31st International Workshop

Biomolecular conformations and dynamics of nucleic acids

Victoria Birkedal

Department of Chemistry, Aarhus University, Denmark

Nature controls the folding of nucleic acids to store information, regulate genes, and build complex functional structures. We do not yet fully understand this fantastic ability to program molecules with precise folding, which is guided by sequences of bases and environmental and biological triggers.

Nucleic acids folding is often dynamic and I will show how we use fluorescence spectroscopy and resonance energy transfer to probe conformations, molecular interactions and dynamics. The combination of these techniques with single molecule microscopy offers unique views of static and dynamic population heterogeneities and insights into the folding ’machinery’.

Integrating DNA-PAINT with electron microscopy for ultrastructural molecular imaging

Johannes Stein

Emmy Noether Group Leader Max Planck Institute for Molecular Genetics

DNA-PAINT enables highly multiplexed molecular imaging in cells, yet the resulting maps remain largely disconnected from cellular ultrastructure. Electron microscopy resolves cellular architecture at nanometer resolution but lacks molecular specificity. Bridging these views has been a longstanding challenge.
We present a correlative workflow combining kinetically enhanced DNA-PAINT (tkPAINT) with transmission electron microscopy (TEM). Cellular ultrastructure is preserved during cryosectioning, labeling, and multiplexed Exchange-PAINT imaging, followed by TEM processing of the same sections. This allows nanometer-scale registration of single-protein localizations directly onto defined ultrastructural features.
Biomolecules can thus be assigned to specific membranes and organelle subdomains within the same specimen, enabling molecular organization to be interpreted in their structural context. Our approach links highly multiplexed single-molecule imaging with electron microscopy and provides a promising route toward ultrastructure-resolved cell biology.

Biology’s brightest: Single fluorescent protein emission studies with the Luminosa confocal microscope

Julien Goldenberg¹, Klaudia Filipek², Evangelos Sisamakis², Claudio Flores¹, Robert Strutt¹

¹Mimotype Technologies GmbH, Friedrichstraße 17, 10969 Berlin, Germany
²PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany

Over the past few decades, fluorescent protein (FP) bioengineering has established a distinct class of fluorescent molecules. Designed principally for bioimaging, purified FPs now possess a range of attractive photophysical and practical properties (1). Studying purified FPs reveals that such properties enable applications that extend beyond genetic encodability, supported by advances in AI-enabled protein design and recombinant protein production.

This talk details a cross-company project between Mimotype and PicoQuant. We present an in-depth sample production and analysis workflow of single FP emission using TCSPC and FCS, focusing on β barrel red fluorescent proteins such as mScarlet3, with record brightness and photostability (2). By improving the photon budget in the solid state, antibunching curves comparative to small molecule dyes may be achieved with FPs (3). To this end, we explore surface coating, functionalization and waveguide implementation (4). Numerous FPs are analyzed including synthetically enhanced FPs and recent highly engineered mutants, determining relevant rate constants. This work establishes PicoQuant’s Luminosa confocal microscope as an essential device for FP engineering, closing the loop between FP in silico design, and experimental quantification at the single protein level. This engineering loop is expected to unlock new applications at the intersection of protein design, bioimaging, photonics and quantum biology (5).

1. Menchicchi B, Stiel AC, Nieddu M, Fuenzalida-Werner JP. Fluorescent proteins: A journey from the cell to extreme environments in material science. Photochem Photobiol. 102: 290-302 (2026) https://doi.org/10.1111/php.70011

2. Gadella, T. W. J., van Weeren, L., Stouthamer, J. et al. mScarlet3: a brilliant and fast-maturing red fluorescent protein. Nat. Methods 20, 541–545 (2023). https://doi.org/10.1038/s41592-023-01809-y

3.Kim, Y., Puhl, H. L., Chen, E., Taumoefolau, G. H., Nguyen, T. A., Kliger, D. S., Blank, P. S. & Vogel, S. S. VenusA206 dimers behave coherently at room temperature. Biophys. J. 116, 1918–1930 (2019). https://doi.org/10.1016/j.bpj.2019.04.014

4. Khelidj, H., Gourdin, A., Ozerov, I. et al. High-throughput fabrication of zero-mode waveguide nanoaperture arrays with sol–gel nanoimprint lithography for enhanced single-molecule fluorescence detection. Small (2026). https://doi.org/10.1002/smll.202510587

5. Burd, S. C., Bagheri, N., Condon, A. F. et al. Magnetic resonance control of spin-correlated radical pair dynamics in vivo. Nature 651, 940–945 (2026). https://doi.org/10.1038/s41586-026-10282-4

THE “BLISS” OF OBSERVING SINGLE-MOLECULE BURSTS FROM CLUSTERS IN LIVE CELL BIOMOLECULAR CONDENSATE

Khalil Joron¹, Eden Mishne³, Eran Meshorer^3,4, Eitan Lerner^1,2

¹Dept. Biological Chemistry, The Alexander Silberman Institute for Life Sciences, The Faculty of Sciences, The Hebrew University of Jerusalem
²The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem
³Dept. Genetics, The Alexander Silberman Institute for Life Sciences, The Faculty of Sciences, The Hebrew University of Jerusalem
⁴Edmond and Lilly Center for Brain Sciences (ELSC), The Faculty of Sciences, The Hebrew University of Jerusalem

For many years, the dream was to collect fluorescence-based data from single biomolecules in live cells. Recently, this has been achieved. The desire remains to acquire fluorescence-based data, one biomolecular assembly at a time, using endogenously expressed proteins tagged with canonically used monomeric fluorescent proteins (FPs) in highly concentrated cellular foci. Here, I present a rather simple approach, dubbed BLISS, for acquiring clusters of mCherry-tagged heterochromatin protein 1α (HP1α) within heterochromatin condensates in live embryonic stem cells. Through this approach, I will show that such mCherry-HP1α clusters exhibit lower fluorescence lifetimes than smaller mCherry-HP1α species and lower-than-average fluorescence lifetimes in FLIM pixels, revealing the source of heterogeneity that is averaged out in diffraction-limited fluorescence microscopy. 

Photon-Resolved Laser-Scanning Microscopy Meets Single-Molecule Microscopy

Giuseppe Vicidomini

Italian Institute of Technology (IIT)

Recent advances in asynchronous-readout Single-Photon Avalanche Diode (SPAD) array technology are enabling a new generation of photon-resolved laser-scanning microscopes. Unlike conventional single-element detectors, SPAD arrays provide spatially resolved detection while retaining single-photon sensitivity and picosecond timing capabilities, enabling each detected fluorescence photon originating from the sample region addressed by the focused excitation beam to be characterized by a series of precise spatial and temporal signature. These capabilities are particularly valuable in single-molecule microscopy.  Photon-resolved detection offers new opportunities for studying molecular dynamics through fluorescence fluctuation spectroscopy and single-molecule tracking, while also enabling advanced imaging strategies to investigate nanoscale molecular organization.

In this contribution, I will focus on the latter aspect and discuss how SPAD-array detection can enhance single-molecule imaging in laser-scanning microscopes thorough its combination with the image-scanning microscopy (ISM) concept. I will first present ISM-FLUX, a localization strategy that combines concepts from ISM and MINFLUX to achieve nanometric localization precision using spatially resolved photon detection. I will then introduce Single-Molecule Localisation  ISM (SML-ISM), where part of the reconstruction strategies developed for ISM-FLUX are leveraged to reconstruct large field-of-view and robust super-resolved single-molecule images .

