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Single Molecule Workshop

27th International Workshop on
“Single Molecule Spectroscopy and Super-resolution Microscopy”

September 7 - 9, 2022, Berlin, Germany

Image Single Molecule Workshop

Meet the single molecule community in Berlin!

It is a great pleasure to announce that the 27th edition of the Single Molecule Workshop will take place from September 7 – 9, 2022 as an in-person event.  Note: contrary to most previuos years, the workshop will start already in the morning of Wednesday, September 7.

Additionally we will be hosting a free special event during the workshop. Check out the tab Special Event for more details.

As the health and safety of attendees, staff, and community are our top priorities, we will continuously monitor and asses current CoVID-19 developments on a regular basis.

We are looking forward to welcome you in Berlin this fall. Join us for an exciting and stimulating conference by either giving a talk, presenting a poster or even without any presentation. As always, we will be awarding a “Best Student Talk” prize worth 750 Euro.


Aim and purpose

Audience during an oral presentation

The focus of PicoQuant’s long-standing workshop lies on ultrasensitive optical detection down to the single molecule level as well as beyond the classical diffraction limit. The event provides an interdisciplinary platform for exchanging ideas and recent results between researchers and professionals working in the fields of physics, chemistry, biology, life and materials science.

During the workshop, talks and posters are presented that cover a wide range of applications and methods revolving around the challenging field of Single Molecule Spectroscopy.

Covered topics include:

  • Fluorescence Lifetime Imaging (FLIM)
  • Single molecule Förster Resonance Energy Transfer (smFRET)
  • Polarization and Anisotropy based techniques
  • Quantitative imaging methods
  • New fluorescence sensors and labeling schemes
  • Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Correlation Spectroscopy (FLCS)
  • Stimulated Emission Depletion (STED) microscopy
  • Single Molecule Localization Microscopy (e.g., PALM, STORM, dSTORM, GSDIM, PAINT)
  • Open source data analysis solutions
  • Big Data and Machine Learning approaches to superresolution and single molecule techniques

Both widefield and confocal fluorescence microscopy techniques are covered as well as in vivo and in vitro measurements with single molecule sensitivity.


Important dates

  • Deadline for abstract submission: June 24, 2022
  • Deadline for early bird registration: June 24, 2022
  • Deadline for fee waiver application: June 24, 2022
  • Deadline for post deadline abstracts (posters only): August 7, 2022
  • Final deadline for workshop registration: August 7, 2022
  • Notification on acceptance of abstracts: mid/end of July 2022
  • Program available: end of July 2022


Student award

As nurturing young scientists is important to PicoQuant, we host a competition for the “Best Student Talk” with an award worth 750 Euro. Undergraduate and graduate students were invited to submit their contributions.

Submission is now closed.


Conference on Single Molecule Spectroscopy at BiOS 2023

Within the Biomedical Optics Symposium BiOS, PicoQuant is co-organizing the special conference "Single Molecule Spectroscopy and Superresolution Imaging XVI" (BO503). Exciting presentations with the latest findings and developments can be expected.
As a special motivation for young researchers, PicoQuant is presenting the "Young Investigator Award" as part of this conference. Young scientists (age 30 or below and not yet full faculty members) are encouraged to participate in this best paper competition, which offers a $750 USD cash award. For more details and submissions, please go to the BiOS website.


Next workshop (preliminary date)

  • September 13-15, 2023 or September 20-22, 2023



Workshop coordinator: Jana Bülter and Claudia Bergemann

Tel: +49-30-1208820-87
Fax: +49-30-1208820-90
Email: workshop@picoquant.com

Please note: schedule and content of this event is subject to change without notice.

Worldwide premiere: First live demo of our new confocal microscope

Included in this year's program is a special session on Wednesday afternoon ("Young stars and news from the community"), in which we will reveal our latest solution for confocal microscopy: Luminosa. In a live demo, our specialists will show you how easily Luminosa can explore your samples and reveal new insights.

Explore new paths in confocal microscopy

A precise and trustworthy companion for advancing your research

"Measurements we could not do before."

Dr. Daniel Nettels, University of Zurich, Switzerland

"Extremely valuable."

Prof. Dr. Claus A. M. Seidel, Heinrich Heine University Düsseldorf, Germany

"Absolutely great!"

Prof. Dr. Jörg Enderlein, Georg-August-Universität Göttingen, Germany


Attendance and registration

Participation is included when you are registered for the 3-day Single Molecule Workshop.

Further opportunities to learn more about Luminosa

There are further opportunities to learn more about Luminosa. Visit our Luminosa page.


Invited speakers and presentation titles

  • Sophie Brasselet Sophie Brasselet
    Institut Fresnel, France
    "Imaging of proteins’ organization in 3D using Single Molecule Orientation and Localization Microscopy (SMOLM)"
  • Ulrike Endesfelder Ulrike Endesfelder
    Rheinische Friedrich Wilhelms University Bonn, Germany
    "Visualizing cellular life: From single cell imaging to in vivo single-molecule biochemistry and (micro-)biology"
  • Viktorija Glembockyte Viktorija Glembockyte
    Ludiwg-Maximilian-University München, Germany
    "DNA origami tools for single molecule sensing"
  • Enrico Gratton Enrico Gratton
    University of California Irvine, USA
    "Single cell physiological characterization in living tissue. Determination of cell fate"
  • Mike Heilemann Mike Heilemann
    Goethe University Frankfurt am Main, Germany
    "Integrative Super-resolution Fluorescence for Structural Cell Biology"
  • Stefan Jakobs Stefan Jakobs
    Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    "Focusing on Mitochondria"
  • Dong Li Dong Li
    Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
    "Multi-modality structured illumination super-resolution microscopy development and applications"
  • Gail McConnell Gail McConnell
    University of Strathclyde, Glasgow, United Kingdom
    "Prospects for single molecule mesoscopy with the Mesolens"
  • David Walt David Walt
    Harvard Medical School, Boston, Massachusetts, USA
    "Precision Medicine by Counting Single Molecules"

Abstract guidelines

Abstract submission for oral presentations is closed.

Post deadline abstracts for poster presentations only are still possible: deadline for submissions is August 7.

  • Abstracts can be submitted for contributed (talks and/or) poster presentations
  • Abstracts can only be submitted along with a registration for the workshop
  • Abstracts must be submitted in English and be no longer than 200 words (body text) with no graphics
  • Abstracts will be made available to workshop participants through the online program overview and the abstract book. If you do not want to have your abstract displayed in the online program overview, please please indicate so during the registration process
  • Notification on acceptance of abstracts will occur in mid/end of July 2022.

Program (preliminary) as of July 25

09:30 - 10:30REGISTRATION
10:30 - 10:45Rainer Erdmann, Berlin, Germany
Opening Remarks
Session: Super-resolution microscopy chair: Rainer Erdmann
10:45 - 11:15
Mike Heilemann, Frankfurt, Germany (Invited Talk)

Integrative Super-Resolution Fluorescence Microscopy For Structural Cell Biologoy

Mike Heilemann

Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, 60438 Frankfurt, Germany

Super-resolution fluorescence microscopy has evolved into a a powerful tool for optical structural cell biology. Providing a spatial resolution at the protein size scale, it is beginning to revolutionize our understanding of cell biology.

Our work focuses on two main challenges: the extraction of quantitative molecular information from imaging data of densely-packed protein complexes that are so-far inaccessible to direct visualization; the contextualization of biomolecular networks and structures in a cell through multi-target imaging. For the first part, we developed kinetics-assisted quantitative single-molecule localization microscopy (qSMLM), which retrieves molecule numbers from fluorescence “blinking” kinetics. We validate this approach for various fluorophore labels and flavors of SMLM, and apply it to reveal the function-dependent oligomerization of membrane receptors TNFR1, TLR4 and MET. For the second part, we integrate non-covalent protein labels into various super-resolution microscopy methods. This dynamic labeling approach minimizes photobleaching, and enables multi-color, 3D, and live cell imaging, as well as imaging of large fields of view.

