImaging Sciences (CBIS), Faculty of Science, National University of Singapore, Singapore 117557, Singapore
³Mechanobiology Institute (MBI), National University of Singapore, Singapore 117411, Singapore

Chromatin remodeling, carried out by multi-subunit remodeler complexes, alleviates topological constraints posed by nucleosomes to regulate genome access. Although mutations in the SWI/SNF subfamily of remodelers are implicated in >20% of human cancers, how misregulation of their intranuclear dynamics could underpin cancer remains poorly understood. Hence, we characterized the intranuclear diffusion and DNA-binding dynamics of wildtype and mutant BRG1, the catalytic active subunit of the human SWI/SNF remodeler complex. By combining live-cell FCS and SMT, we revealed temporally distinct modes of free vs. chromatin-associated diffusion and mutant-specific chromatin binding signatures. Quantifying residence times of bound remodelers further resolved shorter- and longer-lived fractions, likely corresponding to non-specific and specific binding to DNA targets, respectively. Importantly, we showed that the enhancement of remodeler binding dynamics in a DNA-accessibility-dependent manner is modulated by the bromodomain. Moreover, super-resolution density maps constructed from SMT trajectories revealed heterogeneously distributed binding “hotspots""". In summary, our findings shed insight into the multi-modal landscape regulating the spatio-temporal organizational dynamics of SWI/SNF remodelers to selectively modulate genome accessibility.

Madhavi Krishnan

University of Oxford

The desire to trap atoms and molecules in experiment dates back over about 200 years to the diaries of Lichtenberg. From radio-frequency ion traps to optical tweezing of colloidal particles, methods to trap matter in free space or solution rely on the use of external fields that often strongly perturb the integrity of a macromolecule in solution. We recently demonstrated how the ‘electrostatic fluidic trap’ exploits equilibrium thermodynamics to realise stable, non-destructive confinement of a single macromolecule in a room temperature fluid, representing a paradigm shift in a nearly century-old field. The spatio-temporal dynamics of a trapped molecule reveals both its size and electrical charge, which we are now able to measure with very high precision. Our measurement approach not only enables a read out of molecular 3D structure and conformation in solution, but also enables binding affinity measurements and the detection of post-translational modifications in proteins. I will present our most recent advances in this emerging area and demonstrate how high-throughput single-molecule trapping maybe opening up a new path towards a generalized molecular analytics platform based on molecular size and charge measurement under native conditions.

Aleksandra Adamczyk¹, Teun A.P.M. Huijben², Miguel Sison³, Andrea di Luca⁴, Stefano Vanni⁴, Sophie Brasselet³, Kim Mortensen², Fernando D. Stefani⁵, Mauricio Pilo-Pais¹, Guillermo P. Acuna¹

¹Department of Physics, University of Fribourg, Fribourg CH-1700, Switzerland.
²Department of Health Technology, Technical University of Denmark, 2800 Lyngby, Denmark
³Institut Fresnel, Domaine Universitaire St Jerome, Cedex 20,13397 Marseille, France.
⁴Department of Biology, University of Fribourg, Fribourg CH-1700, Switzerland.
⁵Departamento de Física, Universidad de Buenos Aires, C1428EHA Buenos Aires, Argentina.

Over the last decade, DNA nanotechnology has been increasingly used to self-assemble functional nanostructures. One of the main advantages of this approach is that different species including colloidal nanoparticles and single photon emitters such as fluorophores can be positioned with nm precision and stoichiometric control¹. This has been exploited for a growing number of nanophotonic applications². While the relative distance between the hybrid species has been controlled up to the nanometer range, no control over the relative orientation has been exerted. We present a method to both position and orient single photon emitters within DNA origami constructs. In particular, we exploit the ability of DNA origami to exert forces in order to “stretch” covalently incorporated dyes and deterministically align them with the orientation of the double-stranded DNA helix they are located at (see figure 1). We study the dye ́s three-dimensional orientation and wobbling using three independent techniques: polarization-resolved excitation measurement, point-spread function (PSF) analysis³ and the four-polarization image splitting method⁴ combined with a super-resolution (nanoscopy) measurement using the DNA-PAINT technique⁵ to retrieve the orientation of the DNA origami “host” structure. Our results show that by simply removing a number of nucleotides adjacent to both ends of the doubly-linked fluorophore, the dye transitions from a non-predictable orientation given by a combination of external factors to an orientation aligned with the predesign direction of the host ds-DNA helix⁶. We believe this work shows a simple way to deterministically orient dyes which constitute the last degree of freedom required to manipulate the interaction of single photon emitters and fully control the coupling to other species.

Florian Steiner¹, Rute Fernandes¹, Ines Amersdorffer¹, Manuel Nutz^1,2, Michael Förg^1,2, Jonathan Noé^1,2, Thomas Hümmer^1,2

¹Ludwig-Maximilians-University Munich, Department of Physics, Schellingstrasse 4, 80799 Munich, Germany
²Qlibri GmbH, Maistrasse 67, 80337 Munich

Isolated nanoscale systems provide only weak interaction with light due to their small size and therefore often escape direct observation in conventional light microscopy. This limits insights into individual nanosystems and slows down research in the fields of nanotechnology, material science, drug design, and pharmaceutical diagnostics.

To overcome these limitations, our group continuously developed optical micro-resonators, a technology pioneered in quantum optics [1]. In these resonators, light passes a sample up to 100.000 times and thereby enhances weak absorption signals tremendously. By means of micro-cavities with a small mode waist a scanning microscopy approach, i.e. ultra-sensitive spatially resolved absorption measurements near the diffraction limit, can be performed [2]. By optimizing the mechanical stability and by developing integrated electronics, extinction cross section of 1 nm² can be imaged in real time.

The potential of this new type of microscopes is illustrated by imaging of individual carbon nanotubes [3], 2D-materials [4,5], and label free imaging of ultrathin biological sections [6] as a first step towards the label free absorption spectroscopy of single molecules.

Dragomir Milovanovic

German Center for Neurodegenerative Diseases (DZNE) at Charité Berlin, Berlin, Germany

Brain functioning critically relies on neuronal communication that mainly occurs by chemical signaling at the specialized contacts known as synapses. At synapses, messenger molecules are packed into synaptic vesicles (SVs), which are secreted upon the arrival of an action potential. Indeed, loss of SVs and synaptic deficits are associated number of neurodegenerative diseases. Hundreds of SVs accumulate at each synaptic bouton. Despite being held together, SVs are highly mobile, so that they can be recruited to the plasma membrane for their rapid release during neuronal activity. However, how such confinement of SVs corroborates with their motility remains unclear. To bridge this gap, we employ ultrafast single-molecule tracking (SMT) in the reconstituted system of native SVs and in living neurons. SVs and synapsin 1, the most highly abundant synaptic protein, form condensates with liquid-like properties. In these condensates, synapsin 1 movement is slowed in both at short (i.e., 60-nm) and long (i.e., several hundred-nm) ranges indicating that the SV-synapsin 1 interaction raises the overall packing of the condensate. Furthermore, two-color SMT and super-resolution imaging in living axons demonstrate that synapsin 1 drives the accumulation of SVs in boutons. Even the short intrinsically-disordered fragment of synapsin 1 was sufficient to restore the native SV motility pattern in synapsin triple knock-out animals. In the most recent line of experiments, we employ fluorescence correlation spectroscopy to quantify the diffusion properties of synapsin 1 and alpha-synuclein, another major synaptic protein implicated in pathology of Parkinson’s Disease. Here, the data indicates a strong effect of condensate-to-membrane interface in regulating the motion of molecules in the condensates. Thus, synapsin-driven condensation is sufficient to guarantee reliable confinement and motility of SVs, allowing for the formation of mesoscale domains of SVs at synapses and functional neurotransmission.

Victoria Birkedal

Department of Chemistry and iNANO center, Aarhus University, Denmark

Cellular DNA can adopt other structures than double stranded B-DNA, such as G-quadruplexes. The latter require guanine rich DNA found, for example, in telomeres and promoter regions. Formation of G-quadruplexes can play regulatory roles and provide interesting structural handles for drug targeting. RecQ helicases unwind a variety of substrates and have affinity for G-quadruplex structures, that must be resolved to avoid unsuccessful DNA replication. Using single molecule fluorescence tools and a range of biophysical and biochemical assays, we have investigated DNA folding, intrinsic conformational dynamics and unfolding by the Werner RecQ helicase of different G-quadruplex motifs. Overall, we find that the intrinsic G-quadruplex folding/unfolding dynamics is an important factor for RecQ helicases unwinding of these structures.

David Li

University of Strathclyde, Glasgow, United Kingdom

Fluorescence lifetime imaging is a powerful tool to reveal biomolecular interactions that are related to disease progression or treatment. However, most live-cell or in-vivo imaging applications require real-time acquisition and processing. In this talk, we will present a deep-learning approach to speed-up FLIM acquisition with a few photons.

Johan Hummert¹, Max Tillmann¹, Felix Koberling¹, Tino Roehlicke¹, Michael Wahl¹, Ivan Michel Antolovic², Cyril Saudan²,Harald Homulle², Rainer Erdmann¹

¹PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany
²Pi Imaging Technology SA, EPFL Innovation Park, 1015 Lausanne, Switzerland

Confocal microscopy is an essential tool in many academic disciplines due to its intrinsic sectioning capability. The combination with time-resolved single photon detectors and time-correlated single photon counting (TCSPC) devices has established it as the leading platform for time resolved investigation methods such as fluorescence lifetime imaging (FLIM). Recently, high-performance SPAD-arrays featuring few tens of pixels have become available.

In this work we present the two central hardware building blocks: PicoQuant’s latest multi-channel TCSPC device and a cooled high-performance 23-pixel SPAD-array developed jointly with Pi Imaging Technologies. We discuss how these open up new possibilities in time-resolved confocal microscopy.

Johannes Broichhagen

Leibniz-Forschungsinstitut für, Molekulare Pharmakologie (FMP), Robert-Roessle-Str. 10, 13125 Berlin, Germany

Labelling, visualization, and functional manipulation of biomolecules is at the forefront of chemical biology.^[1,2] However, selective and quantitative interrogation and analysis of biomolecules remains a challenge in the field. We employ approaches from molecular biology and fluorophore design to tackle such issues with unconventional strategies for super-resolution imaging in living cells. In one of our latest studies, we installed carbon-deuterium bonds in fluorophores to yield dyes with increased fluorescent lifetimes, higher photostability, and enhanced brightness.^[3] With this in hand, we focus on the glucagon-like peptide 1 receptor (GLP1R), a class B GPCR that is involved in glucose homeostasis and satiety and a blockbuster target to treat patients suffering from type 2 diabetes. We highlight GLP1R in its endogenous context with fluorescently labelled antagonists, the LUXendins, allowing super resolution imaging, 2-photon imaging, single particle tracking and intravital microscopy^[4,5] We next genetically engineered an enzyme self-label onto GLP1R to interrogate its localization and behavior in its native context on the endogenous level.^[6] This allows the tracking of GLP1R in complex tissue settings treated with different type of drugs. Taken together, we aim to use chemistry as a flashlight to shine a spotlight on the invisible in biological systems.