Together, these approaches demonstrate how photon-resolved laser-scanning microscopy can leverage the unique capabilities of SPAD arrays to bridge single-molecule localization, super-resolution imaging, and quantitative photon-by-photon analysis within a unified experimental framework. More broadly, I will show how SPAD-array detection transforms a laser-scanning microscope into a tunable-resolution imaging platform, spanning the range from diffraction-limited imaging, through the twofold resolution enhancement of ISM, down to single-molecule resolution with SM-ISM.

Quantitative Kinetics and Model Selection from PET-FCS Measurements by Bayesian Inference

Annke de Maeyer, Helmut Grubmüller, Lars V. Bock

Department of Theoretical and Computational Biophysics, Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany

Photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) is a powerful technique for probing fluorescence fluctuations caused by changes in the quenching of fluorescent dyes, providing insight into biomolecular conformational dynamics. Here, we present an approach for extracting maximal information from PET-FCS data by directly inferring kinetic Markov models. We derived an analytical closed-form expression for the autocorrelation function that explicitly incorporates transition rates and accounts for both fluorescent and non-fluorescent (quenched) conformational states. Using Bayesian inference, our method identifies the underlying kinetic model and provides transition rates with rigorous uncertainty estimates. We validated our approach on synthetic data and applied it to experimental PET-FCS data of the cotranslational folding of the small alpha-helical domain of the HemK protein. By combining PET-FCS data from multiple experiments, we obtained kinetic models that reveal conformational transitions occurring on microsecond to millisecond timescales.

Interplay of Depletion-Interaction and Viscosity Modulates DNA G-Quadruplex-Ligand Binding in Crowded Environments

Pramod Kumar¹, Ajay Kumar Chand¹, Harsh Sahu¹, Shubham Sharma¹, Sobhan Sen¹, ²

¹Jawaharlal Nehru University, New Delhi-110067, India
²

G-quadruplex DNA (G4-DNA) structures, formed by the folding of guanine-rich single-stranded DNA in the presence of monovalent cations (K⁺ or Na⁺), are commonly found in telomeric and promoter regions and play key roles in biological processes [1,2]. These structures offer specific binding sites for small molecules (ligands), making them attractive targets for anti-cancer therapies. Understanding the thermodynamics and kinetics of ligand/G4-DNA interactions under cell-like crowded conditions is crucial, as molecular crowding can significantly alter ligand-binding affinity and reaction rates. Here, we use fluorescence correlation spectroscopy (FCS), along with complementary techniques, to examine the thermodynamics and kinetics of binding of a porphyrin derivative (TMPyP4) with parallel c-MYC G4-DNA, both in the absence and presence of EG/PEG crowders of varying sizes. We show that smaller crowders reduce ligand binding affinity due to viscosity-induced deceleration in ligands’ association rates and an increase in the dissociation rates due to soft hydrogen bonding interaction with the ligand [3-5]. However, larger crowders enhance ligand binding affinity to G4 via the depletion interaction (excluded volume) effect. This competition between viscosity and depletion-interaction effects depends on crowder size, with TMPyP4 showing an enhanced depletion response relative to viscosity effects on the thermodynamics and kinetics of ligand interaction with G4 as the crowder molecular weight (size) increases. These findings align well with the modified Collins-Kimball-Debye theory of polymer physics on diffusion-controlled kinetics [6] and highlight the importance of selecting appropriate crowders when mimicking intracellular conditions, especially in the context of designing G4-targeted therapeutics.

1.       Spiegel, J. et al. Trends Chem. 2020, 2, 123.

2.       Rhodes, D. et al. Nucleic Acids Res. 2015, 43, 8627.

3.       Alam, P. et al.  J. Phys. Chem. B 2025, 129, 2958.

4.       Clovis, N.S. et al. J. Photochem. Photobiol. A Chem. 2023, 437, 114432.

5.       Alam, P. et al. Methods Appl. Fluoresc. 2024, 12, 045002.

6.       Zosel, F. et al. Proc. Natl. Acad. Sci. 2020, 117, 13480.

High-throughput single-molecule DNA flow-stretch assays for quantitative fluorescence mapping of DNA–protein interactions

Ayush Kumar Ganguli¹, Mohammad Nour Alsamsam², Ugne Bagdonaite¹, VanTruc Vu³, Chun-Jen Huang⁴, Polina Kuzhir⁵, Mindaugas Zaremba¹, Aurimas Kopustas², Marijonas Tutkus¹

¹Institute of Biotechnology, Life Sciences Center, Vilnius University, Lithuania
²Department of Molecular Compound Physics, Center for Physical Sciences and Technology, Vilnius, Lithuania
³Department of Chemical & Materials Engineering, National Central University, Taoyuan, Taiwan
⁴School of Materials Science and Engineering, The University of New South Wales, Sydney, Australia
⁵Department of Physics and Mathematics, Center for Photonics Sciences, University of Eastern Finland, Joensuu, Finland

Single-molecule fluorescence imaging provides direct access to the dynamic behavior of DNA–protein interactions, but its broader application is often limited by low throughput and variability across experiments.¹
Here, we establish a high-throughput single-molecule DNA flow-stretch assay that enables quantitative fluorescence mapping of DNA–protein interactions on extended DNA substrates. Using hydrodynamic alignment of surface-tethered DNA molecules combined with total internal reflection fluorescence (TIRF) microscopy, the platform allows simultaneous observation of hundreds of individual DNA molecules within a single field of view, substantially increasing statistical robustness while retaining single-molecule resolution.^2-4
We systematically optimize surface functionalization, DNA substrate design, and flow conditions to achieve reproducible DNA immobilization and uniform extension. This enables accurate spatial localization of protein binding events along DNA, as well as real-time observation of binding, dissociation, and flow-dependent dynamics. Importantly, defined DNA constructs allow direct correlation between the position of the fluorescence signal and the underlying sequence coordinates.^2-4
To support quantitative analysis, we implement image-based workflows for extracting DNA length distributions, localizing binding sites, and characterizing interaction dynamics across large molecular populations. As a demonstration, we resolve sequence-specific binding of catalytically inactive Cas9 (dCas9), achieving precise localization consistent with the designed target site.²
This work establishes a scalable single-molecule fluorescence platform that combines throughput, spatial precision, and quantitative analysis, providing a robust framework for studying DNA–protein interactions beyond the limitations of conventional single-molecule assays

1.Finkelstein IJ and Greene EC ,Supported lipid bilayers and DNA curtains for high-throughput single molecule studies , Methods Mol Biol ,745, 447–461.(2011)
2.Ganguli AK, Alsamsam MN, Bagdonaitė U, Vu VT, Huang CJ, Kuzhir P, Zaremba M, Kopūstas A and Tutkus M, Single-molecule DNA flow-stretch assays for high-throughput DNA–protein interaction studies, FEBS Open Bio, e70211 (2026).
3.Kopustas A, Zaremba M and Tutkus M , DNA flow-stretch assays for studies of protein-DNA interactions at the single-molecule level, Applied Nano, 3,16–41.(2022)
4.Kopustas A, Ivanovait_ e SN, Rakickas T, Poceviciu Te E, Paksaite J, Karvelis T, Zaremba M, Manakova E and Tutkus M, Oriented soft DNA curtains for single-molecule imaging .Langmuir , 37, 3428–3437.(2021)

Label-free Fluorescence lifetime microscopy for metabolic imaging across scales

Chiara Stringari

Laboratory for Optics and Biosciences (LOB), CNRS, INSERM, École polytechnique, IP Paris, 91128 Palaiseau, France