11:15 - 11:45
Dong Li, Beijing, China (Invited Talk, virtual)

Multi-modality structured illumination super-resolution microscopy development and applications

Dong Li

Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China, lidong@ibp.ac.cn

The key challenge when imaging delicate bioprocesses is how to acquire the most spatiotemporal information with least invasiveness possible. In this regard, current super-resolution techniques, however, usually involve tradeoffs between high spatial and temporal resolution, and low photobleaching and phototoxicity, each of which is equally important for understanding the bioprocesses. Computational approaches, especially deep neural network (DNN), have demonstrated impressive capabilities of image super-resolution and restoration, but still suffer from ill-posedness, model-uncertainty, spectral bias, and demands for high-quality ground-truth (GT). In this talk, I will present our recent advances in multi-modality structured illumination microscopy (Multi-SIM) and high-speed lattice light sheet microscopy (LLSM). Moreover, we synergize the developments in both optical front end and algorithmic back end to overcome the respective challenges. Multi-SIM and LLSM allow us select the optimum imaging mode for specific bioprocesses according to their different properties, e.g., subcellular location, dynamic, and duration, etc., which enable investigating the fine spatial details, rapid kinetics, and long-time dynamics of a wide variety of bioprocesses.

11:45 - 12:05
Fernando D. Stefani, Buenos Aires, Argentina

An alternative to MINFLUX that enables nanometre resolution in a confocal microscope

Luciano A. Masullo1,2, Alan M. Szalai1, Lucía F. Lopez1, Mauricio Pilo-Pais3, Guillermo P. Acuna3, Fernando D. Stefani1,2

11Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2390, C1425FQD Ciudad Autónoma de Buenos Aires, Argentina
22Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Güiraldes 2620, C1428EHA Ciudad Autónoma de Buenos Aires, Argentina
3Department of Physics, University of Fribourg, Chemin du Musée 3, Fribourg CH-1700, Switzerland

Among the numerous methods for single molecule localization microscopy, MINFLUX outstands for achieving a ~10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts. Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups. Here, we present RASTMIN, a single-molecule localization method based on RASTer scanning a light pattern comprising a MINimum of intensity. RASTMIN delivers a localization precision equivalent to MINFLUX while it is easily implementable on any standard scanning (confocal, or multi-photon) microscope with few modifications, namely a sample-drift stabilization system and phase modulation of the excitation beam to obtain a focus with an intensity minimum. We describe the concept of RASTMIN using a framework common to all methods of single-molecule localization using sequential structured illumination [1] and demonstrate its performance experimentally by imaging of DNA-origami structures [2].

[1] Luciano A. Masullo, Lucía F. Lopez, Fernando D. Stefani, Biophysical Reports, 2, 100036  (2022)

[2] Luciano A. Masullo, Alan M. Szalai, Lucía F. Lopez, Mauricio Pilo-Pais, Guillermo P. Acuna, Fernando D. Stefani, Light: Science & Applications,  just accepted (2022)

12:05 - 12:25
Shannan Foylan, Glasgow, United Kingdom

MesoTIRF: a novel Total Internal Reflection Fluorescence illuminator for axial super-resolution imaging on the mesoscale

Shannan Foylan, William Bradshaw Amos, John Dempster, Michael Shaw, Gail McConnell

SF, WBA, GM - [1] Department of Physics, SUPA, University of Strathclyde, Glasgow, UK, JD - [2] Strathclyde Institute for Pharmacy & Biomedical Sciences, University of Strathclyde, Glasgow, UK,MS - [3] Department of Chemical and Biological Sciences, National Physical Laboratory, Teddington, UK, [4] Department of Computer Science, University College London, London, UK

Total Internal Reflection Fluorescence (TIRF) microscopy is a staple imaging modality in many imaging facilities. The characteristic contrast and nanometre thin optical section of TIRF has allowed extensive examination of cell membranes. TIRF has proved useful for photoactivable fluorophore based Single Molecule Localisation Microscopy (SMLM), as the out of focus fluorescence reduction allows the microscopist to achieve single molecule resolution.

However, the lateral Field Of View (FOV) of high NA TIRF objective lenses restricts imaging to a handful of cells. Therefore, the super-resolution detail and image quality afforded by TIRF and SMLM is limited to a small population, requiring sequential imaging to improve the statistical significance of imaging data.

We have developed a TIRF modality for the Mesolens, a custom giant objective lens with a 4X/0.47NA prescription allowing for imaging over a 4.4 mm x 3.0 mm FOV with sub-cellular lateral resolution. The optical section of our developed modality is below the depth of field of the Mesolens in widefield and we will present proof of principle imaging data to illustrate the functionality of the MesoTIRF modality. While this project is relatively new, it presents the possibility of SMLM across 1000 mammalian cells in a single image.

12:25 - 12:45
Michael Isselstein, Planegg-Martinsried, Germany (Student Award)

Linker Molecules Convert Commercial Fluorophores into Tailored Functional probes during Bio-Labeling

Michael Isselstein1, Lei Zhang1,2, Jens Köhler3, Nikolaos Eleftheriadis4, Nadia M. Huisjes1,4, Miguel Guirao-Ortiz55, Alessandra Narducci1, Jochem H. Smit4, Janko Stoffels3, Hartmann Harz5, Heinrich Leonhardt5, Andreas Herrmann3, Thorben Cordes1,4

1Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152 Planegg-Martinsried, Germany
2Institute of Advanced Synthesis, School of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 211816 (China)
3(DWI) Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52056 Aachen (Germany) and Institute of Technical and Macromolecular Chemistry, (RWTH) Aachen University, Worringerweg 2, 52074 Aachen (Germany)
4Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen (The Netherlands)
5Human Biology & Bioimaging, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2–4, 82152 Planegg-Martinsried (Germany)

Many life-science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super-resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of labelling. For this, we present the design and synthesis of ‘linker’ compounds, that bridge biotarget, fluorophore and a functional moiety via well-established labeling protocols. With our strategy, we show that the same commercial dye can become a photostable self-healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self-healing linker–fluorophore conjugates and demonstrated their applications in super-resolution imaging and single-molecule spectroscopy.

Reference: Zhang, Isselstein & Köhler et.al., Angew. Chem. Int. Ed. 61 (2022) e202112959

12:45 - 13:05
chair: Felix Koberling
Poster flash talks 1
(Presenters tba)
13:05 - 14:15LUNCH BREAK
Session: Young stars and news from the communitychair: Rainer Erdmann
14:15 - 14:20Rainer Erdmann, Berlin, Germany
14:20 - 14:50
Viktorija Glembockyte, Munich, Germany (Invited Talk)

DNA origami tools for single molecule sensing

Viktorija Glembockyte1, Lennart Grabenhorst1, Martina Pfeiffer1, Kateryna Trofymchuk1, Cindy Close1, Renukka Yaadav1, Thea Schinkel1, Florian Steiner1, Alexander Murr1, Birka Lalkens2, Philip Tinnefeld1

1Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstr. 11, 81369 München, Germany
2Institut für Halbleitertechnik, Laboratory for Emerging Nanometrology LENA, TU Braunschweig, Langer Kamp 6a/b, Braunschweig, Germany

Advancements in fluorescence imaging and microscopy techniques have provided the ability to detect and monitor biomolecules and interactions between them at the ultimate sensitivity of a single molecule. In this talk I will discuss how single-molecule fluorescence imaging can be synergistically combined with a technique that allows one to also place and position single molecules on the nanoscale, namely, DNA origami, in the pursuit of building new single-molecule sensors. I will discuss how using DNA as a building material we can build light antennas on the nanoscale and amplify fluorescence signals of single molecules up to few hundred-fold, enabling their detection on a smartphone camera. I will also discuss how one can construct a modular sensing platform decoupling the different elements of a biosensor to improve its signal contrast, tune its dynamic range or improve its selectivity. Finally, I will touch on our efforts to build molecular assays with the help of DNA origami that could extend the scope of these single-molecule sensing platforms beyond the detection of nucleic acids and enable the recognition of antibodies, nucleases, and proteases.

[1] V. Glembockyte, L. Grabenhorst, K. Trofymchuk, P. Tinnefeld, Accounts of Chemical Research, 54, 3338 (2021).

[2] K. Trofymchuk and V. Glembockyte et al., Nature Communications 12, 950 (2021).

[3] C. Close, K. Trofymchuk, L. Grabenhorst, B. Lalkens, V. Glembockyte, P. Tinnefeld, Advanced Materials and Interfaces, 2200255 (2022).

[4] M. Pfeiffer, K. Trofymchuk, S. Ranallo, F. Ricci, F. Steiner, F. Cole, V. Glembockyte, P. Tinnefeld,  iScience, 24, 103072 (2021).