Haggai Shapira¹, Breveruos Sheheade¹, Samrat Basak², Eyal Nir¹

¹Department of Chemistry and Ilse Katz Institute for Nanoscale Science & Technology, Ben Gurion University of the Negev, Beer Sheva, Israel
²Third Institute of Physics, Biophysics, Georg-August-Universität, Göttingen, Germany

Inspired by the fast and processive biological F1-ATPase, we developed a high-performance DNA origami rotary motor operated by a microfluidic device¹ and monitored by single-molecule defocused orientation-imaging of gold nanorods². The microfluidic device enables computer-controlled fast and reliable introduction and removal of the DNA strands that facilitate the chemical reactions that power the motor. The bright light scattered from the gold nanorod, free of bleaching or blinking, allows analysis of the motor’s 3D orientation with high angular and temporal resolutions over days. The rotary motor consists of two origami disks that are connected via a single-stranded swivel that allows free rotation but prevents dissociation and is powered by two sets of bipedal walkers that stride on two asymmetric sets of 6 footholds organized in a circular fashion. We assembled the motor and introduced it to the microfluidic device and characterized its Brownian rotation and response to DNA fuels and anti-fuels. The motor successfully performed several revolutions consisting of dozens of steps, during which it displayed an ability to recover from chemical errors. In future work we will try to minimize these chemical errors, with the aim of achieving dozens of revolutions at high speed.

Alexandre Fürstenberg, Jimmy Maillard, Liza Briant, Ekaterina Bestsennaia

Department of Physical Chemistry and Department of Inorganic and Analytical Chemistry, University of Geneva, Geneva, Switzerland

Most biological processes take place in compartmentalized aqueous environments. Nonetheless, water diffusion across membranes and its dynamics in response to changes in osmolarity, membrane composition, membrane tension, or membrane protein conformation is not understood in detail. We have recently shown that some oxazine fluorophores are selectively sensitive to water and can quantitatively report on the number of water molecules in their contact sphere by measurement of their fluorescence lifetime,[1, 2] while mechanosensitive flipper probes displaying ground-state planarization are designed to specifically sense variations in membrane tension and are compatible with single-molecule imaging.[3] We report in this contribution how such quantitative fluorescent probes can be used to investigate local hydration, membrane tension, and its dynamics at the nanometer scale using fluorescence spectroscopy, fluorescence lifetime imaging, and super-resolution microscopy.

Ralf Jungmann

LMU Munich, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

I will discuss our group’s vision of converting standard off-the-shelf fluorescence microscopy hardware into a tool for spatial omics.

To enable this, I will first introduce technical advancements in DNA-PAINT including approaches that achieve sub-nanometer spatial resolution and spectrally unlimited multiplexing in whole cells. I will then discuss protein labeling probes such as Slow Off-rate Modified Aptamers (SOMAmers) that could allow DNA-barcoded labeling of much of the proteome in a cellular environment. Next, I present approaches to increase DNA-PAINT’s traditionally slow imaging speeds to achieve throughputs necessary for network-wide molecular interrogation. To this end, we employed rationale DNA sequence design and demonstrate 100-times faster imaging without compromising quality or resolution. Finally, I will present cell surface receptor quantification at thus far elusive resolutions and levels of multiplexing, which could yield insights into the molecular architecture of receptor interactions and enable the future development of “pattern”-based therapeutics.

Gregor J. Gentsch¹, Bela T.L. Vogler^1,2, Pablo Carravilla^1,2,3, Dominic A. Helmerich⁴, Teresa Klein⁴, Katharina Reglinski^1,2, Markus Sauer^4,5, Christian Eggeling^1,2,6, Christian Franke^1,6,7

¹Faculty of Physics and Astronomy, Institute of Applied Optics and Biophysics, Friedrich Schiller University Jena, Jena, Germany
²Leibniz Institute of Photonic Technology e.V., Jena, Germany, member of the Leibniz Centre for Photonics in Infection Research (LPI), Jena, Germany
³Present address: Science for Life Laboratory, Department of Women’s and Children’s Health, Karolinska Institutet, Solna, Sweden
⁴Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, Würzburg, Germany
⁵Rudolf Virchow Center, Research Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
⁶Jena Center for Soft Matter, Friedrich Schiller University Jena, Jena, Germany
⁷Abbe Center of Photonics, Friedrich Schiller University Jena, Jena, Germany

We introduce a set of novel multiplexing approaches using organelle-specific nanotextures called NanTex for monochromatic, super-resolved image data. Firstly, we demonstrate ad-hoc multiplexing of spectrally identical labeled organelles in single SMLM images based on nanoscale Haralick-feature [1] extraction. Next, we introduce AI-enabled textural demixing, AI-NanTex with supervised Unet [2] feature generation, trained on publicly available (Shareloc [3]) and dedicated experimental single-organelle SMLM data. AI-NanTex enables the regressive extraction of multiple complex structures (e.g., microtubules, clathrin, endosomes, ER, and actin) from single-channel grayscale SMLM images. This context-agnostic texture recognition relies on probabilistic demixing rather than classical image segmentation, accurately identifying multiple organelles even when heavily overlapped. Furthermore, our method is readily applicable to monochromatic, multi-organelle MINFLUX [4] data without requiring additional training, showcasing the broad relevance of the nanotextural concept across different super-resolution imaging modalities. Texture-sensitive nanoscopy expands the possibilities of multi-color imaging by enabling straightforward multiplexing of the best-performing dyes in complex biological contexts.

Niels Radmacher¹, Oleksii Nevskyi¹, José Ignacio Gallea¹, Jan Christoph Thiele³, Ingo Gregor¹, Jörg Enderlein^1,2

¹Third Institute of Physics - Biophysics, Georg August University
²Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells'' (MBExC), Universitätsmedizin Göttingen
³Department of Chemistry, University of Oxford

In recent years, Image Scanning Microscopy (ISM) has emerged as a powerful technique for achieving super-resolution bio-imaging across various applications. Particularly noteworthy is the implementation of a single-photon detector array, enabling the utilization of Lifetime Image Scanning Microscopy, which has proven to be highly effective. In our study, we present a novel approach that combines ISM with direct Stochastic Optical Reconstruction Microscopy (dSTORM), resulting in a doubling of the localization precision in Single Molecule Localization Microscopy (SMLM). Additionally, we capitalize on the valuable lifetime information provided by ISM, allowing for multilabel fluorescence measurements without the detrimental effects of chromatic aberration, even at resolutions significantly surpassing the diffraction limit.
Moreover, we introduce a freely available add-on to previously employed open-source tools for single particle tracking and localization, enhancing the accessibility and utility of our methodology. This add-on serves as a valuable resource for the research community, facilitating the adoption and further advancement of the combined ISM and dSTORM technique.

Susanne C. M. Reinhardt^1,2, Luciano A. Masullo¹, Isabelle Baudrexel^1,3, Philipp R. Steen^1,2, Rafal Kowalewski^1,2, Alexandra S. Eklund^1,3, Sebastian Strauss^1,2, Eduard M. Unterauer^1,2, Thomas Schlichthaerle^1,2, Maximilian T. Strauss^1,2, Christian Klein^3,4, Ralf Jungmann^1,2

¹Max Planck Institute of Biochemistry, Planegg, Germany
²Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany
³Department of Chemistry and Biochemistry, Ludwig Maximilian University, Munich, Germany
⁴Roche Innovation Center Zurich, Roche Pharma Research and Early Development, Schlieren, Switzerland

Super-resolution fluorescence microscopy routinely reaches 15-20 nm resolution in intact cells [1]. Recent approaches achieved localization precisions in the Ångström regime and - under in vitro conditions - resolved targets spaced 5 nm apart [2,3,4]. However, Ångström resolution with optical microscopy has never been demonstrated in vitro or in cells. Thus, the study of direct molecular interactions at sub-10 nm distances has been inaccessible even with super-resolution microscopy.

We introduce Resolution Enhancement by Sequential Imaging (RESI), a DNA-barcoding method to expand the resolution of fluorescence microscopy down to the Ångström scale using a conventional inverted microscope resulting in label-size-limited resolution for molecules in whole intact cells.

We demonstrate the routine visualization of individual Nup96 proteins in nuclear pore complexes at a localization precision of 1 nm. For the first time in optical microscopy, we resolve Nup96 pairs at 11.9 nm in xy and 5.4 nm in z direction in a model free average. Importantly the acquisition of the 67 μm x 67 μm field of view was conducted in 100 min, thus making RESI applicable as a sufficiently high-throughput tool for cell biology at localization precisions of ca. 1 nm.

Finally, we test the ultimate resolution capability of RESI by measuring the backbone distance of single bases in DNA origami. The achieved average precision of 1.3 Å allows us to measure their distance at 9.5 ± 2.6 Å, validating Ångström resolution via the most fundamental and strict definition as the ability to spatially distinguish point objects.

Philipp R. Steen, Eduard M. Unterauer, Luciano A. Masullo, Ralf Jungmann

Max Planck Institute of Biochemistry

DNA-PAINT [1] is a super-resolution technique enabling sequentially multiplexed, nanometer-resolution studies of previously inaccessible biological systems [2,3]. Recent advances have yielded faster acquisition times via optimized DNA sequences [4] and even sub-nm resolutions [5]. However, a systematic investigation of (1) which fluorescent dyes and buffers to use for best performance as well as (2) which combinations facilitate optimal spectral multiplexing are currently missing. Furthermore, there are no clearly defined standards as to what parameters comprise optimal performance in the first place.

To resolve these questions, we first designed a robust, controlled and reproducible dye evaluation platform based on DNA origami and sequential imaging to extract relevant, quantitative parameters such as resolution, binding kinetics and stability for commonly used fluorescent dyes and various buffer conditions. The platform utilizes the specificity of DNA sequences and the controllability of DNA origami to facilitate analysis of individual dyes. Next, we examined dye performance in a cellular environment by imaging NUP96 in the nuclear pore complex. We thus present state-of-the-art DNA-PAINT imaging standards. Finally, we employed the optimal trio of dyes in a custom-built three-color TIRF microscope to achieve six-plex imaging using one imager exchange with three simultaneous acquisitions per imaging round.

Overall, we provide a set of experiments and analysis pipelines that make it possible to quantitatively benchmark different fluorophores for DNA-PAINT and recommend the best performing dyes available. Our method is also extensible to quantitatively and systematically test other relevant parameters of DNA-PAINT experiments such as DNA sequences, any imaging buffers and illumination/detections schemes.

Scott Blanchard

St. Jude Children’s Research Hospital, Memphis, USA

The function and regulation of biological systems is often driven by time-dependent changes in biomolecular interactions and conformations. Ensemble and single-molecule methods have shown, for instance, that protein synthesis is a multistep process whose rate and fidelity is fundamentally defined by time-dependent compositional and conformational changes in the two-subunit ribosome and the factors it transiently interacts with during function. Our team seeks to employ single-molecule fluorescence resonance energy transfer (smFRET) methods to establish robust kinetic and structural models of the protein synthesis mechanism to understand how cellular regulation is achieved and pharmacological regulation may be possible. Progress and challenges associated with these pursuits will be discussed.

Soohyen Jang^1,2, Kaarjel K. Narayanasamy^1,3, Johanna V. Rahm¹, Alon Saguy⁴, Julian Kompa⁵, Marina S. Dietz¹, Kai Johnsson⁵, Yoav Shechtman⁴, Mike Heilemann^1,2

¹Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Max-von-Laue-Str.7, 60438, Frankfurt am Main, Germany
²Institute of Physical and Theoretical Chemistry, IMPRS on Cellular Biophysics, Johann Wolfgang Goethe-University, Max-von-Laue-Str.7, 60438, Frankfurt am Main, Germany
³Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany
⁴Department of Biomedical Engineering, Technion – Israel Institute of Technology, Haifa, 3200003, Israel
⁵Department of Chemical Biology, Max Planck Institute for Medical Research, Jahnstr. 29, 69120 Heidelberg, Germany

Single-molecule localization microscopy (SMLM) achieves nanometer spatial resolution by localizing single fluorophores separated in space and time. A major challenge of SMLM are long acquisition times, leading to low throughput, as well as to a poor temporal resolution that limits its use to visualize the dynamics of cellular structures in live cells. Another challenge is photobleaching, which reduces information density over time and limits throughput and the available observation time in live-cell applications. To address both challenges, we combine two concepts: first, we integrate the neural network DeepSTORM to predict super-resolution images from high-density imaging data, which increases acquisition speed. Second, we employ a direct protein label, HaloTag7, in combination with exchangeable ligands (xHTLs) for fluorescence labeling. This labeling method bypasses photobleaching by providing a constant signal over time and is compatible with live-cell imaging. The combination of both a neural network and a weak-affinity protein label reduced the acquisition time up to ~25-fold. Furthermore, we demonstrate live-cell imaging with increased temporal resolution, and capture the dynamics of the endoplasmic reticulum over extended time without signal loss. 