Fluorescence lifetime imaging microscopy (FLIM) is emerging as a transformative tool in biophotonics. It enables the high-resolution, non-invasive imaging of metabolic processes in live tissues by measuring the lifetime of endogenous biomarkers (such as NAD(P)H and FAD) that are naturally present in cells and tissues, providing a quantitative readout of metabolic pathways, such as glycolysis and oxidative phosphorylation [1, 2]. However, traditional FLIM approaches used to measure and quantify metabolism are limited by factors such as slow acquisition speed, poor signal-to-noise ratio (SNR) and high phototoxicity [3, 4]. In our laboratory, we aim to develop nonlinear optics and FLIM techniques that can measure metabolic transitions at the cellular level across spatial and temporal scales with enhanced speed and performance. In this talk, we will highlight recent advances and applications in label-free metabolic imaging using two-photon fluorescence lifetime microscopy (2P-FLIM) of intrinsic metabolic coenzymes. We discuss here how we implement simultaneous two-photon excitation of NAD(P)H and FAD through wavelength mixing in order to acquire FLIM data of the two biomarkers simultaneously and perform multiparametric metabolic imaging in dynamic biological systems, such as during embryo development and in living tissues [5] and we demonstrated that the two endogenous fluorophores are complementary biomarkers [6]. To improve the interpretation of the FLIM data for metabolic imaging we also developed FLUTE, a Python Graphical User Interface [7]. We then demonstrate how FLIM of metabolic coenzymes reveals the complexity of metabolic processes in intact tissues with minimal phototoxicity and we discuss the optimization and the implementation of this methodology for longitudinal in vivo studies. Finally, we present applications for metabolic imaging in neuroscience, developmental biology, and immunometabolism, measuring subcellular metabolic compartmentalization and complex temporal metabolic patters during stem cell differentiation [8], T cell activation [9] and embryo development.

1. Georgakoudi, I. and K.P. Quinn, Label-Free Optical Metabolic Imaging in Cells and Tissues. Annu Rev Biomed Eng, 2023.
2. Datta, R., et al., Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications. J Biomed Opt, 2020. 25(7): p. 1-43.
3. Poudel, C., I. Mela, and C.F. Kaminski, High-throughput, multi-parametric, and correlative fluorescence lifetime imaging. Methods Appl Fluoresc, 2020. 8(2): p. 024005.
4. Park, J. and L. Gao, Advancements in fluorescence lifetime imaging microscopy Instrumentation: Towards high speed and 3D. Curr Opin Solid State Mater Sci, 2024. 30.
5. Stringari, C., et al., Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing. Sci Rep, 2017. 7(1): p. 3792.
6. Ung, T.P.L., et al., Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA-induced metabolic stress in reconstructed human skin. Sci Rep, 2021. 11(1): p. 22171.
7. Gottlieb, D., et al., FLUTE: a Python GUI for interactive phasor analysis of FLIM data Biological Imaging., 2023. 3:e21.
8. Sánchez-Ramírez, E., et al., Coordinated metabolic transitions and gene expression by NAD+ during adipogenesis. J Cell Biol., 2022. 221(12).
9. Paillon, N., et al., Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes. 2024. 35(1): p. ar11.

Assessing the membrane properties of Oli-neu cells using FLIM and labelled lipids

Fabian Ruebsamen¹, Annika Haak², Thomas Günther-Pomorski¹

¹Department of Biochemistry II, Ruhr-Universität Bochum, Bochum, Germany
²RG Nanoscopy, RUBION, Ruhr-Universität Bochum, Bochum, Germany

Oligodendrocytes are the myelinating cells of the central nervous system, which insulate neurons. During the first three postnatal weeks, oligodendrocytes undergo a tightly regulated differentiation process from oligodendrocyte precursor cells into mature, myelin-producing cells. This transition is accompanied by drastic changes in plasma membrane composition and membrane properties required to enable the formation of the highly specialised myelin-containing membranes. In this study, the secondary mouse oligodendroglial cell line Oli-neu was employed as an in-vitro model to investigate membrane dynamics during oligodendrocyte maturation: Membrane viscosity and lipid behaviour were examined using fluorescence lifetime imaging microscopy (FLIM)-based viscosity probes and fluorescently labelled lipid derivatives. These complementary techniques enabled the characterisation of membrane microenvironment properties and lipid movement.

Mapping Structurally Variable DNA Constructs with FRET Nanoscopy and Optical Pythagoras

Noah H. Salama¹, Nicolaas T. M. van der Voort¹, Jan-Hendrik Budde¹, Michelle P. Rademacher², Suren Felekyan¹, Christian A. Hanke¹, Alexander Henrichs¹, Ralf Kühnemuth¹, Anders Barth³, Claus A. M. Seidel¹

¹Chair for Molecular Physical Chemistry, Heinrich-Heine-University Düsseldorf, Germany
²Institute for Physical Chemistry II, Heinrich-Heine-Universität Düsseldorf, Germany
³Department of Bionanoscience, Delft University of Technology, Delft, Netherlands

Recently, the lateral resolution of super-resolution microscopy reached the size of individual molecules.[1] Nevertheless, obtaining precise information on 3D orientation of structures remains challenging.
We established an approach combining colocalization stimulated emission depletion (cSTED) microscopy with concurrent analysis of Förster resonance energy transfer (FRET) to identify heterogeneities in DNA systems.[2] We obtained precise lateral and axial distances using the Optical Pythagoras by comparing the localization distance between two fluorophores from cSTED to the Euclidean distance based on a structure model or obtained from FRET analysis. We simultaneously localize both fluorophores of single FRET pairs, yielding sample orientation in 3D, with sub-nanometer localization precision for individual dyes. Scanning 1x1 µm in a few seconds, we reached an average distance precision of 1.4 nm for a donor and acceptor label coupled to a single molecule. Studying immobilized dsDNA rulers, bound to the surface from one or both ends, we measure a mean inclination angle of 64° and 34°, respectively. The latter angle correlates well with the local roughness of the surface found by AFM measurements.
We apply our workflow on the level of single particles, enabling us to map conformational exchange within the 3D structure of DNA Holliday junctions.

[1] Hell, S. W. et al., Science, 355, 606-612 (2017).

[2] Budde, J.-H. et al., arXiv preprint, doi.org/10.48550/arXiv.2108.00024 (2022).

Integrating DNA-PAINT with electron microscopy for ultrastructural molecular imaging

Johannes Stein

Emmy Noether Group Leader Max Planck Institute for Molecular Genetics

DNA-PAINT enables highly multiplexed molecular imaging in cells, yet the resulting maps remain largely disconnected from cellular ultrastructure. Electron microscopy resolves cellular architecture at nanometer resolution but lacks molecular specificity. Bridging these views has been a longstanding challenge.
We present a correlative workflow combining kinetically enhanced DNA-PAINT (tkPAINT) with transmission electron microscopy (TEM). Cellular ultrastructure is preserved during cryosectioning, labeling, and multiplexed Exchange-PAINT imaging, followed by TEM processing of the same sections. This allows nanometer-scale registration of single-protein localizations directly onto defined ultrastructural features.
Biomolecules can thus be assigned to specific membranes and organelle subdomains within the same specimen, enabling molecular organization to be interpreted in their structural context. Our approach links highly multiplexed single-molecule imaging with electron microscopy and provides a promising route toward ultrastructure-resolved cell biology.