14:50 - 15:10
Oliver Stach, Zürich, Switzerland (Student Award)

Dissecting the complex dynamics of protein-RNA interactions with single-molecule fluorescence

Oliver Stach, Sebastian L.B. König, Andrea Holla, Daniel Nettels, Benjamin Schuler

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

Many organisms use double-stranded RNA binding proteins (dsRBPs) to specifically recognize RNA in the context of posttranscriptional gene regulation. dsRBPs usually consist of multiple copies of folded RNA binding domains separated by long disordered linkers. Interestingly, dsRBPs remain remarkably dynamic in their RNA-bound state and diffuse along double-stranded RNA [1]. However, understanding the molecular details of these dynamic interactions remains challenging.

In this study, we used single-molecule fluorescence spectroscopy to decipher the complex dynamic interactions of the protein TRBP with RNA. Using fluorescence correlation spectroscopy and 2-color and 3-color single-molecule FRET experiments, we were able to resolve and characterize the kinetics of processes occurring over a broad range of timescales from microseconds to hours, including the diffusive motion of TRBP along the RNA; the movement of its domains relative to each other; the flipping of orientations on the RNA; and the global dissociation of the protein from RNA.

Combining these data with structural information from NMR spectroscopy [2] and extensive kinetic modeling allowed us to reduce these complex interactions to basic principles of single-domain dynamics. Our work thus provides a framework for studying a variety of protein-nucleic acid interactions.

[1] Koh, H.R., Kidwell, M.A., Ragunathan, K., Doudna, J.A. & Myong, S. ATP-independent diffusion of double-stranded RNA binding proteins. Proc. Natl. Acad. Sci. USA 110, 151-6 (2013).

[2] Masliah, G.Maris, C. König, S. L. Yulikov, M. Aeschimann, F. Malinowska, A. L. Mabille, J. Weiler, J. Holla, A. Hunziker, J. Meisner-Kober, N. Schuler, B. Jeschke, G. Allain, F. H. Structural basis of siRNA recognition by TRBP double-stranded RNA binding domains”, EMBO J 37(6) (2018).

15:10 - 15:30
Aleksandra Bednarz, Aarhus, Denmark (Student Award)

Ion-mediated structural integrity and reconfigurability of DNA origami molecular breadboards

15:30 - 15:50
chair: Felix Koberling
Poster flash talks 2
(Presenters tba)
15:50 - 16:20
Evangelos Sisamakis, Berlin, Germany

Luminosa - a new confocal microscope covering the needs of the expanding community of single molecule fluorescence

Evangelos Sisamakis, Felix Koberling

PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany

The last few years the single molecule fluorescence community has been constantly expanding.

As the methodologies are moving away from the hands of a few groups who were responsible for their development, new challenges regarding reproducibility, accuracy and in general "good practice and ease-of-use" have appeared.

The single-molecule FRET community for example has provided important guidance with two seminal papers:

a) Hellenkamp et al (2018) , Precision and accuracy of single molecule FRET measurements - a multi-laboratory benchmark study

b) Lerner et al (2021),  FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

 Luminosa, the new single photon counting confocal microscope was conceptualized from the beginning with the vision to address the new challenges of an expanding community.

16:25 - 16:45COFFEE BREAK
Session: FLIMchair: Mike Heilemann
09:00 - 09:35
Sophie Brasselet, Marseille, France (Invited Talk)

Imaging of proteins’ organization in 3D using Single Molecule Orientation and Localization Microscopy (SMOLM)

Sophie Brasselet

Aix Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, F-13013 Marseille, France, sophie.brasselet@fresnel.fr

Imaging molecular orientation at the nanoscale in live cells and tissues is fundamental in the understanding of proteins’ organization, which is driven by their structural and conformational properties. Measuring fluorescent molecules’ orientation is a way to approach this problem, providing that the label is rigidly attached to the protein of interest. Despite the great progresses in fluorescence imaging down to nanometric scales with Single Molecule Localization Microscopy (SMLM), orientation imaging is still only at its early stage. Measuring single molecules’ 3D orientations in addition to their 3D spatial localization is a challenge due to the difficulty to disentangle spatial and orientational parameters in the SM point spread function (PSF) image formation, nevertheless the search for optimal methods to solve this challenge is rapidly progressing. We present examples of polarized fluorescence microscopy methods that are able to report both orientational and spatial information from single molecules in a non-ambiguous way. These methods, based on phase manipulation [1] or polarized splitting imaging or Fourier plane polarization [2], give access to orientation parameters in combination with high spatial localization precision. We will present the potential and limits of these approaches for the imaging of the nanoscale organization of proteins in cells.

[1] V. Curcio et al. Nat. Communications 11 (1) (2020) DOI: 10.1038/s41467-020-19064-6

[2] C. Rimoli et al. Nat. Communications 13, 301 (2022). DOI: 10.1038/s41467-022-27966-w

09:35 - 09:55
Marta Pilz, Warsaw, Poland

Method to measure drug uptake time into living cells on the ultrashort timescale 

09:55 - 10:15
German Chiarelli, Fribourg, Switzerland (Student Award)

Super-resolved FRET imaging by confocal fluorescence-lifetime single-molecule localization microscopy

German Chiarelli1, Cecilia Zaza2, Ludovit Zweifel3, Mauricio Pilo-Pais1, Fernando Stefani2, Guillermo Acuna1

1University of Fribourg, Switzerland
2CIBION, Buenos Aires, Argentina
3University of Basel, Switzerland

Super-resolution microscopy typically relies on measurements of the fluorophores´ emission intensity. However, organic fluorophores have additional information encoded on their fluorescence lifetime. Thus, a combined super-resolution FLIM/FRET imaging holds unique advantages, for example to monitor biomolecular processes taking place in separate but adjacent sub-diffraction biological structures, or to visualize with high precision the distribution of energy transfer in systems such as light-emitting devices and nanostructured semiconductors. However, while these benefits were clear upon the onset of super-resolution microscopes, and although FRET has been used to enhance or supplement super-resolution methods, obtaining super-resolution FLIM/FRET methods has been challenging [1].

Here, we show that SMLM can be combined with FLIM using a commercial fast scanning microscope. We characterize the performance of the combined techniques by means of DNA nanotechnology. In particular, we self-assemble DNA origami nanosized structures with three binding sites for SMLM based on DNA PAINT. One of the sites is modified with acceptor dyes, so that binding events on that site are accompanied by an energy transfer which is detected based on lifetime measurements. Our results demonstrate that SMLM-FLIM/FRET can be readily implemented in fast scanning microscopes even in sparse labeled samples with a high concentration of imager strands [2].

[1]    Thiele et al. ACS Nano 14 (10), 14190-14200 (2020)

[2]    K. Chung et al Nature Methods 19, 554-559 (2022)

10:15 - 10:35
Johannes Wieland, Ulm, Germany (Student Award)

Fluorescence lifetime imaging and electron microscopy: a correlative approach

Johannes Wieland1,2, Nilanjon Naskar1, Paul Walther2, Angelika Rück1

1Core Facility Confocal and Multiphoton Microscopy, Ulm, University, 89081 Ulm, Germany
2Central Facility for Electron Microscopy, Ulm University, 89081 Ulm, Germany

Fluorescence lifetime imaging microscopy (FLIM) allows the characterization of cellular metabolism by quantifying the rate of free and unbound nicotinamide adenine dinucleotide hydrogen (NADH). This study delineates the correlative imaging of cells with FLIM and electron microscopy (EM). Human fibroblasts were cultivated in a microscopy slide bearing a coordinate system and FLIM measurement was conducted. Following chemical fixation, embedding in Epon and cutting with an ultramicrotome, tomograms of selected cells were acquired with a scanning transmission electron microscope (STEM). Correlative imaging of antimycin A-treated fibroblasts shows a decrease in fluorescence lifetime as well as swollen mitochondria with large cavities in STEM tomography. To our knowledge, this is the first correlative FLIM and EM workflow. Combining the high sensitivity of FLIM with the high spatial resolution of EM could boost the research of pathophysiological processes involving cell metabolism, such as cancer, neurodegenerative disorders, and viral infection.