Eitan Lerner, Yair Razvag, Paz Drori

The department 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, ISRAEL

Super-resolution light microscopy techniques paved the way to observe biomolecules in the cell. Attaining super-resolved information requires either using fluorescent dyes flickering between bright and dark states, immediate depletion of a well-defined region of dye excited states or using the knowledge of the illumination profile to track single biomolecules. We developed a technique dubbed FRET-sensitized acceptor emission localization (FRETsael), in which we gain nanometer localization accuracy of biomolecular interactions in FRET-FLIM. We show that the localization accuracy is 20-30 nm for all true-positive detections no matter what are the underlying experimental conditions. We also report the dynamic range in which the false discovery rate is minimal, and the true positive rate is maximal. Furthermore, we show the performance of the algorithm on more realistic simulations of Actin-Vinculin and ER-Ribosomes pairs of interactions. Finally, we explore the performance of the FRETsael approach on cells with the nuclear pore protein Nup96. The FRETsael imaging approach paves the way towards studying biomolecular interactions with improved spatial resolution from alternating laser excitation scanned frames in confocal microscopy without the use of blinking dyes or special optics.

Eva Schmid

Universitätsstraße 31, 93053 Regensburg, Deutschland

To prove the quantum nature of reactions involving radical pairs (RP) one can look at spin quantum beats. However, this beating between singlet and triplet manifold cannot be measured directly by fluorescence, because it takes place in a charge-separated state (CSS), which does not emit light.

However Mims et al. developed an optical readout technique involving a push pulse, which excites the CSS and transfers it into the S₁ or T₁ state, respectively. The time delay between the push and excitation pulse controls, whether the CSS is excited predominantly in its singlet or triplet manifold, which is subsequently converted to either delayed fluorescence or a triplet state, i.e. dark state. These measurements were done on a molecular dyad comprising a triarylamine electron donor (TAA), perylene diimide acceptor (PDI), and dihydroanthracene (An) bridge in solution. [1]

We will demonstrate that this measurement is feasible on the single molecule level by measuring the push pulse induced delayed fluorescence by TCSPC and the formation of T₁-states by FCS. Further, we show that the spin state can be influenced by an external magnetic field. This pump-push scheme holds potential for detecting resonantly driven transitions between the singlet and triplet states on a single molecule.

Bidyut Sarkar¹, Kunihiko Ishii^1,2, Tahei Tahara^1,2

¹Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan
²Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics, 2-1 Hirosawa, Wako 351-0198, Japan

Two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), ^1-4 developed in our group, is a powerful method to study biomolecular structural dynamics with high time resolution. 2D FLCS distinguishes the conformers of a biomolecule based on their fluorescence lifetimes and enables us to investigate their interconversion dynamics with a microsecond time resolution. Notably, this time resolution is two-three orders of magnitude higher than the conventional single-molecule Förster resonance energy transfer (smFRET) methods. In the first part of my talk, I report an application of 2D FLCS to study prequeuosine (preQ₁) riboswitch,⁵ a therapeutically important noncoding RNA that regulates transcription in bacteria. By investigating the ligand-binding aptamer domain of this riboswitch, we observe that the aptamer domain undergoes three-state folding-unfolding dynamics in a wide time range from a microsecond to >10 milliseconds. We also observe that the ligand, preQ₁, binds to the aptamer domain with the induced-fit mechanism and accelerates its microsecond dynamics. Based on these results, we determine the folding energy landscape of the aptamer domain and propose a molecular mechanism of transcription regulation by the riboswitch through microsecond structural change of the aptamer domain and its acceleration upon ligand binding. ⁶ In the second part, I introduce a new extension of 2D FLCS, which significantly improves its accuracy for FRET-labeled samples by implementing pulsed interleaved excitation (PIE),⁷ i.e., alternate excitation of the donor and acceptor using two temporally interleaved pulses of separate colors. The major advantage of using PIE is to clearly distinguish FRET-labeled species from single dye-labeled species, which are typically present as impurities in FRET samples. Because 2D FLCS based on a single color excitation utilizes only donor fluorescence lifetime^1-3 (with donor-excited acceptor fluorescence intensity in some cases), ^4,6 it cannot distinguish a very lowor zero-FRET species from only donor-labeled molecules. PIE 2D FLCS overcomes this problem by utilizing two different color pulses for exciting the donor and acceptor. Based on the acceptorexcited acceptor fluorescence signal, we can readily distinguish a FRET-labeled species from only donor-labeled species. In addition, PIE 2D FLCS also detects only acceptor-labeled molecules which are invisible in 2D FLCS experiments exciting only the donor.⁸

Julius Trautmann, Christian Eggeling

Institute of Applied Optics and Biophysics (IAOB), Zentrum für Angewandte Forschung, Philosophenweg 7, 07743 Jena (Germany)

Fluorescence microscopy is a powerful imaging technique that plays a vital role in various fields of science. Special techniques such as Fluorescence Correlation Spectroscopy (FCS) can provide valuable information about the dynamics and interactions of molecules. In biological applications such investigations often require deep penetration into samples, leading to deteriorated microscopy data due to optical aberrations. In recent years adaptive optics have become a remedy. Active optical elements such as deformable mirrors (DMs) and spatial light modulators (SLMs) are employed to dynamically correct for aberrations.

We here present our investigations on the influence of aberrations on fluorescence correlation spectroscopy measurements where we have deliberately induced different aberrations using a deformable mirror in a confocal microscope with single molecule detection.

Sara Illodo^1,2, Flor Rodríguez-Prieto¹, Wajih Al-Soufi², Mercedes Novo²

¹Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Departamento de Química Física, Universidade de Santiago de Compostela, E-15782 Santiago de Compostela, Spain
²Departamento de Química Física, Facultade de Ciencias, Campus Terra, Universidade de Santiago de Compostela, E-27002 Lugo, Spain

β-amyloid studies have been increasing since its relationship with neurodegenerative diseases such as Alzheimer's disease (AD) was first reported. To this day, both tau protein and β-amyloids (AB) are believed to be responsible for the appearance of said illness.¹

It is known that AB aggregation leads to fibrillation and that those fibrils are found on AD patients. However, literature suggests that the neurotoxic species are the oligomers formed on the early stages of aggregation.² Thus, a characterization of these species is of great importance.

Most reported studies on AB aggregation focused on β-amyloid 1-42 (AB42) as it was thought to have greater impact on AD, due to its higher hydrophobicity and higher aggregation tendency.² However, β-amyloid 1-40 (AB40) represents the 80-90% of all AB in our organism.² Additionally, recent studies found that there are significant differences between homogeneity and aggregation of AB42 and AB40,^3,4 evidencing the need to study AB40 aggregation as well.

Our group studied AB42’s early aggregation using Fluorescence Correlation Spectroscopy (FCS), which allowed us to determine the critical aggregation concentration and to characterize in shape and size the oligomers.^5,6 In this work, we study the early aggregation of AB40 and analyze the discrepancies found in the literature.^4,7

Tao Chen¹, Jörg Enderlein^1,2

¹Drittes Physikalisches Institut - Biophysik, Georg August University, Göttingen, Germany
²Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), Georg August University, Göttingen, Germany

Out-of-plane fluctuations, also referred to as stochastic displacements, are crucial for regulating essential biological processes within cells and organelles. However, accurately quantifying the dynamics of complex membrane systems, such as mitochondria, with rapid and subtle fluctuations poses a significant challenge. Here, we present a novel methodology that combines metal/graphene-induced energy transfer (MIET/GIET) with fluorescence correlation spectroscopy (FCS) to accurately quantify out-of-plane fluctuations of membranes. This approach provides simultaneous spatiotemporal resolution at unprecedented scales of approximately one nanometer and one microsecond. To validate the technique and assess its spatiotemporal resolution, we measured bending undulations of model membranes. Furthermore, we demonstrate the versatility and applicability of MIET/GIET-FCS in studying diverse membrane systems. This includes the investigation of widely studied fluctuating membrane systems, such as human red blood cell membranes, as well as previously unexplored membrane systems characterized by tiny fluctuations, such as pore-spanning membranes and mitochondrial inner/outer membranes.

Moreover, we expand on the capabilities of MIET/GIET by utilizing it to investigate various membrane biophysics phenomena. This includes the quantification of fluctuations in the endoplasmic reticulum (ER) membrane and plasma membrane, analysis of leaflet-specific diffusion, and revealing the transmembrane dynamics of different molecules within the phospholipid bilayer membrane. Overall, our study demonstrates the potential of MIET/GIET-FCS as a powerful tool for understanding membrane dynamics and investigating a wide range of membrane biophysics phenomena in various biological systems.

Sudipta Maiti

Tata Institute of Fundamental Research, Mumbai, India

Many human diseases have molecular origins. Alzheimer’s and Type-II diabetes are associated with protein misfolding and aggregation, while depression and drug addiction are associated with signalling molecules in the brain. A disease with a molecular origin should in principle be understandable in terms of the basic physical chemistry of the molecule, though in practice most of these diseases have remained poorly understood. Over the years, we have developed various biophotonic tools, spanning from single-molecule fluorescence to cellular imaging, which have allowed us to solve some of the most intricate puzzles in this area. New single molecule methodologies have enabled us to tease apart the roles of individual types of oligomers, which had so far remained a big challenge in understanding the toxic pathway in amyloid diseases. I will talk about our recent discoveries regarding how Alzheimer’s amyloid beta changes its shape to become toxic, how the interaction of signalling molecules modulates the mechanical properties of lipid membranes,  and how these two may be connected.

Thorben Cordes

Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in vivo. We recently performed an international blind study in collaboration with the Seidel and Lamb labs[1] to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. While this provided confidence in the use of smFRET for both mechanistic biochemical studies and structural biology, the design of smFRET assays and the selection of suitable labelling positions remains rather unsystematic. There are sensible 'rules of thumb' for identification of fluorophore labelling-sites, however, there is no approach that allows systematic and quantitative prediction thereof in proteins. Based on a large literature screen and bioinformatics analysis, we identified a set of four parameters, which we combined into a label score to rank residues for their suitability to serve as label site. We show the predictive power of the score with literature data and new experiments. The 'labelizer' package performs an analysis of a pdb-structure (or structural models), label score calculation, and FRET assay scoring in a script or via publicly available webserver (https://labelizer.bio.lmu.de/) to conveniently apply our approach.[2]

Freja Frederikke Pinderup^1,2, Daniel Gudnason^1,2, Mikael Madsen^1,2, Rikke Hansen^1,2, Kurt V. Gothelf^1,2, Victoria Birkedal^1,2

¹iNANO center, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark
²Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark

Conjugated polymers are a multi-chromophoric material with interesting light absorption, emission, energy transfer and light harvesting properties. They are intrinsically highly heterogeneous, which makes single molecule fluorescence a powerful tool for their study. To obtain further morphological control, polymers of the type poly-phenylene-vinylene (PPV) were functionalized with short DNA strands (9 bp and 15 bp) to allow their attachment to DNA origami platforms [1].

Probing the fluorescence of PPV polymers on individual DNA origami platforms allowed uncovering polymer aggregate heterogeneity as a response to either mono- or divalent ions and suppressing aggregation by constraining the polymers through control of attachment points on the DNA origami.

Furthermore, we investigated light harvesting properties by incorporating one or multiple fluorophores, with absorption spectrally overlapping polymer emission, onto the DNA origami platform close to the polymer. Excitation through the polymer made the fluorophore much brighter compared to direct excitation. Observations were quantified by the antenna effect. Our approach allows light harvesting studies with exquisite control of polymer aggregation and distance between polymer and fluorophores.