DNA-PAINT super-resolution microscopy for carbon nanotube identification

Nicco Corduri¹, Alan Szalai², Karol Kołątaj¹, Guillermo Acuna¹

¹University of Fribourg
²Centro de Investigaciones en Bionanociencias, CIBION / University of Fribourg

Super-resolution microscopy has become indispensable for investigating nanoscale architectures beyond the diffraction limit of conventional optical systems, ultimately reaching 1-10 nm spatial resolution. However, fluorescent based super-resolution imaging of carbon nanotube (CNT)-based assemblies has mostly relied on inducing blinking on the CNT’s NIR photoluminescence, with a resolution limited to several tens of nanometers [1],[2]. Here, we present a DNA-PAINT [3],[4] imaging strategy developed for DNA-functionalized CNTs and apply it to the quantitative characterization of both isolated nanotubes and complex assemblies organized on DNA origami platforms. Remarkably, DNA-PAINT offers nanometer-scale localization precision combined with multiplexing capabilities. We first validate our method on isolated CNTs, reliably super-resolving individual nanotubes. The achieved localization precision of 2- 3 nm allows direct detection of individual docking sites along the CNTs. Notably, we observe a direct dependence of the spacing between docking sites on the length of the functionalizing DNA strands. Building on these results, we investigate DNA origami-directed assemblies [5],[6], where large rectangular templates position multiple CNT populations carrying different DNA sequences. Sequential DNA-PAINT imaging allows selective visualization of each CNT population. This approach allows the accurate reconstruction of two-dimensional arrays containing up to nine parallel CNTs with center-to-center separations as small as 14 nm.

[1] Lambert, B. P., Kerkhof, H., Flavel, B. S. & Cognet, L. ACS Nano 18, 30728–30736 (2024).
[2] Otsuka, K., Ishii, A. & Kato, Y. K. Opt. Express 27, 17463–17473 (2019).
[3] Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Nat. Protoc. 12, 1198–1228 (2017).
[4] Jungmann, R. et al. Nano Lett. 10, 4756–4761 (2010).
[5] Rothemund, P. W. K. Nature 440, 297–302 (2006).
[6] Maune, H. T. et al. Nat. Nanotechnol. 5, 61–66 (2010).

MINFLUX-enabled 3D single-particle tracking reveals live-cell neuronal membrane topography

Bela T. L. Vogler^1,2, Christian Eggeling^1,2,3

¹Institute of Applied Optics and Biophysics, Friedrich-Schiller University Jena, Jena, Germany.
²Department of Biophysical Imaging, Leibniz Institute of Photonic Technologies e.V., Jena, Germany, Member of the Leibniz Centre for Photonics in Infection Research (LPI), Jena, Germany
³Jena Center for Soft Matter, Jena, Germany.

MINFLUX fluorescence microscopy enables nanometer-precision localization at microsecond temporal resolution, offering unique opportunities for three-dimensional (3D) single-particle tracking (SPT). Here, we implement 3D MINFLUX tracking in live neurites to resolve the motion of quantum dot–labelled GPI-GFP molecules on curved tubular membranes. Kilohertz sampling permits analysis of membrane dynamics across timescales from microseconds to seconds. The true 3D tracking capability allows us to quantify diffusion along the tubular axis and to detect periodic confinement with a characteristic spacing of ~200 nm. This spacing is consistent with diffusion barriers imposed by the submembrane actin–spectrin lattice. Our results demonstrate that 3D MINFLUX SPT provides robust and quantitative access to nanoscale membrane organization in complex cellular geometries.

A photometric pinhole: event-based optical sectioning without demodulation

Adrian Platz, Gabriel Baum, Andreas Stark, Christian Franke

Institute of Applied Optics and Biophysics, Friedrich-Schiller Universität Jena

Optical sectioning in widefield fluorescence microscopy currently relies on physical apertures or computational demodulation of structured illumination. Demodulation methods fail catastrophically in scattering tissue when the pattern model breaks down in 3D cell cultures, tissue sections, and organoids.

Here, we introduce the event-based photometric pinhole. Therefore, homogeneous illumination with distinct intensity minima (holes) is projected onto the sample, and fluorescence is detected with an event-based camera. The camera's hardware log-contrast threshold θ acts as an axial gate: in focus, the hole produces contrast exceeding θ and triggers events; out of focus, diffraction reduces contrast below θ. The gate is pattern-agnostic: each pixel evaluates its local contrast independently. Scattering can blur a hole but cannot invert it: a hole stays a hole, and sectioning degrades gracefully with depth rather than producing artefacts.

At dhole = 3.5 µm we achieve ~600 nm optical sectioning at 640 nm illumination, comparable to Airyscan- and confocal microscopy. Furthermore, we present a forward model that predicts sub-250 nm optical sectioning for an optimized illumination system.

We demonstrate sectioning in fixed cell cultures, thick brain sections, and glioblastoma tumoroids. The method requires no scanning and no pattern knowledge.

Quantitative mapping of nanoscale protein assemblies by DNA-PAINT

Alexandra Kaminer, Yunqing Li, Jasmin Janzen, Marina Dietz, Mike Heilemann

Activation of receptor tyrosine kinases begins with ligand-induced receptor engagement at the cell surface, leading to the formation of membrane-associated signaling assemblies that activate intracellular signal transduction[1]. Determining the molecular composition and spatial organization of these assemblies in situ remains challenging due to their nanoscale dimensions and intrinsic heterogeneity. Here, we present a single-molecule super-resolution imaging (DNA-PAINT) and analysis workflow that enables the identification and quantitative characterization of individual signaling assembly sites in cells. Using this approach, we examine the nanoscale reorganization of the epidermal growth factor receptor (EGFR) together with its adaptor protein Grb2 following stimulation with the physiological ligand EGF[2]. Furthermore, we apply this workflow to determine the molecular architecture of MET-receptor assemblies at the plasma membrane in a ligand-stimulation dependent manner[3], [4].

[1] Lemmon, M. A. & Schlessinger, J. Cell 141, 1117–1134 (2010).

[2] Kaminer, A., Li, Y., Barth, H.-D., Dietz, M. S. & Heilemann, M. ChemPhysChem 27, e202500915 (2026).

[3] Niemann, H. H. European Journal of Cell Biology 90, 972–981 (2011).

[4] Li, Y., Arghittu, S.M., Dietz, M.S. et al. Nat Commun 15, 9486 (2024).

Chemigenetic tools for live-cell fluorescence microscopy

Michelle S. Frei

Institut für Chemie und Angewandte Biowissenschaften ETH Zürich

Synthetic fluorophores, in combination with self-labeling protein tags, have become indispensable tools in fluorescence microscopy. Compared to fluorescent proteins, synthetic fluorophores show enhanced brightness, photostability, and are available in the far-red to near-infrared spectral region, which is beneficial for advanced fluorescence microscopy applications including super-resolution microscopy. Moreover, fluorogenic fluorophores, which become fluorescent only upon target binding, are particularly well suited for live-cell applications due to their high signal-to-background ratios. While synthetic chemistry allows fine-tuning of a fluorophore’s photophysical properties, the protein environment surrounding the fluorophore binding site also plays a crucial role. Here, we demonstrate how protein engineering of the self-labeling protein HaloTag can be harnessed to develop innovative tools for live-cell fluorescence microscopy.

First, we present a series of engineered HaloTag variants with altered brightness and fluorescence lifetime characteristics that enable multiplexed imaging of up to three distinct targets with in a single spectral channel using fluorescence lifetime imaging microscopy (FLIM). Second, we explore the use of HaloTag beyond its conventional role as a labeling tag by integrating it into functional biosensors. Specifically, we sandwiched a circularly permuted version of HaloTag7 between a phosphoamino acid binding protein and a protein kinase A (PKA) specific peptide to get access to HaloTag-based kinases activity reporters (HaloKARs). Through protein engineering, we optimized the biosensor’s performance ultimately reaching a 12-fold change upon PKA stimulation. The best HaloKAR was successfully applied in advanced multiplexing experiments and functional super-resolution microscopy measurements.