Wieland, J.G., Naskar, N., Rück, A. et al. Fluorescence lifetime imaging and electron microscopy: a correlative approach. Histochem Cell Biol 157, 697–702 (2022). https://doi.org/10.1007/s00418-022-02094-0

10:35 - 10:45GROUP PICTURE
Session: Instrumentationchair: Sophie Brasselet
11:20 - 11:50
Gail McConnell, Glasgow, United Kingdom (Invited Talk)

Prospects for single molecule mesoscopy with the Mesolens

Gail McConnell

University of Strathclyde, Glasgow, United Kingdom

We have developed a large, complex objective with a magnification of 4x and a numerical aperture of just less than 0.5 which we call the Mesolens. We originally specified this lens for mammalian embryology, and we have shown that it can image every cell of a 6mm-long embryo 3mm thick with sub-cellular resolution if the tissue is cleared appropriately. A by-product of the high numerical aperture is that the optical throughput is approximately 20x greater than a conventional 4x objective. The pupil size of the lens is so great that it cannot be used with a conventional microscope frame, so we have built the imaging system around the lens.

I will present an update of recent technology developments relating to the Mesolens, and I will give details on our latest biological and clinical imaging applications that use existing and new mesoscale technologies.

11:50 - 12:10
Jörg Enderlein, Göttingen, Germany

Nanocavity-based electric charge measurements of biomolecules in solution

Jörg Enderlein1, Alexey I. Chizhik1, Narain Karedla2

1Third Institute of Physics – Biophysics, Georg August University, Göttingen, Germany
2The Rosalind Franklin Institute, Harwell Campus, Didcot OX11 0FA, United Kingdom

Structure and electrical charge are fundamental characteristics of biological molecules. Electrostatic effects are ubiquitous and dominate the activities and properties of many biomolecules. Established techniques of structural biology such as x-ray crystallography, small-angle x-ray scattering (SAXS), small-angle neutron scattering (SANS), nuclear magnetic resonance spectroscopy (NMR), and cryo-electron microscopy (cryoEM) are capable of delivering detailed atomic information on biomolecules, but cannot directly measure electric charges. Measuring electric charges of biological molecules in ion-containing buffer solutions at single molecule level is far from trivial. Methods which have been used for this purpose are watching the translation of molecules through (solid-state) nanopores [1-2], or to use a geometry-induced electrostatic fluidic trapping [3-4]. However, these approaches are complex, error-prone and not easy to implement. Here, we present the new idea of using conducting planar nano-cavities for measuring electric charges. The nano-cavity is comprised of two glass slabs covered with thin metal electrodes. Applying the same voltage to both electrodes leads to a redistribution of charged molecule within the nano-cavity, which can be read out optically. We discuss to approaches for optical read out: Watching the mean drift velocity of the fluorescently tagged charged molecules in pressure-driven Poiseuille flows [5], or measuring their cavity-modulated fluorescence lifetime.  

[1]       C. Plesa, S. W. Kowalczyk, R. Zinsmeester, A. Y. Grosberg, Y. Rabin, and C. Dekker, “Fast translocation of proteins through solid state nanopores,” Nano Lett. 13, 658-663 (2013).

[2]       K. J. Freedman, M. Jürgens, A. Prabhu, C. W. Ahn, P. Jemth, J. B. Edel, and M. J. Kim, “Chemical, thermal, and electric field induced unfolding of single protein molecules studied using nanopores,” Anal. Chem. 83, 5137-5144 (2011).

[3]       M. Krishnan, N. Mojarad, P. Kukura, and V. Sandoghdar, “Geometry-induced electrostatic trapping of nanometric objects in a fluid,” Nature 467, 692–695 (2010).

[4]       F. Ruggeri, F. Zosel, N. Mutter, M. Różycka, M. Wojtas, A. Ożyhar, B. Schuler, and M. Krishnan, “Single-molecule electrometry,” Nat. Nanotechnol. 12, 488–495 (2017).

[5]          J. Enderlein, D. Sakhapov, I. Gregor, M. Croci, N. Karedla “Charge Separation in Charged Nanochannels for Single-Molecule Electrometry“ J. Chem. Phys. 156, 105104 (2022).

12:10 - 12:30
Gabriel Moya, Munich, Germany (Student Award)

Compact and versatile 3D-printed microscopy platform (CAV-3D-MP)

12:30 - 12:50
Barbora Špačková, Prague, Czech Republic

Potential of Nanofluidic Scattering Microscopy in quantitative imaging of single biomolecules

Barbora Špačková

Institute of Physics, Prague, Czech Republic

Imaging of a single biomolecule in its natural state—freely diffusing in the solution, without any chemical modification or labelling—has long presented an ultimate challenge. The light scattering efficiency of a protein is very small, and the fast Brownian motion only permits the accumulation of the scattered light for an extremely short time that the protein spends in a diffraction limited spot. Nanofluidic Scattering Microscopy (NSM) [1], a recently developed method, overcomes the limitations of currently available techniques. NSM enables to image single biomolecules, without the need for labeling or surface attachment, and with a lower detection limit of 60 kDa in molecular weight. Moreover, by tracking the Brownian motion and from the optical contrast, molecular weight and hydrodynamic radius of individual biomolecules can be determined. In this contribution, I will discuss the limits of the method and its applications for real-time label-free characterization of the dynamics of freely moving biomolecules and their interactions.

B. Špačková, J. Tenghamn, H. K. Moberg, G. Sjösten, H. Šípová-Jungová, F. Westerlund, D.Midtvedt, G. Volpe, M. Käll, J. Fritzsche, C. Langhammer: Nanofluidic Scattering Microscopy for label-free weight and size screening of single diffusing biomolecules, Nature Methods, 2022. 3

12:50 - 13:10
chair: Felix Koberling
Poster flash talks 3
(Presenters tba)
Session: Biological Applications 1chair: Gail McConnell
14:40 - 15:10
Ulrike Endesfelder, Bonn, Germany (Invited Talk)

Visualizing  cellular  life:  From  single  cell  imaging  to  in  vivo  single-molecule  biochemistry  and (micro-)biology

Ulrike Endesfelder

Institute for Microbiology and Biotechnology, University of Bonn, Germany

Microbes as unicellular organisms are important model systems for studying cellular mechanisms and functions. In the last decade, immense progress has been made in our understanding of the life and inner workings of bacteria with the help of modern fluorescence microscopy techniques. By visualising single molecules and the molecular architecture of subcellular structures in living cells, we can now look at bacteria based on their molecular interactions and assemblies with molecular  resolution.  In  particular,  we  can  generate  detailed, quantitative,  spatially  and  temporally  resolved molecular maps and decipher dynamic heterogeneity and subpopulations at the subcellular level. Here, we will present some examples from our work and give an insight into our visions for the future.

15:10 - 15:30
Jessica Angulo-Capel, Castelldefels, Spain (Student Award)

Spatiotemporal dynamics of secretory proteins in the Golgi apparatus

Jessica Angulo-Capel, Maria F. Garcia-Parajo, Felix Campelo

ICFO - Institute of Photonic Sciences, Av. Carl Friedrich Gauss, 3, Castelldefels, Spain

Single particle tracking (SPT) is a fluorescence microscopy technique that allows to monitor the dynamics of individual molecules in living cells with a very high spatiotemporal resolution (~10s nm and ~10s ms, respectively) [1]. Although SPT has been widely used to investigate protein diffusion at the cell surface, its application to the study of intracellular processes has been limited due to a number of technical challenges. Here, I will present our SPT-PALM-based experimental and computational pipeline and how we use it to monitor the intracellular dynamics of single secretory proteins along the secretory pathway.

Interestingly, the formation at the Golgi of transport carriers destined for secretion has been recently shown to require the establishment of functional and dynamic interactions between Endoplasmic Reticulum (ER) and Golgi membranes: the so-called ER-Golgi membrane contact sites (MCS) [2,3]. However, the mechanism for this still remains unclear. Aiming at understanding how ER-Golgi MCS regulate protein sorting and carrier formation for secretion, we used intracellular SPT-PALM to monitor the spatiotemporal dynamics of secretory proteins with respect to MCS. I will present our results on the dynamics of secretory cargoes in the Golgi, and how that depends on the existence of functional ER-Golgi MCS.