Susann Zelger-Paulus, Besim Fazliji, Mélodie C.A.S. Hadzic, Richard Börner, Roland K.O. Sigel

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

"Seeing is believing": imaging biomolecules has revolutionized the study of various biological processes, e.g., binding interactions or folding pathways. Here, we focus on the visualization of the folding and splicing of a catalytic active RNA involved in precursor mRNA maturation, specifically the group II intron Sc.ai5γ derived from S. cerevisiae. 

This RNA undergoes a complex multi-step folding, leading to a well-defined three-dimensional structure while catalyzing self-cleavage. We aim to comprehensively understand the folding and splicing mechanisms by tracking dynamics via distance-dependent Förster Resonance Energy Transfer (FRET) on a single-molecule level. Therefore, we employ different RNA labeling techniques such as covalent end labeling or using hybridization probes¹. 

We encapsulate the RNA into surface-immobilized vehicles, enabling total internal reflection (TIR) microscopy². This approach allows us to look at freely diffusing RNA molecules and show the influence of substrate, protein cofactors, or Mg²⁺ ions on RNA folding³. Our research uncovers the molecular mechanisms underlying the interplay between folding and splicing of the Sc.ai5γ group II intron, providing valuable insights into RNA-mediated catalysis and pre-mRNA maturation. 

Paul Wiseman

McGill University, 801 Sherbrooke St. West, Montreal H3A 0B8, Canada

I will introduce k-space image correlation spectroscopy (kICS) as a fluorescence fluctuation method applicable to standard fluorescence and super-resolution images. I will explain how kICS analysis can separate correlations from time dependent photophysical processes and space-time dependent transport processes and illustrate with application of kICS to simultaneously measure the diffusion and blinking of dronpa labeled actin in live cells. In the second part, I will discuss using STED super-resolution imaging to visualize how NADPH oxidase homolog dual oxidase 1 (DUOX1) and carbonic anhydrase 12 (CA XII) are organized in the apical membranes of HBE cells, revealing their localization in ring domains at the base of motile cilia, identified with the ciliary marker centrin 2 (CEN2). DUOX1 is a vital component of the hydrogen peroxide/lactoperoxidase antimicrobial system, a major bacterial killing mechanism in the airways, while CA XII is heavily involved in maintaining the pH level needed for effective antimicrobial response in the airway lumen. I will show characterization of the size of these ring domains with image correlation analysis in k-space (spatial frequency), presenting a new approach for evaluating the size of circular clusters in super-resolution images.

Marius Glogger¹, Dongni Wang¹, Julian Kompa², Ashwin Balakrishnan¹, Julien Hiblot², Hans-Dieter Barth¹, Kai Johnsson^2,3, Mike Heilemann¹

¹Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany
²Department of Chemical Biology, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany
³Institute of Chemical Sciences and Engineering (ISIC), École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland

Stimulated emission depletion (STED) microscopy is capable of imaging cellular structures and providing structural information at the nanometre scale. A necessity of the technique is the need to balance excitation and emission spectra of the fluorophores used, thereby limiting the number of observable targets of interest in a cell. In our work, we overcome this “spectral barrier” by utilising exchangeable labels instead of covalent labels with distinct spectral properties. This theoretically allows imaging an unlimited number of proteins in tandem. We show how different labelling strategies can be combined based on the utilisation of exchangeable ligands (DNA-PAINT, PAINT, HaloTag), thereby increasing the multiplexing capacity of STED microscopy, letting us image multiple structures in live and fixed cells¹.

Steffen J. Sahl¹, Jessica Matthias^2,7, Kaushik Inamdar^3,4, Taukeer A. Khan¹, Michael Weber¹, Stefan Becker⁵, Christian Griesinger⁵, Johannes Broichhagen^6,8, Stefan W. Hell^1,2

¹Max Planck Institute for Multidisciplinary Sciences, Department of NanoBiophotonics, 37077 Göttingen, Germany
²Max Planck Institute for Medical Research, Department of Optical Nanoscopy, 69120 Heidelberg, Germany
³Max Planck Institute for Multidisciplinary Sciences, Research Group Structure and Dynamics of Mitochondria, 37077 Göttingen, Germany
⁴University Medical Center Göttingen, Department of Neurology, 37075 Göttingen, Germany
⁵Max Planck Institute for Multidisciplinary Sciences, Department of NMR-based Structural Biology, 37077 Göttingen, Germany
⁶Max Planck Institute for Medical Research, Department of Chemical Biology, 69120 Heidelberg, Germany
⁷Present address: Abberior Instruments America, Bethesda, MD 20814, USA
⁸Present address: Leibniz-Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin, Germany

Estimations of molecular distances <10 nm by optical means have long been the almost exclusive domain of Förster resonance energy transfer (FRET) methods [1,2]. By site-specifically labeling molecular subunits with at least two fluorophores, single-molecule FRET [3-5] has also allowed for the monitoring of distances within macromolecules. We present the direct determination of such intra-molecular distances based on the MINFLUX localization concept [6]. We measured intra-macromolecular spacings of photoactivatable small-molecule fluorophores in well-characterized molecular systems, with simple polypeptides and proteins serving as initial examples. Our experiments indicate that the widely employed dark-state photoblinking of cyanines reaches an impasse for small dye separations, as the dyes couple and no longer behave independently. The complex photophysics of such dye-dye communication [7] impedes the reliable, isolated detection of their signals. As we show, sequential photoactivation - providing the required fluorophore state transitions in a more orthogonal way - is a viable strategy to read out dye coordinates even for smallest separations, beyond any perceived remaining barriers of resolution. Our results [8], with direct optical resolution of intra-molecular distances and conformations, show that it is possible to perform fluorescence-based analyses of structural biology at room or physiological temperature relying on direct positional measurements rather than on indirect FRET readouts.

Marina Santana-Vega¹, Carlos J. Bueno-Alejo^2,3, Andrea Taladriz-Sénder⁴, Amanda K. Chaplin^3,5, Chloe Farrow^2,3, Alexander Axer⁴, Hesna Kara^3,5, Vasileos Paschalis^3,5, Sumera Tubasum^3,5, Zhengyun Zhao⁴, Cyril Dominguez^3,5, Ian C. Eperon^3,5, Andrew J. Hudson^2,3, Alasdair W. Clark¹

¹James Watt School of Engineering, University of Glasgow, Advanced Research Centre, Glasgow, G11 6EW, United Kingdom
²School of Chemistry, University of Leicester, University Road, Leicester, LE1 7RH, United Kingdom
³Leicester Institute of Structural & Chemical Biology, Henry Wellcome Building, University of Leicester, Lancaster Road, Leicester, LE1 7HB, United Kingdom
⁴WestCHEM & Department of Pure & Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, United Kingdom
⁵5Department of Molecular and Cellular Biology, Henry Wellcome Building, University of Leicester, Lancaster Road, Leicester, LE1 7HB, United Kingdom

One of the current limitations in single-molecule imaging is the lack of substrates capable of eliminating non-specific binding at high concentrations. This prevents the real-time observation of biomachinery processes with dissociation constants above the nanomolar range. Here, we demonstrate a new strategy which overcomes this issue using the fluorous effect. The fluorous effect describes the tendency of perfluorinated phases to favour fluorous-fluorous interactions over aqueous and organic phases. Thanks to the fluorous effect, perfluorinated monolayers are excellent anti-fouling coatings, while allowing the selective immobilization of fluorous-tagged biomolecules with high specificity.¹ We have characterised perfluorinated monolayers at the single-molecule level by observing adsorption and desorption events using interferometric scattering microscopy (iSCAT).² Our results prove that perfluorinated monolayers eliminate irreversible adsorption of proteins and lipid vesicles, while, crucially, still ensuring that molecules interacting with the surface have a residence time that allows them to be detected by iSCAT. We believe the use of fluorous chemistry for single-molecule imaging has the potential overcome limits of current passivation methods, allowing the study of biomachinery processes in real time and at biologically relevant concentrations.

Dirk-Peter Herten^1,2,4, Jonas Euchner^1,2, Klaus Yserentant^1,2,3, Felix Hild⁴, Daniel Patten⁵, Johan Hummert^1,2,8, Shishir Shetty⁵, Ronald Curticean⁶, Irene Wacker⁶, Rasmus R. Schröder^6,7

¹Institute of Cardiovascular Sciences & School of Chemistry, University of Birmingham, Birmingham, UK
²Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, UK
³School of Pharmacy, University of California, San Francisco, USA
⁴Institute of Physical Chemistry, Heidelberg University, Heidelberg, Germany
⁵Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, UK
⁶BioQuant, Heidelberg University, Heidelberg, Germany
⁷Heidelberg University Hospital, Heidelberg, Germany
⁸PicoQuant GmbH, Berlin, Germany

Cells maintain homeostasis by tightly regulating cellular processes and spatial compartmentalization. Visualisation of both, the cellular ultrastructure as well as the localisation of functional proteins, requires correlative light and electron microscopy (CLEM) approaches. However, array tomography by mean of serial sectioning of resin embedded samples to combine fluorescence super-resolution and electron microscopy remains challenging with many shortcomings in fluorophore photostability and structural preservation as well as ease of handling. A comprehensive evaluation of existing embedding conditions revealed that the degree of fluorescence preservation strongly depends on resin composition and polymerization parameters. Here, we present novel resin formulations which achieve excellent fluorophore preservation compatible with dSTORM imaging and scanning electron microscopy (SEM) array tomography. Serial ultrathin sectioning is not only a necessary step for 3D SEM but is also beneficial for dSTORM imaging due to the absence of out-of-focus signal and premature bleaching. As a proof of concept, liver sinusoidal endothelial cells were studied in presence and absence of a senescence associated secretory phenotype in 3D. Combining the presented workflow with well-defined fluorescent labels using protein tags will enable near quantitative super-resolution fluorescence microscopy in a CLEM context and thereby make full use of information obtainable from3D CLEM.

Farzaneh Alipoor Azizollahi, Simona Šalčiūnaitė, Mindaugas Zaremba, Algirdas Toleikis

Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius, Lithuania

Cas3, also called a “DNA shredder”, is an ATP-dependent nuclease-helicase. It is the key effector protein of type I CRISPR systems. Cas3, along with targeting complex Cascade, destroys the invading bacteriophage DNA. They must act quickly and efficiently, otherwise, phage invasion will lead to the destruction of the host. Although the initiation of this process is relatively well understood, the mechanism of Cas3 DNA unwinding and shredding remains largely unknown.

To study the mechanism of Cas3 activity at a single-molecule level, we are using a custom-built magnetic tweezers setup (MT). This single-molecule approach will reveal a dynamic view of different subpopulation events, which otherwise would be lost through an averaged-out view of classical biochemical methods. Also, it will enable assisting or hindering loads and torque, thus mimicking various situations that might arise in cells. Furthermore, the DNA hairpin substrate which we designed for Cas3 activity studies, will allow observation of Cas3 helicase activity in real-time. We anticipate that the mechanical properties obtained in this study will provide valuable insights into the mechanism of Cas3.