Overall, our work underscores the power of combining protein engineering with synthetic fluorophores to create imaging tools with outstanding properties. We expect these HaloTag-based tools to facilitate the widespread use of advanced microscopy techniques.

Towards quantitative predictions of lifetime and anisotropy values of fluorophores on biomolecules

Daria Maksutova, Thomas-Otavio Peulen, Thorben Cordes

Biophysical Chemistry, Department of Chemistry and Chemical Biology, Technische Uni­ver­si­tät Dort­mund, Germany

Fluorescent labelling enables powerful fluorescence-based approaches, including single-molecule detection and Förster resonance energy transfer (FRET), to investigate biomolecular structure and function [1,2]. Quantitative interpretation of such experiments depends on the photophysical properties of attached dyes, which are modulated by local biomolecular and solvent environments and remain difficult to assess and predict [3]. Our work aims to establish a predictive framework for fluorescence parameters of dyes on biomolecules, accounting for local environmental effects.
To disentangle dye–protein and dye–solvent interactions, we investigate dyes from different structural classes attached to E. coli maltose-binding protein, a well-characterized test system. In multiparametric experiments down to the single-molecule level, we combine time-resolved and anisotropy-resolved fluorescence with fluorescence correlation spectroscopy to probe quenching processes from the pico- to millisecond range [4]. Solvent-mediated quenching contributions [5] are assessed simultaneously for red and far-red dyes by comparing measurements in aqueous and deuterated buffers.
By correlating fluorophore and protein structural features with experimental fluorescence parameters [3], we establish the basis for a coarse-grained dye model that captures solvation- and amino-acid-mediated quenching via photoinduced electron transfer [4]. Our results reveal pronounced site-dependent variations in fluorescence lifetime and anisotropy, underscoring the need for predictive models to guide fluorescence experiment design.

[1] G. Agam et al., Nature Methods, 20.4, 523–535 (2023).
[2] C. Gebhardt et al., Nature Communications, 16.1, 4147 (2025).
[3] T.-O. Peulen, D. Maksutova, and T. Cordes, arXiv:2602.21931 (2026).
[4] T.-O. Peulen, O. Opanasyuk, and C. A. M. Seidel, The Journal of Physical Chemistry B, 121.35, 8211–8241 (2017).
[5] J. Maillard et al., Chemical Science, 12.4, 1352–1362 (2021).

Parallel stopped-flow interrogation of diverse biological systems at the single-molecule scale

Scott C. Blanchard^*1,2, Roman Kiselev⁺¹, Ryan A. Brady⁺¹, Arnab Modak¹, Jose L. Alejo¹, Aaron Cruz-Navarrete¹, Daniel S. Terry¹, Roger B. Altman¹, Wesley B. Asher^3,4, Jonathan A. Javitch^3,4,5

¹Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA
²Department of Chemical Biology & Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN, USA
³Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA
⁴Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA
⁵Department of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA
^*Corresponding Author
⁺These authors contributed equally

Single-molecule imaging techniques have provided unprecedented insights into functional changes in composition and conformation across diverse biological systems. As with other biophysical methods, single-molecule fluorescence and Förster resonance energy transfer investigations are typically limited to examination of one sample at a time. Consequently, experimental throughput is restricted, and experimental variances are introduced that can obscure functional distinctions in closely related systems. Here, to address these limitations, we introduce parallel rapid exchange single-molecule fluorescence and single-molecule Förster resonance energy transfer to enable simultaneous steady-state and pre-steady-state interrogations of diverse systems. Using this approach, we elucidate the timing of distinct conformational events underpinning β-arrestin1 activation, unmask antibiotic-induced impacts on messenger RNA decoding fidelity and demonstrate that endogenously encoded ribosomal RNA sequence variation modulates antibiotic sensitivity. This generalizable and scalable method promises to broaden the scope and reproducibility of quantitative single-molecule interrogations of biomolecular function.

Kiselev R, Brady RA, Modak A, Cruz-Navarrete FA, Alejo JL, Terry DS, Altman RB, Asher WB, Javitch JA, Blanchard SC. Parallel stopped-flow interrogation of diverse biological systems at the single-molecule scale. Nat Methods. 2026 Jan;23(1):78-87. doi: 10.1038/s41592-025-02944-4. Epub 2025 Dec 2. PMID: 41331139; PMCID: PMC12791014.

Metal-induced energy transfer uncovers activation-induced axial reorganization of signaling complexes inside cells

Eike Wienbeuker¹, Arthur Felker¹, Oleksii Nevskyi², Steffen T. Harms¹, Daniel Marx², Kevin Tanzusch¹, Anna Chizhik², Rainer Kurre¹, Changjiang You¹, Jörg Enderlein², Jacob Piehler¹

¹Department of Biology/Chemistry and Center for Cellular Nanoanalytics, Osnabrück University, Barbarastr. 11, Germany
²Third Institute of Physics (Biophysics), Georg August University, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany

Transmembrane signaling mediated by cytokine receptors orchestrates key cellular processes such as proliferation, differentiation, and immune responses. While numerous high-resolution structures of cytokine receptor ectodomains are available, the structural organization of the largely disordered intracellular domains (ICD) has remained unclear. Here, we interrogate the axial organization of cytokine receptor signaling complexes at the plasma membrane by metal-induced energy transfer (MIET). For this purpose, we leveraged biofunctionalized nanodot arrays (bNDAs) to capture cell surface receptors at a defined distance from the substrate. This approach enables axial distance measurements with ~1 nm resolution at both ensemble and single-molecule levels via fluorescence lifetime imaging microscopy (FLIM). Using the prototypic class I cytokine receptor GP130 as a model system, we uncover by MIET that the ICD extends randomly into the cytosol in the resting state but undergoes an axial rearrangement upon signal activation. These results highlight the potential of bNDA-supported MIET for resolving the axial architecture of signaling complexes within the cellular context.

DNA Origami-Based Plasmonic Nanoantennas for Amplification-Free Single-Molecule microRNA Detection

Kristina Djordjevic, Julian Bauer, Renukka Yaadav, Philip Tinnefeld

Department of Chemistry and Center for NanoScience, Ludwig-Maximilians- University, Butenandtstraße 5−13, 81377, Munich, Germany

DNA origami-based plasmonic nanoantennas have emerged as a powerful platform for amplification-free single-molecule fluorescence detection by combining programmable DNA nanotechnology with the electromagnetic field enhancement of metallic nanoparticles.1 Here, we present DNA origami nanoantenna architectures for ultrasensitive nucleic acid detection, with a particular focus on microRNA (miRNA) sensing for future point-of-care diagnostics. The structures are based on DNA origami “Fork” geometries that enable nanometer-precise positioning of silver nanoparticles and nucleic acid assay components within plasmonic hotspots.2,3 Upon target hybridization, fluorescent imaging strands localize in the hotspot region, resulting in strongly enhanced fluorescence signals detectable at the single-molecule level by confocal and TIRF microscopy. Different assay configurations were systematically investigated to evaluate sensitivity, specificity, kinetics, and structural stability. In addition to proof-of-concept nucleic acid detection, this work explores amplification-free miRNA sensing motivated by the diagnostic relevance of short regulatory RNAs in diseases such as cancer and endometriosis. Future studies will focus on systematic mismatch investigations to evaluate sequence discrimination and single-base mismatch sensitivity under plasmon-enhanced conditions. Furthermore, nanopatterning strategies will be expanded toward multiplexed nanoantenna arrays for high-density single-molecule biosensing.3,4

[1] Viktorija Glembockyte, Lennart Grabenhorst, Kateryna Trofymchuk et al., Acc. Chem. Res. 54, 3338-3348 (2021).