[1] Manzo C, Garcia-Parajo M. F., Reports on Progress in Physics, 78, 124601 (2015)

[2] Wakana, Y., et al., Molecular Biology of the Cell, 26, 4686 (2015)

[3] Wakana, Y., et al., Journal of Cell Biology, 220(1) (2021)

15:30 - 15:50
Hendrik Sielaff, Singapore, Singapore

Quantifying and Mapping Intranuclear Dynamics of Human SWI/SNF Chromatin Remodelers in Single Living Cells

Hendrik Sielaff1,2, Wilfried Engl1,2, Aliz Kunstar1,2, Siyi Chen1,2, Winston Z. Zhao1,2,3

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 is a critical process that regulates genome access by alleviating the topological constraints posed by nucleosomes, and is carried out by multi-subunit chromatin remodeler complexes. Mutations in the SWI/SNF family of chromatin remodelers are implicated in >20% of all human cancers. However, our quantitative understanding of their intranuclear dynamics remains limited. Using FCS and single-molecule tracking (SMT), we probed the diffusion and DNA-binding dynamics of three key subunits of the human SWI/SNF remodeler complex in live cells, revealing temporally distinct molecular fractions with diffusion coefficients characteristics of fast/slow diffusion and DNA-binding. Quantifying the residence time of bound remodelers further resolved shorter- and longer-lived fractions, likely corresponding to non-specific and specific binding to DNA targets, respectively. Enhancing DNA accessibility reduces the residence time of stable DNA-binding, suggesting more efficient sampling and translocation of nucleosomes by remodelers. Moreover, super-resolution density maps constructed from SMT trajectories revealed spatially heterogeneous binding “hotspots” across the nucleoplasm, and the relative frequency with which remodelers reside at these “hotspots” is DNA accessibility-dependent. Our findings constitute the first live-cell measurement of human SWI/SNF remodeler dynamics, and hold promise in revealing insights into the potential implications of the misregulation of SWI/SNF-mediated remodeling dynamics in cancer cells.

15:50 - 16:10
Bosong Ji, München, Germany (Student Award)

SmFRET and real-time dynamic behaviour of Endonucleases IV under DNA tension

Bosong Ji, Philip Tinnefeld, Andres M. Vera

Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, 81377 München, Germany

The DNA base excision repair (BER) system is responsible for removing DNA base lesions from the genome throughout the cell circle, and it is crucial for maintaining the cell viability and genetic stability. All the initial enzymes of BER pathway, such as the Abasic site Endonuclease IV, need to bend the DNA before removing the abnormal base and cleaving the DNA backbone. In the cellular context, the ubiquitous physiological mechanical strain in the DNA would oppose bending and thus affect the activity of the enzymes. However, neither the effect of forces on the activity of Endonuclease IV nor the real-time kinetics of the bending process is known for Endonuclease IV. 

Here, we use smFRET (single-molecule Fluorescence resonance energy transfer) to visualize the bending of DNA molecules by Endonuclease IV by labelling the damaged DNA with donor and acceptor dyes on either side of the enzyme-binding-site. We successfully monitored the conformational changes on the DNA by Endonucleases IV by the increased of FRET efficiency upon bending and the real-time kinetics of the process was studied. In addition, we used a newly-developed DNA origami-based force clamp to study the effect of DNA tension on the bending process.

16:10 - 16:30
chair: Felix Koberling
Poster flash talks 4
(Presenters tba)
16:35 - 16:50COFFEE BREAK
19:30 - 22:30DINNER
Session: Correlation Spectroscopychair: David Walt
9:00 - 09:35
Stefan Jakobs, Göttingen, Germany (Invited Talk)

Focusing on Mitochondria

Stefan Jakobs

University Medical Center of Göttingen, Clinic for Neurology &, Max Planck Institute for Multidisciplinary Sciences, Dept. of NanoBiophotonics &, Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Translational Neuroinflammation and Automated Microscopy, Am Fassberg 11, 37077 Goettingen, E-mail: sjakobs@gwdg.de

Mitochondria, the ‘powerhouses of the cell’, are double membrane organelles that are essential for eukaryotic life. Because of their inner-cellular mobility, their small size and their complex architecture, they are notoriously challenging objects for high-resolution light microscopy. We employ STED super-resolution microscopy and other microscopies to investigate the inner mitochondrial architecture. We aim at understanding how mitochondria develop and maintain the complex folding of the inner membrane and how this is related to mitochondrial gene expression. This talk will summarize our recent progress in investigating inner-mitochondrial protein distributions and cristae dynamics.

09:35 - 09:55
Zunhao Wang, Braunschweig, Germany (Student Award)

Surface-controlled adsorption of plasmonic DNA origami nanostructures for single-molecule detection using polar surface array substrates

Zunhao Wang1,2, Zhe Liu2,3, Julia Molle1, Stefan Wundrack1,2, Markus Etzkorn2,3, Rainer Stosch1

1Physikalisch-Technische Bundesanstalt (PTB), Bundesallee 100, 38116 Braunschweig, Germany
2TU Braunschweig LENA, Langer Kamp 6a/b, 38106 Braunschweig, Germany
3TU Braunschweig Institute of Applied Physics, Mendelssohnstraße 2, 38106 Braunschweig, Germany

The detection of specific single molecules in liquids, adsorbed on surfaces or in the gaseous state, is a long-desired goal in spectroscopy and requires spectroscopic techniques offering high spectral sensitivity. Single-molecule surface-enhanced Raman spectroscopy (SM-SERS) is one technique that has been significantly developed in recent decades and has the potential to detect the spectroscopic fingerprint of individual molecules using plasmonic-active materials. The gain of spectral sensitivity in SM-SERS for single-molecule detection can be achieved by utilizing DNA origami structures with plasmonic metal nanoparticles, forming complex nanostructures for the targeted capture of single molecules. However, such measurements are challenging since the nanostructures easily agglomerate when attached to surfaces.

Here, we introduce a promising approach to precisely control the adsorption position of DNA origami nanostructure in the form of a polar surface array (PSA). The PSA surface is fabricated by deposition of specific organic molecules on a hydrophilic substrate, followed by electron-beam lithography for surface patterning with nanometer precision. The polar surface pattern of the PSA substrate drastically reduces the agglomeration of DNA nanostructures, as shown by electron microscopy studies. Furthermore, we show the impact of various experimental parameters to improve the isolation of a single DNA nanostructure for future SM-SERS measurements.

[1] Kershner, R. et al. Nature Nanotech. 4, 557–561 (2009).

[2] Tapio, K. et al. ACS Nano 4 7065-7077 (2021).

[3] Shetty. R.M. et al. ACS Nano 7, 11441-11450 (2021).

09:55 - 10:15
Hendrik Utzat, Mountain View, United States

Interferometric Photon-Correlation Tools for Spectral Diffusion Measurements of Emerging Single-Photon Emitters

Hendrik Utzat1,4, Weiwei Sun2, Alex Kaplan2, Boris Spokoyny3, Moungi Bawendi2

1Stanford University, Department of Materials Science and Engineering, Stanford, USA
2Massachusetts Institute of Technology, Department of Chemistry, Cambridge, USA
3Exponent Inc. Boston, USA
4University of California, Berkeley, College of Chemistry, Berkeley, USA

Spectral diffusion is a ubiquitous process caused by bath fluctuations which randomizes the spectral mode of single-photon emitters at cryogenic temperatures. Accurately measuring spectral diffusion on a single-emitter level is still a challenging task owing to the required high spectral and temporal resolution with an additionally high temporal dynamic range. As a direct consequence, spectral diffusion and the underlying exciton-bath interaction are poorly understood for most emerging single-photon emitters.

In this talk, we highlight our recent progress towards understanding spectral diffusion in nascent quantum emitters using photon-correlation Fourier spectroscopy (PCFS). PCFS can measure the bandwidth and kinetics of spectral fluctuations down to nanosecond timescales. Using PCFS, we show how quantum emitters in 2D hexagonal boron nitride exhibit multi-timescale discrete spectral jumping that can be attributed to a bath with at least two characteristic fluctuation relaxation times.[1] Analysis of colloidal perovskite quantum dots at low temperatures reveals that different emissive fine-structure states are coupled to the same bath fluctuations and exhibit correlated diffusion dynamics.[2] Broadly, we propose PCFS as a particularly suitable tool for the detailed study of decoherence processes and spectral diffusion occurring over many orders of magnitude in the temporal domain. Finally, I introduce a new theoretical concept for interferometric analysis spectral diffusion in the presence of relaxation dynamics in single optical emitters that can delineate the effects of optical fine-structure on bath-induced diffusion dynamics.[3] Our technique is further applicable to biological samples with complex condensed matter dynamics.

[1] B. Spokoyny*, H. Utzat*, H. Moon, G. Grosso, D. Englund, & M.G. Bawendi Effect of Spectral Diffusion on the Coherence Properties of a Single Quantum Emitter in Hexagonal Boron Nitride. Journal of Physical Chemistry Letters, 11, 4, 1330–1335 (2020).