Samrat Basak¹, Roman Tsukanov¹, Nazar Oleksiievets¹, Nikolaos Mougios², Jan Christoph Thiele¹, Felipe Opazo^2,3, Jörg Enderlein^1,4

¹III. Institute of Physics - Biophysics, Georg August University, 37077 Göttingen, Germany
²Center for Biostructural Imaging of Neurodegeneration (BIN), University of Göttingen Medical Center, 37075 Göttingen, Germany
³NanoTag Biotechnologies GmbH, 37079 Göttingen, Germany
⁴Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), Georg August University, Göttingen, Germany

DNA-PAINT is one of the most powerful Single-Molecule Localization Microscopy (SMLM) techniques. It is capable of imaging with nanometer spatial resolution and is a perfect fit for multi-target super-resolution imaging of cells. The most common approach for this is Exchange-PAINT, which uses sequential imaging of targets. Exchange-PAINT does not have chromatic aberration, as the same fluorophore is used for imaging of all targets. However, its main disadvantage is its relatively long acquisition time, which scales linearly with the number of imaged targets, and the need for extensive sample washing during buffer exchange cycles. The elegant solution for fast multi-target super-resolution imaging is Fluorescence Lifetime DNA-PAINT (FL-PAINT) [1]. This technique enables parallel target acquisition and uses fluorescence lifetime information for target identification. To do this, targets are imaged by a mix of different imagers labeled with fluorophores emitting in the same spectral region but having different lifetimes. FL-PAINT can be implemented both using wide-field and confocal FL-SMLM [2]. The downside of FL-PAINT is a high background fluorescence level due to the high concentration of imagers in the mix solution, which affects the achievable resolution. To address this limitation, we have implemented a speed-optimized DNA-PAINT (Fast-PAINT) introduced by Jungmann lab in 2020 [3]. This approach uses a reduced concentration of imagers due to optimized imager/docking DNA sequences, thus reducing the background signal, and facilitating imager-docking binding kinetics thanks to concatenated docking strands. The combination of Fast-PAINT with FL-PAINT allows for fast and simultaneous acquisition of multiple targets with improved resolution and minimal cross-talk between targets. Overall, fluorescence lifetime SMLM is a powerful tool that opens up a new world of exciting capabilities, including multiplexed imaging, environmental sensing, FRET imaging, and much more.

Timothy J D Bennett^1,2, Konstantin Zouboulis^1,2, Justin Benesch^1,2, Yujia Qing^1,2, Madhavi Krishnan^1,2

¹Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, United Kingdom
²The Kavli Institute for Nanoscience Discovery, Oxford OX1 3QU, United Kingdom

Post-translational modifications (PTMs) are essential to the operation of cells. PTMs regulate the function of proteins within cells through control of conformation, activity and charge. Sulfonation, for example, improves solubility of dopamine and other endogenous substrates as a detoxification pathway in chemical metabolism[1], while S-glutathionylation plays a crucial role in cell signalling, triggering apopotic and proliferative pathways in cells unders oxidative or nitrosative stress[2]. The importance of these modifications is most evident when dysregulation of PTMs occur, which can cause catastrophic breakdown of normal cellular processes resulting in cancerous growth and disease state formation. For instance, hyperphosphorylation of tau protein causes aggregation similar to that found in Alzheimer's brain disease[3].

Here we use escape-time electrometry (ETe) to demostrate a rapid and high-throughput method to detect individual PTMs on a protein[4]. We exploit the sensitivity of ETe measurements to the effective charge of biomolecules in solution to distinguish the difference between several modifications of a thioredoxin mutant. By measuring the effective molecular charge across a pH range we identify multiple different PTMs, including phosphorylation, sulfonation and glutathionylation. We also show the sensitivity of ETe to PTM postion and demonstrate the ability to distinguish a modification in folded vs unfolded domains.

Özlem Urcan¹, Ranbir Kaur¹, Ingo Köhne², Rudolf Pietschnig², Johann Peter Reithmaier¹, Mohamed Benyoucef¹

¹Institute of Nanostructure Technologies and Analytics (INA), CINSaT, University of Kassel, Germany
²Institute of Chemistry, CINSaT, University of Kassel, Germany

The control of light-matter interaction is an enabling technique for many emerging quantum technology applications. The use of photonic crystals (PhCs) is especially interesting since they can confine light to small mode volumes and enhance light emission. Owing to their visible and near-infrared luminescence properties, lanthanide (Ln) molecules with their narrow linewidth characteristics and their immobilization on PhC cavities will offer the potential for scalable quantum systems.

The aim of this work is to fabricate telecom wavelength PhCs by electron beam lithography, inductively coupled plasma reactive ion etching, and selective wet etching techniques, and to investigate how different process parameters affect the quality of PhCs. The optical and morphological properties of PhCs are studied using micro-photoluminescence spectroscopy and scanning electron microscopy. The immobilization of molecules emitting in the telecom O-band on PhC cavities and their optical properties are discussed.

This work is supported by the state of Hesse in the frame of LOEWE priority project SMolBits and the DFG grant-BE 5778/4-1.

Miriam Gerstel¹, Ingo Köhne², Paul Mertin³, Bernd Witzigmann³, Johann Peter Reithmaier¹, Rudolf Pietschnig², Mohamed Benyoucef¹

¹1 Institute of Nanostructure Technologies and Analytics (INA), CINSaT, University of Kassel, Germany.
²Institute of Chemistry, CINSaT, University of Kassel, Germany
³EEI Department, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

Lanthanide ion exhibits characteristic narrow emission bands ranging from VIS to NIR along with relatively long emission lifetimes, which makes them attractive for applications in lighting, sensing, and display technologies. We investigate the optical characteristics of phosphonate ester-supported nitrite and chloride neodymium(III) complexes as solid bulk material and in solution. Optical properties of Nd complexes are determined by photoluminescence (PL) spectroscopy, which reveals three strong and two less pronounced emission bands of Nd(III) ions in the NIR region. The emission lines of the emission band centered around 880 nm are labeled with the help of temperature-dependent PL measurements. Investigation of equimolar solution reveals information about the emission strength of the complexes with different ligand types. For light enhancement, molecules are immobilized on photonic crystal cavities (PhCs). The fabrication of PhCs by electron-beam lithography, inductively coupled plasma reactive ion etching, and selective wet etching techniques are discussed. This work is supported by the state of Hesse in the frame of LOEWE priority project SMolBits and the DFG grant-BE 5778/4-1.

Anupam Bhoi, Felix Müller-Planitz

Institute of Physiological Chemistry, Technical University of Dresden, Dresden, Germany

The genetic material in eukaryotes is compacted by wrapping DNA around histone proteins, forming nucleosomes that inhibit nuclear processes. Access to genetic information requires the repositioning of nucleosomes which involves mechanistically complex processes of breaking and reforming DNA-histone contacts to move histones along DNA without dissociation. ATP-dependent nucleosome remodeling enzymes can control nucleosome positions, but their mechanism is poorly understood.

To better understand chromatin remodelers, my project aims to delineate the structural, kinetic, and thermodynamic pathway of the Drosophila ISWI remodeler, which can slide nucleosomes and establish even spacing between them. The multi-domain ISWI protein consists of catalytic ATPase motor domains, a regulatory N-terminal region (NTR), and a C-terminal Hand-Sant-Slide (HSS) domain. I aim to visualize the large-scale conformational changes occurring in ISWI during nucleosome binding and remodeling. I monitor ensemble and single-molecule Fluorescence Resonance Energy Transfer (FRET) changes between ISWI’s different domains by site specifically introducing two compatible fluorophores into ISWI. In parallel, I measure intermolecular FRET changes between ISWI and its substrates (DNA and nucleosomes) to coherently determine the thermodynamics and kinetics of ISWI’s mechanism. Revealing ISWI's conformational dynamics would provide valuable insights into the catalytic strategies of complex remodelers, aiding drug design for enzymes involved in malignancies.

Tuyen Bujnicki, Karen Hänel, Anneliese Cousin, Victoria Kraemer-Schulien, Melanie Schwarten, Dieter Willbold

Forschungszentrum Jülich GmbH, Institute of Biological Processing, Structural Biochemistry (IBI-7), 52428 Jülich

The SuFIDA technology is a single molecule counting technology. It is an advanced ultrasensitive immunoassay technology that enables the detection of biomarkers that were previously undetectable down to lower femtomolar concentrations. SuFIDA is the evolution of the sandwich ELISA with greater selectivity, higher sensitivity and the digital readout using an optical imaging technique.

In SuFIDA, capture antibodies are immobilized on a glass surface and bind the analyte from the sample. A fluorescence-labeled antibody is used as the detection probe, which recognizes an epitope on the target molecule that is not occupied by the capture antibody. Using highly sensitive TIRF microscopy, all individual fluorescence-labeled probes on the surface are detected and virtually counted individually. By using an in-house developed analysis software, the huge amounts of images are evaluated in a short time using various analysis functions (e. g. automatic artefact detection).

Diego Cora¹, Sara Illodo^1,2, Flor Rodríguez Prieto², Wajih Al-Soufi¹, Mercedes Novo¹

¹Departamento de Química Física, Facultade de Ciencias, Campus Terra, Universidade de Santiago de Compostela, 27002 Lugo, Spain.
²Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Departamento de Química Física, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.

Alzheimer's disease (AD) is a neurodegenerative condition that currently affects over 50 million people worldwide.^1,2 The exact mechanisms underlying the onset of AD are still not fully understood, however, the amyloid cascade hypothesis suggests that AD is related with the accumulation of amyloid (Aβ) peptides in the brain.³ There are two major types of Aβ peptides: Aβ40 (composed of 40 amino acids) and Aβ42 (composed of 42 amino acids), the latter being the most neurotoxic and prone to aggregation.

Recently, significant therapeutic advances in AD have been made. Plasma exchange (PE) with albumin replacement is being investigated as a potential therapeutic approach for AD. This treatment takes advantage of the association between human serum albumin (HSA) and the Aβ peptides and it’s showing encouraging results.⁴

The aim of this work is to characterize the association between the HSA and the Aβ peptides in their monomeric state, as a first step to understand the underlying mechanisms of the PE treatment. We also try to clarify the inconsistencies in the literature, considering that previous studies have yielded disparate results.^5–10 For that, we used conventional fluorescence techniques in steady-state and time-resolved conditions and microscopy techniques like Fluorescence Correlation Spectroscopy, using fluorescently-labelled Aβ peptides.

Alicia Damm^1,2, Su Jin Paik¹, Raj Kumar Sadhu¹, Aurélie Di Cicco¹, John Manzi¹, Maxime Dahan¹, Pierre Sens¹, Daniel Lévy¹, Patricia Bassereau¹

¹Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico-Chimie Curie, 75005 Paris, France
²Present address : Laboratoire de Physique de l’Ecole Normale Supérieure, ENS, Université PSL, CNRS UMR 8023, Sorbonne Université, Université de Paris, F-75005 Paris, France

Mechanosensitivity is an intrinsic property of some channels, with a function dependent on the membrane mechanical deformation. However, could other types of transmembrane proteins be sensitive to mechanical stimuli? Transporter proteins are embedded in the membrane and, to properly operate, have to significantly change conformation, being thus potentially influenced by membrane mechanics. Here, we address this question using a bacterial ABC (ATP Binding Cassette) exporter, BmrA, and we study the effect of membrane curvature on its ATP-dependent conformational dynamics at the single molecule level.

The purified protein was reconstituted into small unilamellar vesicles (SUVs), with two different sizes (average diameter of 125 and 20 nm respectively) allowing us to vary the membrane curvature. We found that the protein ATPase activity decreases by a factor 2.8 when increasing membrane curvature. We have then successfully used single molecule FRET to probe the transporter conformation in immobilized liposomes. We have identified two main conformations, respectively open and closed, and observed that the fraction of BmrA in closed conformation decreases in highly curved liposomes, either in the presence of ATP, or of the non-hydrolysable ATP analog AMP-PNP. This demonstrates that the conformational changes of a transporter and its function can depend on membrane curvature.