[2] Cindy Close et al., Adv. Mater. Interfaces, 9, 2200255 (2022).

[3] Renukka Yaadav et al., Adv. Mater., 37, 07407 (2025).

[4] Shetty R. M. et al., J. ACS Nano, 15, 11441-11450 (2021).

Development of smart DNA probes for multiplexed super-resolution microscopy

Florian Schüder

Institute of Bioengineering EPFL, Lausanne

Fluorescence microscopy, an important and widely used tool in biological research, has witnessed a true renaissance since the invention of methods that circumvent the so-called diffraction limit, namely super-resolution techniques. In resonance with these advancements, my work combines advanced high-resolution microscopy with novel approaches from the field of DNA Nanotechnology to push the limits of light microscopy and apply it to biological research. In this presentation, I will describe current advances in DNA-based super-resolution microscopy, including novel immunolabeling strategies, high-throughput imaging, and spectrally unlimited multiplexed imaging.

PhaseFLUX: A simple and robust 3D MINFLUX microscope

Fabian Hauser, Ioannis Pitsios, Jonas Ries

University of Vienna, Center for Molecular Biology, Department of Structural and Computational Biology, Vienna, Austria; Max Perutz Labs, Vienna Biocenter Campus (VBC), Vienna, Austria

MINFLUX [1] achieves superior localization precision over camera-based single-molecule localization microscopy [2] by rapidly probing individual fluorophores with a patterned beam featuring an intensity minimum. However, current implementations [2,3] are complex and expensive, often relying on deformable mirrors or electro-optical deflectors. Here we present PhaseFLUX, a simple, robust, and cost-effective MINFLUX implementation [4].

By combining an electro-optical modulator (EOM) and a binary spatial light modulator (SLM), our experimental setup achieves a localization rate of up to 1.8 kHz and 900 Hz per iteration in 2D and 3D, respectively. The resulting bilobed, high-contrast point-spread functions (PSFs) for x/y and a 3D donut PSF for z enable efficient 3D localization with lower laser powers, larger effective field of view, and improved precision for a given photon budget [4]. Sub microsecond EOM phase updates allow for ultrafast, repeated scanning of patterns by changing the PSFs minimum position, thereby reducing the impact of fluorophore fluctuations and enabling future multiplexed multi-colour extensions. Control and estimation algorithms [5] are implemented on a FPGA, enabling real time feedback for single molecule probing.

Our first prototype of PhaseFLUX demonstrates the feasibility of this approach while maintaining low component costs and provides a practical path toward high performance open source multi-colour MINFLUX instruments.

[1] F. Balzarotti et al., Science, 355, 606–612 (2017).
[2] K.C. Gwosch et al., Nat. Methods, 17, 217–224 (2020).
[3] R. Schmidt et al., Nat. Commun., 12, 1478 (2021).
[4] T. Deguchi et al., Light Sci Appl, 13, 134 (2024).
[5] Z. Marin et al., Nat Commun., 17, 246 (2026).

A unified approach to point-spread function calculations for complex imaging systems

Jörg Enderlein

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

Optical microscopy has undergone dramatic advancements over the past three to four decades, driven by the emergence of super-resolution techniques such as Stimulated Emission Depletion (STED) microscopy and Single-Molecule Localization Microscopy (SMLM). These developments have created a critical need for exact wave-optical modeling of such imaging modalities—specifically, the calculation of precise Point Spread Functions (PSF) required for optimal image reconstruction and deconvolution. For instance, accurate localization in SMLM relies on a detailed understanding of the underlying PSF, particularly when fluorescent emitters are not free to rotate.

Here, we present a unified framework for exact, quantitative PSF calculations applicable to a wide range of imaging modalities. Our approach leverages the principle of reciprocity between excitation and emission, which significantly simplifies the calculation of molecular detection functions, such as those of confocal microscopy.

After discussing the underlying physical principles of our model, we demonstrate its versatility through several applications in non-trivial imaging scenarios. Specifically, we address:

• Wide-field microscopy PSFs incorporating arbitrary optical aberrations;
• Confocal Laser Scanning Microscopy (CLSM), demonstrating the modeling of confocal detection with arbitrary detection-path apertures;
• Focusing through stratified media and compensating for refractive index mismatches;
• Defocused imaging of single molecules, accounting for emission dipole orientation;
• Scanning images of single molecules and handling complex excitation polarizations;
• STED microscopy, including the integration of optical non-linearities;
• Complex pupil functions, exemplified by calculating the PSF of the James Webb Space Telescope;
• Fluorescence Correlation Spectroscopy (FCS) modeling, which requires combining non-trivial excitation and detection volumes;
• Coherent imaging, as demonstrated through Interferometric Scattering (iSCAT) microscopy.

Finally, we introduce a comprehensive, open-source MATLAB package that enables researchers to perform arbitrarily complex PSF calculations for their own imaging applications.

Imaging Lipid species at the nanoscale

Aditya Chhatre¹, Tom Borianne¹, Anastasios Papangelis², Cecilie Martin-Lemaitre¹, Andre Nadler², Alf Honigmann¹

¹Technische Universitaet Dresden, BIOTEC, CMCB, Dresden, Germany.
²Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

Eukaryotic cell membranes are composed of thousands of chemically distinct lipid species whose asymmetric distribution is thought to regulate membrane protein assembly and function. However, directly observing lipid species distributions and lipid–protein interactions within cellular membranes has been severely limited by the lack of sub-organelle spatial resolution and chemical specificity in traditional imaging techniques. Here we show that combining minimally modified bifunctional lipid probes (bf-lipids) with multiplexed 3D DNA-PAINT microscopy overcomes these constraints, enabling the visualization of distinct lipid environments surrounding membrane protein complexes with close to molecular resolution. By implementing an optimized chemical linkage workflow to overcome steric constraints within dense lipid environments, we achieved high-density targeting of multiple lipid classes alongside specific organelle markers. Using sequential imager strand exchange and astigmatic 3D localization, we demonstrate a robust spatial precision of approximately 4 nm in XY and 15 nm in Z under TIRF illumination. These findings establish a versatile super-resolution workflow that bypasses a major experimental bottleneck in membrane biology, opening the door to mapping nanoscale lipid compartmentalization and specific lipid–protein interfaces directly inside cells.

Solid-state Luminescence Intermittency Unlocks in situ Label-free Super-Resolution Imaging of Supramolecular Fiber Growth

Xiaomin Liu¹, Peigen Yao¹, Alvaro Lopez-Acosta², Johnee Neethinathan¹, Márton Gelléri³, Ingo Lieberwirth¹, Thomas Basché⁴, Mischa Bonn¹, Johannes Gierschner², Thomas Hermans²

¹Max Planck Institute for Polymer Research, Mainz, Germany
²IMDEA Nanoscience, Madrid, Spain
³Institute of Molecular Biology, gGmbH, Mainz, Germany.
⁴Johannes Gutenberg-University Mainz, Mainz, Germany

Understanding the complex structures of molecular self-assembly requires imaging methods with high spatial resolution. However, common fluorescence based super-resolution microscopy requires extrinsic fluorescent labelling , which might perturb the assembly pathways. Here, we show a direct visualization of supramolecular fibers from a saccharide derivation (SachCHO), which exhibits solid-state luminescence intermittency (SLI), enabling label-free stochastic optical reconstruction microscopy (STORM). Mechanistic study reveals SLI is highly localized on fibers with structural defects, which favors the reversible photochemical reaction. SLI-STORM is further utilized to visualize the polymerization of fibers in-situ, revealing the non-classical growth and formation of structural defects. Due to the widespread existence of structural defects in supramolecular polymers, amyloid fibril aggregates are further investigated and shown to have similar SLI behavior, demonstrating general application of SLI. By leveraging the material's own photo-physics/chemistry, SLI-STORM demonstrates the ability to probe structural defects and assembly process with minimal invasion for synthetic and natural supramolecular structures.