[2] H. Utzat, W. Sun, A.E. Kaplan, F. Krieg, M. Ginterseder, B. Spokoyny, N. Klein, K. Shulenberger, C. Perkinson, M. Kovalenko, and M.G. Bawendi, Coherent Single-Photon Emission From Colloidal Lead Halide Perovskite Quantum Dots, Science, 363, 6431, 1068-1072 (2019).

[3] H.Utzat, M.Bawendi. Lifetime-resolved photon-correlation Fourier spectroscopy,” Opt. Express 29, 14293-14303 (2021)

10:15 - 10:35
Nicola J. Fairbairn, Glasgow, United Kingdom (Student Award)

Measuring exciton-exciton annihilation in polyfluorene using time-resolved photon antibunching

Nicola J. Fairbairn, Olga Vodianova, Gordon J. Hedley

Hedley Single Molecule Laboratory, School of Chemistry, University of Glasgow, Glasgow, UK, G12 8QQ.

Single molecule spectroscopy (SMS) allows individual molecules to be measured without ensemble averaging effects. This approach becomes particularly interesting for conjugated polymers, where individual chains have different electronic properties. Light emitted by single molecules allows their photon statistics to be analysed as the number of chromophores is small, therefore we can determine how the emitted photons are distributed temporally.

Polyfluorene, (PFO), is a blue-emitting conjugated polymer used in organic light-emitting diodes and sensors. Exciton-exciton annihilation in PFO is a light emission loss-mechanism that is desirable to minimise but difficult to study independently. This novel SMS technique of picosecond time-resolved antibunching (psTRAB) monitors exciton-exciton annihilation in single PFO chains.

Low concentrations of PFO chains in a matrix are measured individually using confocal fluorescence microscopy to detect photoluminescence. Time-correlated single-photon counting reconstructs the PL decay and creates photon antibunching graphs that determine how many independent emitting sites exist in the polymer chain. By altering the time window from which antibunching is constructed, variations in antibunching are observed in PFO, indicating that the number of independent emitters reduces with time - a signature of annihilation. Changing the local environment of the polymer influences the annihilation rate, therefore ways to reduce it are identified.

[1] Hedley, G.J., Schröder, T., Steiner, F. et al. Picosecond time-resolved photon antibunching measures nanoscale exciton motion and the true number of chromophores. Nat Commun 12, 1327 (2021).

Session: Biological Applications 2chair: Stefan Jakobs
11:10 - 11:40
David R. Walt, Boston, United States (Invited Talk)

Precision Medicine by Counting Single Molecules

David R. Walt

Harvard Medical School

Many clinically informative biomarkers are present at extremely low concentrations. We have developed multiple ultrasensitive single molecule detection technologies that convert bulk measurements into digital measurements. By digitizing signals, we convert the gold standard immunoassay ELISA technology into an ultrasensitive protein detection method, which enables proteins to be measured that have never been previously detected in clinical samples. We have used the technology to discover biomarker and biomarker panels for a variety of clinical indications including oncology, neurodegenerative disease, and infectious disease. Measuring proteins at these levels, however, does not necessarily make for better clinical outcomes unless one accounts for individual human variation. The resulting information can be used for precision medicine that can be applied to early disease detection, therapeutic efficacy, and recurrence monitoring.

11:40 - 12:00
Cindy Close, Munich, Germany (Student Award)

Docking-Site-Mediated Photostabilization for Super-Resolution Imaging

Cindy Close, Michael Scheckenbach, Viktorija Glembockyte, Philip Tinnefeld

Department of Chemistry and Center for Nanoscience (CeNS), Ludwig-Maximilians University, Butenandtstr. 11 81377 Munich, Germany

DNA-PAINT is a single-molecule localization microscopy technique, relying on transient hybridization of fluorescently labeled ssDNA imager to complementary docking strands on target molecules.During acquisition, docking sites are imaged over the course of multiple binding, dissociation and photobleaching events. Through constant imager strand exchange, the limited photon budget of a single fluorophores is circumvented, making it possible to extract super-resolution images at high illumination intensities. Over long periods of continuous high-duty cycle excitation of fluorophores, DNA-PAINT binding sites can however, be depleted.2 Fluorophores in triplet excited states may generate singlet oxygen and downstream reactive oxygen species, damaging the docking sites and labelled target structures. The use of triplet state quenchers (TSQ) and enzymatic scavenging systems is further limited to systems insensitive to pH change or high additive concentration. Inspired by fluorophore regeneration and self-repair mechanisms, we link the TSQ cyclooctatetraene to an oligonucleotide sequence.3 Binding  at the docking site directly next to the imager, programmed exchange and self-regeneration of the photostabilizer strand is possible. The presented contribution shows preliminary results, using the described approach to increase the accessible photon budget of the fluorophore, improve the longevity of DNA-PAINT docking sites and reduce possible photodamage to DNA origami and fixed cells.

[1] R. Jungmann et al., Nano Letters 2010, 10, 4756.

[2] P. Blumhardt et al., Molecules 2018, 12, 3165.

[3] M. Scheckenbach et al., Angew. Chem. Int. Ed. 2020, 4931

12:00 - 12:20
Donald Cameron*, Stockholm, Sweden

Regulation of Topoisomerase 2α activity via condensate formation and oncogenic activation by MYC

Donald Cameron*1, Kathryn Jackson*1, Carl Ivar Möller2, Matteo Mazzocca3, Chiara Pederiva1, Evgeniya Pavlova2, Sriram Kesarimangalam2, Fredrik Westerlund2, Davide Mazza3, Laura Baranello1

1Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
2Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
3Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Italy

Topoisomerases (TOPs) are essential enzymes which prevent the DNA from becoming over- or under-twisted as it is transcribed by allowing rotation around transient DNA breaks. If the overtwisted DNA isn't relaxed by TOPs, it can cause DNA damage and cell death. We have shown that the master oncoprotein MYC can recruit and stimulate TOPs at gene promoters to facilitate elevated transcription associated with cancer1. By understanding how this interaction is mediated, we could therapeutically target oncogenic stimulation of TOPs, thereby disconnecting transcription and supercoil relief specifically in cancer cells, thus identifying a new non-genotoxic modality for cancer therapy.

Using a range of techniques including in vivo single molecule tracking (SMT) and in vitro condensate imaging, we have discovered that the essential topoisomerase TOP2α can form condensates both in the nucleolus and locally at DNA substrates. The cell can regulate the equilibrium between these condensates dependent on the need for supercoil relaxation. MYC is also able to hijack this equilibrium to recruit more TOP2α to the DNA. We are also developing a method to directly image supercoiling in live cells in conjunction with TOP2α SMT to understand how TOPs scans the DNA to identify sites that require TOP activity.

[1] Das*, Kuzin*, Cameron et al., Molecular Cell, Vol. 82, p.140 (2022)

12:20 - 12:40
Jörg Fitter, Aachen, Germany

Brightness-gated two-color coincidence detection: a single-molecule tool for characterizing biological processes and high affinity bi-molecular binding

Jörg Fitter, Olessya Yukhnovets, Henning Höfig

I. Physikalisches Institut (AG Biophysik), RWTH Aachen University,52074 Aachen, Germany

Life on the molecular scale is based on a complex interplay of biomolecules under which the ability to form macromolecular complexes is crucial. Fluorescence based two-color coincidence detection (TCCD) is widely used to characterize molecular binding. However, TCCD suffers from an underestimation of coincident events due to a detection volume mismatch of the two different wavelengths involved in the confocal detection. Here, we introduce a brightness-gated TCCD (BTCCD) which overcomes this limitation [1]. We first demonstrate the performance of BTCCD by means of perfectly double labeled DNA-based calibration samples and present then application cases with cell-free protein synthesis assays to quantify, for example, the protein synthesis performance [2]. Since the BTCCD approach is intrinsically related to single molecule detection [3], the concentation of diffusing molecules is typically in the pico-molar regime which makes BTCCD a promising tool to determine bi-molecular binding affinities (in terms of KD-values) in this regime. Therefore, we applied BTCCD to very tight binders, such as antibodies and their related antigens, and measured reliable binding affinities for the biological binding partners with KD-values between 0.1 and 10 pico-molar.         