Felix Erichson¹, Fabio D. Steffen², Richard Börner¹

¹Laserinstitut Hochschule Mittweida, Mittweida, Germany
²University Zurich, Zurich, Switzerland

Tertiary contact interactions between RNA tetraloops and their receptors play a crucial role in ribosome assembly and maturation at lower temperatures [1]. To investigate the binding mode of a particular GAAA tetraloop (TL) and its kissing loop (KL) receptor, identified in the 25S rRNA of baker's yeast, a minimal construct of the tertiary contact was designed [2]. We developed a FRET-assisted modeling pipeline and applied it to a fluorescently labeled model of the bound KL-TL_GAAA construct [3]. The tertiary construct was assembled with PyMOL including the experimentally solved KL-TL_GAAA  motif. FRET histograms were simulated using FRETraj [4] based on MD simulations with explicit dyes and multiple accessible contact volumes (mACV). Dye interactions with the RNA were factored into the FRET prediction by reweighting of the contact volume (CV) relative to the accessible volume (AV) [5] using experimentally determined dynamic anisotropy of the dyes. The MD simulation shows a change in orientation of the bound TL with altered base interaction partners, resulting in an mACV-derived FRET distribution that remarkably matches the experimental FRET distribution. This validated model provides valuable insights into structural aspects of the GAAA binding interaction and shows that integrative modeling helps elucidating RNA structures and further their function.

Blaise Gatin-Fraudet, Sigrid Milles, Johannes Broichhagen

Leibniz-FMP Berlin, Robert-Rössle-Str. 10; 13125 Berlin

Protein structures determine their specific function and the working mechanisms on the molecular level. X-ray crystallography and cryo-EM allow atomic level insights into proteins with static conformations.¹ Actual dynamics can only be addressed by solution state techniques, and an extreme example for dynamic protein systems are intrinsically disordered proteins. Among these techniques, single molecule fluorescence spectroscopy allows real-time observation of protein dynamics in solution. Specific long-range distances between residues can be measured using single molecule Förster Resonance Energy Transfer (smFRET). The choice of the label — ideally bright, photostable, soluble and small — and its covalent attachment to the protein are  critical. In addition, the background in the green spectral regime requests the use of red-emitting dyes, especially for samples with high background, such as the interior of the cell or liquid-liquid phase separating conditions.² Unfortunately, red-emitting dyes are often less bright and more sticky.^3,4 We describe deuterated and sulfonated fluorophores in the red and NIR spectrum with improved quantum yields, reduced bleaching rates and higher solubility. Their improved characteristics warrant undiscovered potential to replace state-of-the-art fluorophores used in the field smFRET.

Swarnali Ghosh¹, Prasun Mandal^1,2

¹Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur, West Bengal, India-741246
²Centre for Advanced Functional Materials, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur, West Bengal, India-741246

Semiconductor quantum dots (QDs) or perovskite nanocrystals (PNCs) are either not very stable or contain toxic metals like Cd/Pb which jeopardizes their sustainable applications.^1-2 Properties of QDs/PNCs depend on the exciton dynamics. Unlike electron trapping/detrapping dynamics, there are very few literature reports of intrinsic hole-trapping (HT) and hole-detrapping (HDT) in QDs/PNCs.^3-7 Hence, there is a strong quest to obtain highly-bright, highly-stable, toxic-metal-free QDs in which intrinsic HT/HDT phenomena can be observed.

As a model toxic-metal-free QD, small-sized (~3.3 nm), highly-bright (PLQY = 96%) CuInS₂/ZnSeS Core/Alloy-Shell (CAS) QD has been synthesized.⁸ No significant decrease of PLQY for one year even under ambient condition signifies very high stability of this QD.⁸ Employing ultrasensitive single particle spectroscopy, intrinsic both HT and HDT, as well as very significant variation of their amplitudes (~16 to ~42% for HT and ~44 to 23% for HDT), have been observed for the first time in this CAS QD⁹ in the entire QD family/PNCs. Unlike, detrimental electron-trapping,¹⁰ intrinsic HT has been noted to be beneficial towards increasing PLQY. Simultaneous trapping of both electron and hole lead to the longest ON duration (>2 minutes)⁹ reported for any toxic-metal-free QD.

All these detailed statistical analyses based interesting results will be elaborated.

Sanne Giezen¹, Ilja Voets¹, Roderick Tas²

¹Department of Chemical Engineering and Chemistry, Laboratory of Self-Organizing Soft Matter, Eindhoven University of Technology, Eindhoven 5612 AP, the Netherlands; and Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven 5612 AP, the Netherlands
²Department of Biomechanical Engineering, Delft University of Technology (TU Delft), Mekelweg 2, Delft 2628 CD, The Netherlands

Antifreeze proteins (AFPs) are unique proteins that bind to growing ice crystals and thereby reduce the freezing temperature, thermal hysteresis,  and/or inhibit ice recrystallization. Cold-blooded organisms produce these proteins to endure the extreme cold of their natural habitat [1]. Although AFPs have been investigated over 50 years, the interfacial dynamics involved during binding remain uncertain. To bridge this gap, our group designed a subzero nanoscopy set-up to investigate AFPs on a single-molecule level. Recently, this technique revealed that irreversible pinning is required for thermal hysteresis but not for ice recrystallization inhibition [2]. In this presentation, I will explain how our group studied the underlying mechanism at the ice-water interface.

Katherina Hemmen¹, Thomas-Otavio Peulen¹, Ashwin Balakrishnan¹, Susobhan Choudhury¹, Ruiqi Liu¹, Mike Friedrich¹, Martin J. Lohse², Katrin G. Heinze¹

¹Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, 97080 Würzburg, Germany
²ISAR Bioscience Institute, Semmelweisstr. 5, 82152 Planegg, München

We study G-protein-coupled receptors (GPCRs) by time-resolved fluorescence spectroscopy and multi-parameter fluorescence image spectroscopy (MFIS) in living cells to understand (a) the translational and rotational diffusion, (b) monitor the conformational changes upon ligand binding and (c) quantify local heterogeneities by fluorescence correlation spectroscopy (FCS), time-resolved anisotropy (TRA) and Förster Resonance Energy Transfer (FRET).

To disentangle GPCR translational and rotational dynamics from fluorophore dynamics in FCS and TRA experiments, we labeled GPCRs with differently sized fluorophores in GPCR experiments in the absence and presence of ligands. Conformational changes in the GPCRs upon ligand are quantified by FRET while MFIS probes for GPCR oligomerisation. Here, we show how to implement a suitable workflow to quantify diffusion, conformational changes and protein interaction, and oligomerisation states of membrane receptors in live cells in open-source software that can be generalized to other use cases.

Christof Hepp^1,2, Qing Zhao^1,2, Nicole Robb³, Ervin Fodor⁴, Achillefs Kapanidis^1,2

¹Kavli Institute for Nanoscience Discovery, University of Oxford, Sherrington Road, Oxford OX1 3QU, Oxford, UK
²University of Oxford Department of Physics, Oxford, UK
³Warwick Medical School, Medical School Building, Coventry CV4 7HL
⁴Sir William Dunn School of Pathology, S Parks Rd, Oxford OX1 3RE

Influenza A, a negative-sense RNA virus, has a genome that consists of eight single-stranded RNA segments. During influenza co-infections, re-assortant virus strains containing gene segments from either strain can occur, occasionally leading to pandemic outbreaks with severe, worldwide consequences for human health.

To better understand the formation of these potentially pandemic re-assortants, we analysed the selective packaging of all eight RNA segments into virions. To this end, we designed a novel multiplexed DNA-PAINT approach capable of a) detecting the presence or absence of all eight gene segments inside of more than 10,000 individual virus particles in one experiment in just 4 hours and b) spatially resolving the individual segments inside complete virus particles with a resolution of better than 10 nm. With its high throughput and the capability of unambiguously identifying specific gene segments, this experiment provides novel structural information complementing electron microscopy studies.

Our results suggest a flexible network of inter-segment interactions that form a robust genome assembly for influenza A. In the long term, we will develop our experimental approach for the structural and functional study of viral nucleoprotein complexes in infected cells to elucidate key elements of the viral life cycle like transcription, replication and sub-cellular transport.

Stella Hernández, Lucas Piñeiro, Mercedes Novo, Wajih Al-Soufi

Departamento de Química Física, Facultade de Ciencias, Campus Terra, Universidade de Santiago de Compostela, E-27002 Lugo, Spain

Thiazole Orange (TO) is a DNA intercalating dye of potential use for double-stranded DNA (dsDNA) detection and quantification. It is nearly non fluorescent in aqueous solution but shows a significant fluorescence when it is associated with dsDNA. High affinity constants have been reported for the complexation between TO and dsDNA and sequence selectivity of TO has been studied to evaluate its potential use in fluorescent intercalator displacement assays.^1,2 Moreover, the interaction of TO with other DNA conformations has been reported.³

The affinity to dsDNA of the intercalating agents may vary with the experimental conditions, especially the charge and concentration of the cations present in the solution.⁴ In this work we use steady-state and time-resolved fluorescence spectroscopy to study the interaction between TO and dsDNA. The effects of salt concentration and cation charge on the binding constant have been systematically studied using NaCl and MgCl₂. Also, the potential influence of the chain length and sequence of dsDNA was analyzed.

Fluorescence Correlation Spectroscopy was used to study the dynamics of TO-dsDNA association and its dependence on salt concentration.

Johan Hummert¹, Max Tillmann¹, Felix Koberling¹, Cyril Saudan², Harald Homulle², Ivan Michel Antolovic², Evangelos Sisamakis¹, Rainer Erdmann¹

¹PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany
²Pi Imaging Technology SA, EPFL Innovation Park, 1015 Lausanne, Switzerland

Time-resolved fluorescence detection is a powerful technique in fluorescence microscopy. The fluorescence lifetime encodes information about the local environment of fluorophores or can be used for single-color multiplexing. Commonly, confocal microscopes with single photon counting hardware are used for lifetime imaging, defining the state of the art of the technique. However, for certain applications widefield imaging can bring unique advantages such as higher throughput and longer observation times.
Here we show how the combination of a powerful picosecond laser and a novel SPAD camera facilitates time-resolved imaging in widefield microscopy. The SPAD512S camera from Pi Imaging Technology enables time-resolved fluorescence detection through gating with picosecond accuracy. We benchmark the camera’s performance with widefield lifetime imaging on commercially available cell samples. Suitable lasers from the PicoQuant portfolio offer sufficient power to saturate the camera across the field of view on these samples. Thus, using the camera`s full dynamic range enables lifetime imaging at video rate acquisition speeds while simultaneously minimizing photobleaching. This could unlock new possibilities for lifetime imaging as the technology matures, especially for applications where imaging speed is essential.

Line Lauritsen¹, Maria Szomek¹, Mick Hornum², Peter Reinholdt², Jacob Kongsted², Poul Nielsen², Jonathan Brewer¹, Daniel Wüstner¹

¹Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
²Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

Subcellular membranes have specific lipid and protein compositions, which give rise to organelle-specific membrane packing and polarity. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Additionally, Nile Red derivatives are essentially non-fluorescent in water, allowing for their use in super-resolution microscopy experiments. Here, we compare the potential of derivatives of Nile Red with functional substituents for spectral stimulated emission depletion (STED) microscopy of membrane packing in living cells. All studied Nile Red derivatives reveal lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C (NPC) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes (LE/LYSs). We find that a Nile Red derivative with a 10-fluoro atom has particularly high environmental sensitivity despite reduced molecular brightness. All Nile Red derivatives are suitable as exchangeable dye for super-resolved spectral imaging of membrane packing in living cells.

Daniel Marx¹, Oleksii Nevskyi¹, David Malsbenden², Dominik Wöll², Jörg Enderlein¹

¹III. Institute of Physics – Biophysics, Georg‐August‐University Göttingen
²Institute for Physical Chemistry – RWTH Aachen University

The ongoing miniaturization of devices and advancements in nanotechnology have led to a notable reduction in structural size, resulting in increased surface-to-volume ratios. As a consequence, the impact of changed physical properties due to interfaces becomes more pronounced. For instance, Bäumchen et al. demonstrated that the impact of polymer-air interfaces is the dominant cause of reduced glass transition temperatures measured in thin polymer layers [1]. In a related study, Wöll et al. employed Single-Molecule Tracking (SMT) experiments to assess heterogeneities within such films. They discovered that the fraction of mobile molecules increased as the polymer film thickness decreased [2]. This behavior can be attributed to the presence of a thin surface layer with significantly enhanced mobility relative to the bulk polymer.