1.             Yao, P.; Lopez-Acosta, A.; Liu, W.; Blaskovits, J. T.; Gelléri, M.; Lieberwirth, I.; Erker, C.; Andrienko, D.; Basché, T.; Bonn, M.; Hermans, T. M.; Liu, X. Solid-state Luminescence Intermittency Unlocks in situ Label-free Super-Resolution Imaging of Supramolecular Fiber Growth. ChemRxiv 2026. https://chemrxiv.org/doi/full/10.26434/chemrxiv.15000757/v1

Single-Particle Hyperspectroelectrochemistry for Operando Studies of Mass, Charge, and Energy Transfer in Plasmonic Nanomaterials

Landes, Christy F.

Department of Chemistry, University of Illinois Urbana-Champaign, Illinois 61801, United States, E-mail:cflandes@illinois.edu

I will discuss our recent use of hyperspectroelectrochemical microscopy to induce and measure mass, charge, and energy transport in individual hybrid plasmonic nanomaterials. Photocatalyzed chemical reactions are potential sustainable alternatives for industrial syntheses that otherwise require high temperatures and pressures. Plasmonic nanoparticles are promising light harvesters because of their high extinction cross-sections, but the challenge remains to harness absorbed light to do precision chemistry before it is transformed into heat. Gold nanorods have the additional advantage that they can both induce and sense local chemical changes through their longitudinal plasmon resonance. I will focus on inducing energy transfer between gold nanorods and molecular adsorbates to initiate local polymerization and the formation of plasmonic-polymer hybrid nanomaterials using operando single-particle hyperspectroelectrochemistry. Energy transfer efficiencies of over 50% are achievable when the spectral overlap between the gold nanorod scattering and monomer absorption is maximized. Single-particle measurements, supported by other spectroscopic evidence and theoretical calculations, help to distinguish among the many competing relaxation processes occurring in plasmonic nanomaterials. We use these measurements to demonstrate efficient energy cascade from photon to plasmon to exciton and finally, to new light-initiated chemistry.

Contact-Free Mechanical Properties Evaluation of Microscale 3D Printed Hydrogels Using Autofluorescence Fluorescence Lifetime Imaging Microscopy

Or Eivgi

Bar-Ilan University Ramat-Gan, Israel 529002

In this work we demonstrate a contact-free strategy to evaluate the mechanical properties of microscale 3D printed PEGDA hydrogels by exploiting their intrinsic autofluorescence using fluorescence lifetime imaging microscopy (FLIM). Cylindrical hydrogel micropillars are fabricated using two-photon 3D laser printing under systematically varied printing laser dose, generating structures with different degrees of crosslinking. The autofluorescence signal of these hydrogel pillars shows a clear dependence on the printing laser dose and the degree of acrylate conversion as obtained using Raman spectroscopy. In order to validate the optical readout mechanically, we develop an adapted nanoindentation protocol for 3D printed hydrogel microstructures in water. The measured reduced modulus of the hydrogel micropillars spans 3–5 MPa across the printed array and correlates linearly with the fluorescence lifetime signal, enabling indirect stiffness evaluation from FLIM data. Further, we show that hydration state strongly affects both photophysical properties and mechanical properties: drying the hydrogel pillars increases autofluorescence intensity and autofluorescence lifetime and stiffness, while rehydration partially restores the pristine behavior but not completely, indicating irreversible network changes. These results establish intrinsic autofluorescence FLIM as a sensitive, non-contact indicator of crosslinking density, mechanical integrity, and dehydration-induced damage in microscale 3D printed PEG based hydrogels.

Eivgi,O.; Vazquez-Martel, C. Catt, S. O.; Blasco, E. Submitted

Single-Molecule FRET Correlations Reveal Rapid Local Diffusion of NEIL1 on DNA

Jan-Philipp Krämer-Günther¹, Soundhararajan Gopi¹, Andrea Holla¹, Lucas Kirchen¹, Robert Best², Daniel Nettels¹, Benjamin Schuler³

¹Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
²Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
³Department of Biochemistry and Department of Physics, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

Efficient target search on DNA is important for a wide range of regulatory processes in the cell. Quantifying this biomolecular diffusion on nanometer length scales and microsecond timescales remains a major experimental challenge. While single-particle tracking resolves long-range motion, local diffusion processes underlying target search on DNA are difficult to access directly. Here, we introduce a framework to extract quantitative diffusion coefficients from single-molecule Förster resonance energy transfer (smFRET) correlation measurements. Using a combination of smFRET and analytical modeling, we show that both the timescales and amplitudes of fluorescence correlations encode information about nanoscale motion. By rigorously correcting correlation amplitudes for photophysical effects and fitting a physical model of diffusion on a cylindrical surface, we directly obtain diffusion coefficients and geometric constraints with minor prior assumptions about the system. We validate this approach using the DNA repair protein NEIL1 as a model system. Coarse-grained molecular dynamics simulations support the smFRET data and provide mechanistic insights into how these processes occur at the molecular level. This smFRET correlation method provides access to a previously inaccessible regime of biomolecular dynamics and establishes smFRET correlation analysis as a general tool for probing nanoscale diffusion processes in DNA–protein interactions.

Development of Sustainable Organic Reactions in Water Through FLIM

Andrea Delledonne, Suzanne A. Blum

University of California, Irvine

Organic chemistry generally requires toxic and flammable solvents derived from petroleum. These solvents account for ~60% of reaction waste and carry environmental and safety costs.¹ Water is a promising alternative, but many organic molecules are neither stable nor soluble in it. Recently, a new approach has emerged: surfactants that can create protected compartments. These compartments are droplets and micelles, where reactions can occur. Yet, these systems are so difficult to study with traditional techniques that their behavior remains poorly understood, slowing down the development of new sustainable and safer reactions.² Here, we study cross-coupling reactions in water, similar to those used to make pharmaceuticals, agrochemicals, and materials. Fluorescence lifetime imaging microscopy (FLIM) and microspectroscopy (with PicoQuant’s FlexLambda) track the in situ formation of a fluorescent product, revealing that micrometer-scale organic droplets act as both the primary reaction sites and reservoirs for organics. These measurements also disprove a previously hypothesized mechanism, wherein micelles become saturated with product inhibiting catalytic turnover. This study highlights how imaging techniques, and FLIM in particular, can support the rational design of sustainable aqueous organic reactions by revealing the actual nature of colloidal objects, identifying reaction sites, and testing mechanistic hypotheses at the microscale.