[1] Höfig, H. et al., Commun Biol. 2, 459. (2019)

[2] Kempf, N., et al. Sci Rep., 7, 46753. (2017) 

[3] Yukhnovets, O., et al. Biomolecules, 12, 468. (2022)

12:40 - 13:00
Kamila Nurmakova, New Haven, United States (Student Award)

Using Fluorescence Correlation Spectroscopy To Characterize Interactions Between Apolipoprotein E and Amyloid-beta Oligomers

Kamila Nurmakova1, Zachary Levine2

1Department of Chemistry, Yale University, New Haven, CT
2Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT

Apolipoprotein E is a major lipid and cholesterol transporter in the central nervous system. It exists as three major isoforms (E2, E3 wt, and E4) associated with different risks of developing Alzheimer’s disease (AD). The E4 allele is the strongest genetic risk of AD, while the E2 allele is regarded as protective. ApoE was shown to influence the synthesis, aggregation, clearance, and degradation of disordered amyloid-beta oligomers (Aβo), which are central to AD pathogenesis. Previous reports on interactions between Aβo and apoE are largely inconclusive because they rarely account for the dynamic nature of each protein. Furthermore, the presence of lipids influences the conformation of apoE and alters the aggregation kinetics of Aβo.
We developed a fluorescence correlation spectroscopy (FCS)-based assay that accounts for these variables and allows us to capture the interactions between stabilized Aβ oligomers and different isoforms of apoE in the presence and absence of lipids. We observed that isoforms of apoE bind Aβo with a similar high affinity. We also observed notable differences in the affinities of lipid-free and lipid-bound apoE to Aβ oligomers. Taken together, this work suggests a complex interplay between lipids, Aβ oligomers, and lipid transporting proteins in the context of AD.

Session: FRETchair: Viktorija Glembockyte
14:35 - 15:05
Enrico Gratton, Irvine, United States (Invited Talk, virtual)

Single cell physiological characterization in living tissue. Determination of cell fate

Enrico Gratton

Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California., Irvine

Optical super-resolution has been around for more than 10 years. Yet, superresolution is mainly applied to produce stunning images at the 10-20 nanometer scale of the interior of cell. This kind of superresolution imaging had limited applications to reveal the dynamics, motion and interactions of molecules at the nanoscale, which is at the basis of life.

In This talk we show work done in our lab to filling this gap by developing enabling technologies that will open the potential of superresolution imaging to dynamic at the microsecond-millisecond- temporal scale.

15:05 - 15:25
Michael Börsch, Jena, Germany

ATP-dependent subunit rotation of single FoF1-ATP synthase trapped in solution

Michael Börsch, Thomas Heitkamp, Iván Pérez

Single-Molecule Microscopy Group, Jena University Hospital, Nonnenplan 2, 07743 Jena, Germany

20 years ago we introduced single-molecule FRET (smFRET) measurements to study subunit rotation and regulatory conformational changes of individual FoF1-ATP synthases in liposomes, either driven by ATP hydrolysis or during ATP synthesis [1]. However, observation times of freely diffusing proteoliposomes in a confocal microscope are limited to tens of milliseconds by Brownian motion. To counteract diffusive motion actively in real time, we have built a fast anti-Brownian electrokinetic trap (ABEL trap, invented by A. E. Cohen and W. E. Moerner [2]) with a laser focus pattern and electrode feedback controlled by a FPGA. Using the ABEL trap for smFRET measurements we recorded fast subunit rotation in FoF1-ATP synthases at different ATP concentrations and revealed variable hydrolysis rates from enzyme to enzyme [3]. Here we show how the ATP/ADP ratio affects the speed of ATP-driven e-subunit rotation and the relative percentage of active FoF1. We will provide insights how phosphate, selenite or sulfite interfere with ADP inhibition of single FRET-labeled FoF1-ATP synthase from E. coli. Finally, we unravel the speed limit of ATP-driven subunit rotation in FoF1-ATP synthase reconsituted in a lipid nanodisc.

[1] M. Börsch, M. Diez, B. Zimmermann, R. Reuter, P. Gräber, FEBS lett, 527, 147-152 (2002).

[2] A. E. Cohen, W. E. Moerner, Proc SPIE, 5699, 293-305 (2005).

[3] T. Heitkamp, M. Börsch, J Phys Chem B, 125, 7638-7650 (2021).

15:25 - 15:45
Michael Schlierf, Dresden, Germany

An open-source framework for automated large throughput single-molecule FRET spectroscopy

15:45 - 16:05
Paul David Harris, Jerusalem, Israel

Multi-parameter photon by photon hidden Morkov modeling for confocal FRET and PIFE

Paul David Harris

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 9190401, Israel.

Photon by photon hidden Markov modeling (mpH2MM)1,2 extends the classical hidden Markov modeling by allowing variable time intervals between successive photon detection events. This makes it ideal for analyzing the photon arrivals detected in confocal-based single-molecule fluorescence experiments, extracting state parameters and transition rates from the data. We demonstrate how this method can detect and quantify transitions as fast as tens of microseconds and as slow as a few milliseconds in a variety of experimental modalities. Using alternating laser excitation with smFRET, it is possible to disentangle photophysical transitions from conformational dynamics in biomolecules. We further demonstrate that mpH2MM assists in detecting fluorescence lifetime-based dynamics as rapid as tens of microseconds in fluorescence lifetime-based measurements where a single dye reports on local events in the vicinity of the fluorophore, such as when using the effect known as PIFE. This shows that mpH2MM is a valuable tool in a variety of contexts, especially when multi-parameter fluorescence detection setups are available. We offer mpH2MM as a fast and efficient python module, H2MM_C, with a backend written in C, along with Jupyter notebooks to make mpH2MM easy to employ in your data analysis pipeline.

[1] Harris, P.D. et al., Nat. Comms., 13, 1000 (2022)

[2] Pirchi, M., et al. J. Phys. Chem., 120,13065 (2016)

16:15 - 16:25CLOSING REMARKS
Flash talk
Aleksandra K. Adamczyk, Fribourg, Switzerland

Orientation control of single molecules in DNA origami 

Kasper Arfman, Wageningen, Netherlands

Microfluidic Force Spectroscopy

Kasper Arfman1, Martijn van Galen2, Hanne van der Kooij2, Joris Sprakel1

1Laboratory of Biochemistry, Wageningen University & Research, Netherlands
2Laboratory of Physical Chemistry and Soft Matter, Wageningen University & Research, Netherlands

Single molecule force spectroscopy is a powerful tool to study a molecule’s stretching behaviour, bonding kinetics, and their relation to force. Most existing methods are however time consuming, costly and/or require expertise. Here we use microfluidic force spectroscopy (µFFS) as a method for multiplexed, simple and accessible molecular mechanics studies. It allows making 10s-100s of measurements in a single experiment. Force measurements can easily be combined with fluorescence imaging and require only very limited technical resources to set-up or perform. In µFFS, analytes are used as tethers connecting micrometre sized beads to the surface of a flow channel. The beads are used as handles to apply tensile forces to the analytes. Forces applied can be as low as ~1 pN and are tuned by flow rate. Using microscopy and tracking software, we can accurately quantify bond lifetime and extension from bead trajectories. We use µFFS to study how plant transcription factors (TFs) bind DNA. Here, the method serves multiple functions. Firstly, it provides forces needed to linearize DNA to facilitate single-molecule fluorescence imaging. Secondly, by measuring extension, the method can reveal how TF binding affects DNA conformation.

Mikayel Aznauryan, Pessac, France

RNA binding by human translation initiation factor 4B

Mikayel Aznauryan

ARNA Laboratory, U1212 INSERM, UMR5320 CNRS, University of Bordeaux
European Institute of Chemistry and Biology, University of Bordeaux, INSERM, CNRS

Translation initiation factor 4B (eIF4B) is an essential co-factor of RNA helicase eIF4A, and is particularly important for translation of mRNAs with long and structured 5’ untranslated regions. EIF4B possesses only one canonical folded RRM domain and the rest of the protein is predicted to be intrinsically disordered. Within the latter there are certain regions of sequence bias, such as the arginine-rich motif at the C-terminal region (CTR) of eIF4B. Both the RRM and the CTR of eIF4B are expected to bind RNA, as part of the key functionality of the protein, however the mechanistic aspects of these interactions are entirely vague nowadays. We used a combination of single-molecule Forster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopies to investigate the conformational and dynamical peculiarities of RNA-binding regions of eIF4B and furthermore elucidated the molecular mechanisms of eIF4B/RNA interactions.