In this research, we employ 3D-SMT techniques with exceptional axial and lateral resolution. Our primary objective is to directly investigate the distance-dependent mobility profiles at both the polymer-air and polymer-substrate interfaces. To achieve the necessary axial resolution, we utilize Metal Induced Energy Transfer (MIET) imaging, which enables sub-5 nm resolution [3]. Furthermore, by measuring the orientations of the molecular probes, we aim to analyze both translational and orientational diffusion characteristics in the proximity of the glass transition temperature.

Gabriel Moya¹, Oliver Brix¹, Eitan Lerner², Niels Zijstra¹ & Thorben Cordes¹

¹Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany
²Department 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

Scientific advances not only depend on new ideas, conceptual leaps, and paradigm shifts, but also on the availability of new Technologies. Here, we introduce a novel micro-spectroscopy and imaging platform based on 3D printing and flexible combinations of microscope components. This compact and versatile 3D-printed microscopy platform, BRICK-MIC, allows for the assembly of various fluorescence imaging techniques, including confocal and video detection. Consequently, it facilitates single-molecule and single-particle detection as well as super-resolution fluorescence imaging.

Our primary aim is to provide a user-friendly and adaptable solution for obtaining molecular insights into sample properties such as conformational states, particle sizes, and biomolecular interactions. This is achieved through cost-effective manufacturing of the required setups, with the overarching goal of enabling more widespread use of established assays in high-throughput screening and diagnostic applications.

Thomas-Otavio Peulen, Katrin G. Heinze

Rudolf-Virchow-Zentrum - Center for Integrative and Translational Bioimaging, Haus D15, Josef-Schneider-Straße 2, 97080 Würzburg

Bacteria vastly outnumber eucaryotic life in number and metabolic activity. Their prime mode of living are bacterial communities. Proteins and DNA networks span these communities to provide structural integrity. I study molecular structures and dynamics of key components by multiparameter microscopy, single-molecule spectroscopy, super-resolution microscopy, and integrative modeling.

To determine in situ structures, I developed software for (i) fluorescence-based modeling, (ii) single-molecule spectroscopy, and (iii) multiparameter image spectroscopy. I integrate fluorescence information with other data sources using BFF, the Bayesian fluorescence framework, software I developed as part of the integrative modeling platform (IMP). Resolving molecular structure of biomolecules that shape bacterial communities, I demonstrate how single-molecule FRET, super resolution microscopy, and cryoEM data are integrated for dynamic structural biology that can determine molecular structures in situ.

Mariia Popova, Benedikt Bauer, Sabrina Horn, Iain Davidson, Jan-Michael Peters

Research Institute of Molecular Pathology (IMP), Vienna, Austria

The Structural maintenance of chromosomes (SMC) complexes play a crucial role in the precise spatial and temporal organization of genomic DNA by folding it into loops and topologically associated domains, a process essential for chromosome assembly, gene regulation, and recombination [1]. Among the SMC complexes, cohesin was proposed to mediate long-range cis interactions by binding to DNA and actively reeling it into loops in interphase cells. [2]. This process, known as loop extrusion, has been recently demonstrated in vitro using single-molecule fluorescence microscopy [3]. However, the precise mechanism by which cohesin entraps and moves the DNA remains poorly understood.

To address this, we propose a system for direct measurement of cohesin-DNA interactions using single-molecule Fluorescence Resonance Energy Transfer (smFRET). Previous studies have identified several DNA binding sites and conformational changes of the cohesin complex that are crucial for loop extrusion [4]. Based on this knowledge, we are currently developing an smFRET assay with fluorescently labeled DNA and cohesin. This assay will enable us to investigate the dynamics and efficiency of DNA binding at different sites on cohesin. Furthermore, this system holds the potential to identify new DNA binding sites of cohesin, opening avenues for further exploration and understanding.

Klara Postulkova, Kristyna Bousova, Jiri Vondrasek

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo namesti 542/2, 160 00 Praha 6, Czech Republic

The expanding use of proteases in therapeutics or research has led to the need for different strategies to engineer them. New protease properties can be achieved by mutagenesis or by fusion with different protein domains.

One of the most studied viral proteases is the HIV-1 protease. Its a homodimeric enzyme that plays a crucial role in viral maturation. One monomer of the HIV-1 protease is anchored in each Gag-Pol polyprotein. To cleave itself out of the precursor, two Gag-Pol moieties must form at least a transient dimeric strucure to form the active site. The maturation of the protease is called autoprocessing and its mechanism is not yet fully understood.

This process can be disrupted by a specific mutations in the sequence of HIV-1 protease and also by fusion with specific proteins at its N-terminus.

In our design, we linked HIV-1 protease to Thioredoxin (commonly used solubilizing protein) at the N-terminus by a linker carrying his-tags and the cleavage site of HIV-1 protease. In addition to monitoring basic properties of the fusion protein, such as expression profile, stability or solubility, we analyzed the effect of N-terminal domain on the activity (FRET assay) and autocatalytic activity (SDS-PAGE) of HIV-1 protease.

Imesha Rathnayaka¹, Santiago Martinez-Lumbreras², Michael Sattler*², Don C. Lamb*¹

¹LMU Munich, Department of Physical Chemistry
²TUM Munich, Department of Chemistry

Proteins are the building blocks of life. Tight regulation of the several steps performed during protein biosynthesis is required to form functional and well-structural proteins. The spliceosome is a massive ribonucleoprotein complex involved in post-transcription regulation and is responsible for removing the noncoding regions and the introns from pre-mRNA. The E and A subcomplexes of the spliceosome are critical components, as these subcomplexes primarily initiate the intron lariat formation. This study aims at revealing the bridging factors that are responsible for formation of the spliceosome subcomplex A with the aid of multicolor DNA PAINT-based Single Particle Tracking. Transient binding of the freely diffusing imager strands facilitates the determination of the localization kinetics of the RNA without being limited by photobleaching. We will then follow the formation of the spliceosome subcomplexes upon the stepwise addition of ribonucleoproteins. These findings will contribute to our understanding of the molecular mechanisms that are associated with alternative splicing and may have implications for the development and enhancement of spliceosome-targeted therapies.

Tobias Rex¹, Theresa Mößer², Carsten Grashoff², Cristian A. Strassert¹

¹Westfälische Wilhelms-Universität Münster, Institute of Inorganic and Analytical Chemistry
²Westfälische Wilhelms-Universität Münster, Institute of Integrative Cell Biology and Physiology

The synthesis and characterization of three novel self-assembling platinum(II) complexes based on tetradentate luminophores with different amphiphilic character is reported. Due to the tailored design of the coordination compound, strong aggregation is promoted leading to the formation of self-assembled structures in water. Therefore, the photophysical properties were studied at different concentrations by steady-state and time-resolved photoluminescence spectroscopy. The concentration-dependent formation of self-assembled structures were confirmed by DLS and SEM. As a proof of principle for biological applications, the compounds were tested on mouse fibroblast cells. All three complexes were taken up by the cells and the localization in different cell compartments including cell membrane, cytoplasm and nucleus was observed, demonstrating their biocompatibility. Possible cytotoxic effects were evaluated by cytotoxicity assays and the generation of ¹O₂ was studied. Furthermore, photoexcited-state lifetimes were obtained in living fibroblast by phosphorescence lifetime microscopy. In summary, these supramolecular systems offer excellent water solubility with potential application in live cell imaging and photosensitization in terms of generation of singlet molecular dioxygen.

Volker Buschmann, Christian Oelsner, Bita Rezania, Maria Loidolt-Krüger

PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany

Second harmonic generation (SHG) imaging microscopy is a nonlinear optical imaging technique that uses SHG as a contrast mechanism to produce high-resolution images. SHG occurs in materials with non-centrosymmetric crystal structures. Therefore, SHG imaging has been applied for characterization of 2D semiconductors, transition-metal dichalcogenides (TMDs) such as WS2 and MoSe2, lithium niobate crystals, PZT thin films, graphene, lanthanides, and even biological tissues. It provides information on the crystal lattice, assesses crystal quality and maps grain boundaries, defects, and mechanical strain. Furthermore, SHG imaging reveals the number of stacked layers as well as their orientation with respect to each other.

SHG imaging is commonly performed with a confocal laser scanning microscope using a high-power fs pulsed laser for excitation. In this study, we establish the use of high power ps pulsed lasers instead, with the aims of decreasing laser safety issues as well as reducing microscope complexity and cost. In addition, ps lasers offer high flexibility and tunability in terms of pulse duration, repetition rates, output power and external triggering.

We investigated two different 2D materials namely WSe2 and MoS2 on PDMS utilizing MicroTime 100 time-resolved confocal microscope. Reflection images and SHG images were acquired using a VisIR-1064 laser with 1064 nm wavelength, dimmed to laser class 3B and time-resolved photoluminescence (TRPL) images with 640 nm excitation.

The results set prove that ps pulsed lasers can replace fs lasers for SHG imaging. Moreover, the combination of ps lasers enables the collection of complementary datasets including reflection, SHG, TRPL and even two photon excitation (TPE) images collected from the same region of the sample using a single microscope setup. Thus, the optical properties of the materials can be characterized locally with several techniques to obtain a comprehensive understanding.

Henry G. Sansom, Steven W. Magennis

School of Chemistry, University of Glasgow, Joseph Black Building, University Avenue, Glasgow, G12 8QQ, United Kingdom

Single-molecule measurements of fluorescent base analogues and amino acids would provide a wealth of information on biomolecules that is not possible with conventional fluorescent labels. However, the short excitation wavelengths of these analogues make this challenging. This problem can be circumvented by employing multiphoton excitation.

We recently demonstrated a home-built multiphoton microscope and showed that an oligonucleotide labelled with a fluorescent base analogue could be detected via two-photon FCS, whereas one-photon excitation caused rapid photobleaching. [1, 2] Multiphoton excitation also provides other benefits over resonant excitation such as a reduction in background and increased penetration depth. It has also been shown that single-molecule detection of FBAs is possible, however none are yet bright enough for routine use when incorporated into oligonucleotides. [3, 4] Here we present recent advances in the measurements of FBAs with the goal of routine single-molecule detection via multiphoton excitation.

Jesús Seijas^1,2, Flor Rodríguez-Prieto², Wajih Al-Soufi¹, Mercedes Novo¹

¹Departamento de Química Física, Facultade de Ciencias, Campus Terra, Universidade de Santiago de Compostela 27002, Lugo, Spain.
²Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Departamento de Química Física, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by amyloid plaques and neurofibrillary tangles in the brain tissue resulting from protein aggregation. The detrimental consequences of amyloid aggregation within the human body are believed to instigate a cascade of events leading to neurotoxicity, synaptic dysfunction, and ultimately, cognitive decline.^1,2

Understanding the underlying mechanisms of amyloid aggregation, especially at the early stages, is vital for the development of effective therapeutic strategies aimed at preventing or halting the progression of AD. Our group has studied this early aggregation using Fluorescence Correlation Spectroscopy, which allows the determination of the fraction of aggregated amyloid of the sample and the characterization of the aggregates formed.^3,4

However, there is a need for detection and quantification methods of the amyloid aggregation degree based on conventional fluorescence techniques, which could be used in standard laboratories. Therefore, the aim of this work is to design a strategy to detect Aβ oligomers based on the autofluorescence of amyloids combined with other fluorescent probes such as Thioflavine T, widely used for the detection of Aβ fibrils.⁵

Ndege Simisi Clovis, Sobhan Sen

Spectroscopy laboratory215, School of Physical Sciences ,Jawaharlal Nehru University, New Delhi-110067
Department of Photonics Science and Engineering Program, 215, Indian Institute of Technology Kanpur, Kanpur-208016

Probing the kinetics of ligand binding to biomolecules is of paramount interest in biology and pharmacology. Measurements of such kinetic processes provide information on the rate-determining steps that control the binding affinity of ligands to biomolecules, thereby predicting the mechanism of the molecular interaction. In this context, ligand binding to G-quadruplex DNA  structures has attracted tremendous attention primarily because of their use in possible anticancer therapy. Although a large number of G-quadruplex-specific ligands have been proposed, probing the kinetics of G-tetrad-selective binding of (multiple) ligands within a G-quadruplex DNA structure remains challenging. Most of the earlier studies focused on the thermodynamics of ligand binding; however, the binding of multiple ligands within a GqDNA structure have not been explored. Here, we propose a simple FCS-based method that measures the G-tetrad-selective association and dissociation rates of ligands within a GqDNA  by correlating the fluorescence fluctuations of a site-specific fluorophore (Cy3) in GqDNA  induced by the ligand binding to the G-tetrads. We show that well-known GqDNA ligands, BRACO19, TMPyP4, Hoechst 33258, and Hoechst 33342, have G-tetrad-selective measured kinetic rates which suggest site-dependent variation of free energy barriers for binding/unbinding of the ligands with G-tetrad site (5' vs 3' end) and ligand/GqDNA structural dependency.