[1] La Sorella, G., Strukul, G., & Scarso, A., Green Chemistry, 17 (2), 644–683 (2015)

[2] Lipshutz, B. H. Green Chemistry, 26 (2), 739–752 (2024)

Prying into Biomolecular Condensates Using Single-Molecule FRET and HomoFRET

Mukhopadhyay, Samrat

Indian Institute of Science Education and Research (IISER) Mohali

Macromolecular phase separation of proteins and nucleic acids into biomolecular condensates is associated with a wide range of cellular functions and deadly neurodegenerative diseases. My laboratory has been actively developing, adapting, and applying a diverse array of emerging methodologies that permit us to delineate the key molecular principles governing macromolecular phase separation. Using site-specific intramolecular single-molecule FRET (Förster resonance energy transfer), we dissected the key molecular events associated with phase separation of the prion-like low-complexity domain of FUS (Fused in Sarcoma), which is linked to the formation of cytoplasmic and nuclear membraneless organelles [1]. Our results showed that a symphony of structural unwinding events converts intramolecular interactions into dynamic crosslinks of multivalent intermolecular contacts, resulting in condensate formation. We also extended our single-molecule FRET studies to investigate condensate-mediated chaperoning of tau that is associated with Alzheimer’s disease [2]. We also demonstrated a unique application of intermolecular homoFRET to study the modulation of emergent material properties of FUS condensates [3]. We further demonstrated that homoFRET imaging can detect and characterize the in-situ formation of cytoplasmic stress granules within mammalian cells. I will also discuss our new results on other RNA-binding proteins involved in stress granule formation

[1] A. Joshi, A. Walimbe, A. Avni, S. K. Rai, L. Arora, S. Sarkar & S. Mukhopadhyay. Nature Communications (2023) 14, 7331. [2] S. K. Rai, R. Khanna, A. Sarbahi, A. Joshi & S. Mukhopadhyay Science Advances (2025) 11, eads1241. [3] A. Joshi, A. Walimbe, S. Sarkar, L. Arora, G. Kaur, P. Jhandai, D. Chatterjee, I. Banerjee, S. Mukhopadhyay.  Nature Communications (2024) 15, 9215.

Single-molecule microfluidics for protein analysis and biomarker sensing

Georg Krainer

University of Graz, Institute of Molecular Biosciences (IMB), Humboldtstraße 50/III, 8010 Graz, Austria

The integration of single-molecule approaches with microfluidics has opened new possibilities for protein analysis and biomarker sensing. By combining the sensitivity of single-molecule fluorescence with the versatility of microfluidic platforms, these methods enable quantitative measurements with rich molecular information. In my talk, I will present our work in this area and introduce recently developed single-molecule microfluidic approaches for protein analysis and biomarker detection. In particular, I will highlight single-molecule microfluidic diffusional sizing (smMDS) [1], which enables quantitative analysis of protein size, interactions, and assembly states in solution at the single-molecule level. I will also present DigitISA [2], a digital microfluidic immunoassay that detects low-abundance protein biomarkers in solution by counting individual immunocomplexes with high sensitivity. Together, these approaches demonstrate the versatility of single-molecule microfluidics for both quantitative protein biophysics and ultrasensitive biomarker detection.

[1] Krainer G, Jacquat RPB, Schneider MM, Welsh TJ, Fan J, Peter QAE, Andrzejewska EA, Šneiderienė G, Czekalska MA, Ausserwoeger H, Chai L, Arter WE, Saar KL, Herling TW, Franzmann TM, Kosmoliaptsis V, Alberti S, Hartl F-U, Lee SF, Knowles TPJ. Single-molecule digital sizing of proteins in solution. Nat Commun, 2024, 15, 7740. DOI: https://doi.org/10.1038/s41467-024-50825-9

[2] Krainer G, Saar KL, Arter WE, Welsh TJ, Czekalska MA, Jacquat RPB, Peter Q, Traberg WC, Pujari A, Jayaram AK, Challa P, Taylor CG, van der Linden L-M, Franzmann TM, Owens RM, Alberti S, Klenerman D, Knowles TPJ. Direct digital sensing of protein biomarkers in solution. Nat Commun, 2023, 14, 653. DOI: https://doi.org/10.1038/s41467-023-35792-x

Noise-Corrected PCA for Improved Analysis of TCSPC-FLIM Data

Alix Le Marois^3,5, Klaus Suhling⁴, Rainer Heintzmann^1,2

¹Leibniz Institute of Photonic Technology, Albert-Einstein Str. 9, 07745 Jena, Germany
²Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-Universität Jena, Helmholtzweg 4, 07743 Jena, Germany
³The Francis Crick Institute, 1 Midland Road, London NW1 1AT
⁴S6.30, Strand Building, Strand Campus, Strand, London, WC2R 2LS
⁵Institute Fresnel, Avenue Escadrille Normandie Niemen, 13013 Marseille, France

Phasor analysis applied to time-correlated single photon counting (TCSPC) data effectively projects fluorescence decays onto a set of linearly independent subspace coordinates, but in a manner that is sub-optimal with respect to noise. Principal Component Analysis (PCA), in contrast, is designed to identify optimal subspace coordinates that capture the true variance of the underlying data. However, when directly applied to FLIM data, Poisson-distributed photon noise distorts this variance structure and limits PCA performance.

This work introduces a Noise-Corrected PCA (NC-PCA) framework that explicitly accounts for Poisson noise, restoring PCA’s ability to extract meaningful components from photon-limited datasets. The method enables identification of distinct fluorescence decay components without prior knowledge of their number or kinetics and operates effectively at significantly lower photon counts than conventional approaches.

We demonstrate that NC-PCA not only outperforms phasor analysis in resolving microenvironments, but can also serve as a powerful preprocessing step, improving downstream phasor projections. Validation on simulated and experimental data, including membrane imaging in HeLa cells, highlights its robustness and sensitivity to subtle biological variations.

A. Le Marois, S. Labouesse, K. Suhling, and R. Heintzmann, Noise-Corrected Principal Component Analysis of fluorescence lifetime imaging data, J. Biophot. 11, 1124-1133, (2016), DOI:10.1002/jbio.201600160

https://github.com/RainerHeintzmann/NCPCA.jl

Open-source platform for the analysis of fluorescence fluctuation spectroscopy data with SPAD array detectors

Eli Slenders¹, Eleonora Perego², Sabrina Zappone³, Giuseppe Vicidomini⁴

¹Optical investigation of Dynamical Systems, Istituto Italiano di Tecnologia, Genoa, Italy
²Faculté de biologie et médecine, Université de Lausanne, Lausanne, Switzerland
³Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
⁴Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Genoa, Italy

Fluorescence fluctuation spectroscopy (FFS) is a family of techniques for measuring the dynamics of (bio)molecules [1] by analyzing the temporal and/or spatial fluctuations in the fluorescence intensity as fluorophores pass through the detection volume of a confocal microscope. However, by integrating the fluorescence over the sensitive area of the detector, information on the spatial distribution of the photons in the image plane is lost. Recently, the introduction of small single-photon avalanche diode (SPAD) array detectors has opened up a new range of spatiotemporal information, allowing for more detailed analysis of system dynamics [2, 3]. However, the lack of open-source software for analyzing high-dimensional FFS datasets currently limits the adoption of FFS techniques to researchers with programming expertise.

We present BrightEyes-FFS [4], an open-source Python environment for FFS analysis with SPAD array detectors. The environment supports raw data import from BrightEyes-MCS, Genoa Instruments PRISM, and PicoQuant Luminosa. Various algorithms are available for computing correlations, and the resulting curves can be fitted to various models. A graphical user interface and an automated Jupyter Notebook writing tool make the analysis fast and user-friendly. In summary, we believe that BrightEyes-FFS will enable a wider community to study molecular diffusion, flow, and interaction dynamics.

[1] D. Magde, W. W. Webb, E. Elson, Phys. Rev. Lett., 29, 1972.
[2] L. Scipioni et al., Nat. Commun., 9, 2018.
[3] E. Slenders et al., Light Sci. Appl., 10, 2021.
[4] E. Slenders et al., BioRxiv., 2026.