Evangelos Sisamakis, Marcelle Koenig, Maria Loidolt-Krueger, Fabio Barachati, Matthias Patting, Marcus Sackrow, Kamil Bobowski, Mathias Bayer, Felix Koberling, Rainer Erdmann

PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany

Single molecule studies and – more specifically – single molecule FRET methodologies have become a standard tool for studying dynamic structural changes in proteins and nucleic acids. These types of measurements can reveal dynamic events on time scales covering several orders of magnitude from ~ns to several seconds. This allows studying e.g., chain dynamics, binding, folding, allosteric events, oligomerization and aggregation. The power of these methodologies is highlighted by the study of Intrinsically Disordered Proteins (IDPs) whose biological relevance has been increasingly studied over the recent years.

In this poster we show how easily these measurements can be performed with Luminosa single photon-counting confocal microscope and how all necessary correction parameters are automatically determined requiring no interaction from the user by employing methodologies benchmarked by the scientific community. We will also show how the variable PSF feature can be used in such measurements to fine-tune the observation window of freely diffusing biomolecules.

Evangelos Sisamakis, Fabio Barachati, Marcelle Koenig, Maria Loidolt-Krueger, Ellen Schmeyer, Matthias Patting, Marcus Sackrow, Felix Koberling, Rainer Erdmann

PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany

Fluorescence Lifetime Imaging (FLIM) has become more attractive in recent years as it offers increased specificity in many assays as well as the possibility of multiplexing the read out of many markers with a small number of detectors.

Here we present how FLIM modalities are implemented in Luminosa, the new single-photon counting confocal microscope by PicoQuant. Thanks to a dynamic binning format and GPU-based algorithms FLIM images of 1024x1024 can be analysed in a few seconds. The FLIM analysis workflow suggests the best fitting model based on statistical arguments and requires minimal user interaction making these modalities become accessible to new users who can then confidently start working with FLIM and incorporate it into their research toolbox combining the strengths of phasor plots with decay fitting.

Arina Svoeglazova, Daniel Scholl, Maud Sigoillot, Rafael Colomer Martinez, Marie Overtus, Chloé Martens, Cédric Govaerts

Structure and Function of Membrane Proteins laboratory, Chemistry department, Faculty of Sciences, Université Libre de Bruxelles, Brussels, Belgium

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians caused by mutations in the cystic fibrosis transmembrane regulator (CFTR): a chloride channel regulating fluid transport in epithelial tissues. The prevalent thermodynamically destabilizing mutation is the deletion of phenylalanine at the 508 position (F508del) in the nucleotide-binding domain 1 (NBD1) of CFTR, which leads to channel degradation and lack of ion transport.

We study conformational dynamics of NBD1 in complex with nanobodies. Nanobodies are the antigen-binding fragments of heavy-chain-only antibodies found in camelids [1]. We developed a collection of nanobodies targeting human NBD1 [2] and identified nanobody T1a, which had “F508del mutation-like” effects on the domain. Such interactions have been shown by means of smFRET experiments in conjunction with X-ray crystallography and hydrogen-deuterium exchange mass spectrometry (HDX-MS) methods. We hypothesize that T1a binding to NBD1 mimics the effect of F508del on the domain, providing a unique tool to investigate the molecular basis of the CF-causing mutation.

In our research, we want to stay more focused on smFRET method usage to trace destabilization changes in NBD1, which occur upon binding to the nanobody since it is a powerful technique for studying single-molecule structural dynamics.

Bikash Chandra Swain, Pascale Sarkis, Sabrina Rousseau, Laurent Fernandez, Ani Meltonyan, Cameron D. Mackereth, Mikayel Aznauryan

University of Bordeaux, Inserm U1212, CNRS UMR 5320, ARNA, Institut Européen de Chimie et Biologie, F-33600 Pessac, France

The eukaryotic translation initiation factor 4B (eIF4B) plays an essential role in translation by enhancing the activity eIF4A helicase in unwinding the structured motifs of mRNA. It is particularly crucial for the translation of mRNAs with long and structured 5’-end untranslated regions, such as those coding for many proto-oncogenes. The eIF4B is predicted to be predominantly disordered, except the folded RRM domain. The disordered C-terminal half of eIF4B (eIF4B-CTR) is essential for RNA binding, as it possesses a non-canonical RNA binding motif, enriched in arginine residues. The exact mechanisms of these interactions are, however, currently unknown.

Hence, we characterized the eIF4B-CTR alone and in complex with RNA, employing a combination of single-molecule Förster resonance energy transfer (smFRET), nanosecond fluorescence correlation spectroscopy (nsFCS) and nuclear magnetic resonance (NMR) spectroscopy. Our results show that eIF4B-CTR is disordered and flexible, with significant presence of intrachain interactions. The RNA binding to eIF4B-CTR is highly dynamic, with fast exchange between the molecules forming the complex, and is highly sensitive to ionic strength, implying an electrostatic mechanism of interactions. Upon RNA binding, the N-terminus of eIF4B-CTR compacts, whereas the C-terminus expands. Overall, our study provides detailed understanding of the RNA interaction with eIF4B-CTR.

Arti Tyagi^1,2, Moritz Engelhardt¹, Roland Kanaar², Maarten W. Paul², Kristin S. Grußmayer¹

¹Grußmayer Lab, Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, Netherlands
²Department of Molecular Genetics, Erasmus MC, Rotterdam, Netherlands

Homologous recombination (HR) is vital for DNA damage repair and genetic diversity. BRCA2 plays crucial role in HR, interacting with several proteins in the damage repair pathway. Many studies explored BRCA2 interactions, occurring in 3D space throughout the nucleus, using two-dimensional or fixed-cell super-resolution imaging. This provides an incomplete picture and may lead to misleading inferences.

An alternative is live cell volumetric imaging at DNA damage sites[2]. We use an easy-to-implement optics comprising of a specially designed prism that splits light into different axial planes[1], and cell lines containing genetically encoded fluorescent tags with the protein-of-interest (like BRCA2). With our setup, we can observe dynamics of BRCA2 upon damage induction in live cells by single molecule tracking. We aim to compare the differences in mobility patterns obtained from assessing 2D versus 3D tracks. Moreover, applying correlative super-resolution imaging with single particle tracking could provide additional insights into BRCA2 dynamics[3].

Using multicolor 3D tracking, we can see that BRCA2 accumulates at damage sites, exhibits confined motion, and forms distinct nanoscale clusters[3]. We, further, aim to understand spatial organization of BRCA2 during different stages of the repair process, its assembly and disassembly within the BRCA complex and its specific role in HR.

Mirko Weber, Felix Erichson, Richard Börner

Laserinstitut Hochschule Mittweida, Mittweida, Germany

Understanding the complex biomolecular mechanisms underlying long-range RNA tertiary contacts [1] is critical for advancing RNA-based therapeutics and drug discovery applications. However, current state-of-the-art structure prediction methods like RNAfold [2] for secondary structures and de novo modeling approaches such as RNAcomposer [3] and Rosetta [4] yield remarkable results, but struggle to compose binding events of spatially separated secondary structure elements, i.e., the formation of RNA tertiary contacts. As a result, the combination of FRET-assisted RNA structure modeling and de novo RNA building with subsequent MD simulations as an integrative modelling approach gained popularity for capturing structural dynamics and unraveling folding pathways [5, 6].

To streamline the time-consuming process of preparing MD simulation-compatible PDB files, we developed an easy-to-use, platform-independent command line tool written in Python. This tool efficiently rearranges and cleans up distorted PDB files derived from RNA building with PyMOL [7]. Further, our tool provides users with the flexibility to choose from three distinct operations: verifying the integrity of chain identifiers, ensuring proper terminators, and promoting consistent usage of RNA building blocks. Additionally, users can also combine these operations to tailor the tool's functionality according to their specific needs enhancing their own FRET-based integrative modeling pipelines.

Tino Röhlicke, Hans-Jürgen Rahn, Nicolai Adelhöfer, Torsten Krause, Florian Weigert, Michael Wahl

PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany

We present a new high resolution design of FPGA-based Time-Correlated Single Photon Counting (TCSPC) and time tagging electronics with multiple synchronized input channels. The new instrument achieves a state-of-the-art digital time resolution of 1 ps and a single channel timing uncertainty of 2 ps rms along with a world record dead-time of only 650 ps. This unprecedented combination enables new high-resolution TCSPC applications and significantly improves the resolution of the established high-speed fluorescence lifetime imaging method RapidFLIM. In oder to support the widest possible variety of single photon detectors the new instrument provides software-configurable input circuitry. For optimal timimg with e.g. Superconducting Nanowire Single Photon Detectors (SNSPD) the inputs can be configured as edge triggers while for best performance with Hybrid Photodetectors (HPD) or Micro Channel Plates (MCP) they can be configured as vertex finding Constant Fraction Discriminators (CFD). Along with the new instrument we developed an elegant and easy to use new Application Programming Interface (API) for Python. In addition to time tagged data acquisition it provides a wide variety of data analysis methods and real-time visualizations for typical requirements in quantum optics and fluorescence measurements. All methods can also be applied on recorded data files. Apart from design features and benchmark results of the instrument as such, we present results from fast fluorescence lifetime imaging and some snapshots of other applications.

Xin Zhu, Rowan Walker Gibbons, Ali Behjatian, Madhavi Krishnan

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, OX1 3QZ, United Kingdom

Inferring the 3D structure and conformation of disordered biomolecules, e.g., single stranded nucleic acids (ssNAs) in solution remains challenging due to both their conformational complexity, and the limited applicability of high resolution structural techniques such as x-ray crystallography. Here, we measure with sub elementary charge precision the effective charge, qeff, of short to medium chain length ssNAs in solution (5-60 bases) using the Escape-Time electrometry (ETe). ETe is a technique based on optical microscopy technique that can report on moelcular 3D conformation in a rapid and facile way. Combining ETe measurements with theoretically calculated qeff values for structures obtained from Molecular Dynamics simulations and analyzed in a Poisson Boltzmann (PB) electrostatics framework, we uncover a dependence between the molecular effective charge of ssNAs in solution and 3D conformational properties of nucleic acid oligomers, shedding light, e.g., on the stacking strength between nucleobases. Such properties of ssNAs of course dramatically depart from their double stranded counterpart. In summary, the ability of ETe to discriminate between effective charges of molecular species at the 1% level holds great promise for a new charge-based biomolecular analytical tool offering readouts of molecular 3D conformation, operating at the single molecule level.