Our results show that although CTR of eIF4B is disordered, it adopts relatively compact conformation, due to intrachain interactions that bring together two extremities of this region. The eIF4B CTR non-specifically binds RNA at relatively high affinity, but in extremely dynamic manner. Upon RNA binding the primary RNA binding region compacts, meanwhile leading to expansion of the overall protein chain. In contrast, the RRM of eIF4B binds RNA weakly, but with peculiar sequence specificity. Altogether, our results uncover the specifics of eIF4B/RNA binding and how different regions synergistically cooperate in this process.

Flash talk
Bryan A. Bogin, New Haven, CT, United States

Studying the sizes and kinetics of oligomer formation on the surface of micelles using fluorescence correlation spectroscopy

Bryan A. Bogin1, Zachary A. Levine2

1Department of Biophysics and Biochemistry, Bass Center, Yale University, 266 Whitney Ave., New Haven, CT 06520
2Department of Pathology, Bass Center, Yale University, 266 Whitney Ave., New Haven, CT 06520

In patients with Type II Diabetes, islet amyloid polypeptide (IAPP) fibrils slowly accumulate in pancreatic β-cells.1 While these fibrils are a hallmark of the disease, soluble oligomeric precursors are most potently linked with its pathogenicity.2 IAPP oligomers (IAos) disrupt homeostasis through a variety of mechanisms, including mitochondrial stress, aberrant Ca2+ signaling, and the formation of membrane pores.3 However, characterization of toxic oligomers has been challenging due to their instability in aqueous buffers. We hypothesize that toxic species are generated at a critical size of IAos, beyond which, leads to the spontaneous growth of amyloid. Since many IAos localize to cell membranes, we predict micelles (a membrane mimic) will slow aggregation rates and stabilize soluble intermediates. We use Thioflavin T (ThT) to monitor rates of fibril formation with and without sodium dodecyl sulfate (SDS) micelles. Additionally, we use fluorescence correlation spectroscopy (FCS) to measure the size of IAos during early stages of aggregation and in the presence of a small-molecule amyloid inhibitor. We monitor the size of IAos that produce a ThT signal to identify species undergoing conformational transitions, which may be associated with its toxic gain-of-function.

1. Raleigh, D.; Zhang, X.; Hastoy, B.; Clark, A. Journal of Molecular Endocrinology Volume 59 Issue 3 (2017). Journal of Molecular Endocrinology 2017, 59 (3), R121–R140.

2. Ferreira, S. T.; Vieira, M. N. N.; Felice, F. G. D. IUBMB Life 2007, 59 (4–5), 332–345.

3. Asthana, S.; Mallick, B.; Alexandrescu, A. T.; Jha, S. Biochimica et Biophysica Acta - Biomembranes 2018, 1860 (9), 1765–1782.

Volker Buschmann, Berlin, Germany

Integration of a Superconducting Nanowire Detector into a Confocal Microscope for TRPL-Mapping: Sensitivity and Time Resolution

Volker Buschmann1, Eugeny Ermilov1, Felix Koberling1, Jürgen Breitlow1, Hugo Kooiman2, Johannes W. N. Los2, Jan van Willigen2, Mario U. Castaneda2, Jessica de Wild3,4,5, Guy Brammertz3,4,5, Evangelos Sisamakis1, Rainer Erdmann1

1PicoQuant GmbH, Rudower Chaussee 29, D-12489 Berlin, Germany, info@picoquant.com
2Single Quantum, Molengraaffsingel 10, 2629 JD Delft, The Netherlands
3Hasselt University, imo-imomec, Martelarenlaan 42, 3500 Hasselt, Belgium
4Imec, imo-imomec, Thor Park 8320, 3600 Genk, Belgium
5EnergyVille, imo-imomec, Thor Park 8320, 3600 Genk, Belgium

Superconducting Nanowire Single Photon Detectors (SNSPDs) were introduced to the market in 2003 and since then have found a niche in multiple quantum optics applications, due to their outstanding properties such as fast instrument response and high quantum efficiency.[1] The high quantum efficiency is especially important for material science applications in the IR-range beyond 1000 nm, where other available single photon detectors have a low sensitivity, high dark noise and slow time response.

We have integrated a SNSPD (Single Quantum) into a standard MicroTime 100 confocal fluorescence lifetime microscope (PicoQuant) in order to compare the performance of different SNSPD designs with a standard IR-PMT for time-resolved photoluminescence (TRPL) measurements and imaging on a CIGS (Cu(InGa)Se2) device.

While one of the used SNSPDs had a classical single mode fiber coupling to guide the light onto the sensor, the other detector used an internal multimode fiber instead [2].

We detected a significant increase in photoluminescence sensitivity of both designs compared to a standard IR-PMT, as well as a several times higher sensitivity of the multimode-fiber coupled nanowire compared to the singe-mode fiber one, in spite of comparable photon quantum efficiencies in this wavelength range for the sensor only. The increased sensitivity combined with the lower dark count rate resulted in an increase of the signal-to-noise ratio by more than 2 orders of magnitude compared to the IR-PMT.

The high sensitivity of SNSPDs combined with high temporal resolution (instrument response function of the overall system was below 100 ps) allows to identify and investigate highly quenched micrometer-sized defect sides of the thin-layer CIGS sample even at low illumination levels.

[1] I.E. Zadeh, J. Chang, J.W.N. Los, S. Gyger, A.W. Elshaari, S. Steinhauer, S.N. Dorenbos, V. Zwiller, Superconducting nanowire single-photon detectors: A perspective on evolution, state-of-the-art, future developments, and applications, Appl. Phys. Lett. 118, 190502 (2021)

[2] J. Chang, I.E. Zadeh, J.W.N.Los, J. Zichi, A. Fognini, M. Gevers, S. Dorenbos, S. F. Pereira, P. Urbach, V. Zwiller, Multimode-fiber-coupled superconducting nanowire single-photon detectors with high detection efficiency and time resolution, Appl. Optics. 58, 36, 9803-9807 (2019)

Clement Cabriel, Paris, France

Single-molecule super-resolution imaging with microfabricated 3D substrates for 3D cell culture

Clement Cabriel1, R. Margoth Cordova Castro1, Erwin Berenschot2, Amanda Dávila-Lezama2,3, Kirsten Pondman4, Severine Le Gac4, Niels Tas2, Arturo Susarrey-Arce2, Ignacio Izeddin1

1Institut Langevin, ESPCI Paris, Université PSL, CNRS, 75005 Paris, France
2Mesoscale Chemical Systems, MESA+ Institute, University of Twente, PO. Box 217, Enschede AE 7500, the Netherlands
3Facultad de Ciencias de la Salud, Universidad Autónoma de Baja California, Blvd. Universitario número 1000, Valle de las Palmas, 22260 Tijuana, México
4Applied Microfluidics for BioEngineering Research, MESA+ Institute for Nanotechnology & TechMed Centre, and Organ-on-chip Centre, University of Twente, Enschede, The Netherlands

The potential of micro-and nanofabricated samples as a platform to modulate cell behavior using 3D physical cues has shown tremendous interest in cell differentiation or integrin endocytosis modulation. Topography can drastically alter cell behavior such that cell-cultured on rigid glass substrates behave as if they are on soft hydrogels. Investigating cell behavior and nanoscale organization requires advanced techniques such as AFM or super-resolution microscopy.

We produced glass fractal pyramids composed of several generations of octahedra of decreasing sizes, compatible with 3D SMLM of biological samples at the molecular scale. We explain the manufacturing process, then we show how these samples can be labelled with fluorescent dyes to perform SMLM characterization of their lateral and axial dimensions. We highlight how the multiscale nature of the fractals can be used to both perform axial calibration in 3D SMLM and measure resolutions.

Having demonstrated the feasibility of SMLM on these substrates, we use them as a platform for super-resolution bioimaging. Indeed, cells cultivated on these 3D substrates exhibit very different growth patterns than on flat glass substrates, close to spheroids even with short culture times. We perform SMLM quantitative assessment of modifications of protein distribution and directionality compared with plain glass substrates.

K. Metavarayuth, P. Sitasuwan, X. Zhao, Y. Lin, and Q. Wang, ACS Biomater. Sci. Eng. 2, 2, 142-151 (2016)

X. Li, L. H. Klausen, W. Zhang, Z. Jahed, C. Tsai, T. L. Li, and B. Cui, Nano Lett. 21, 19, 8518-8526 (2021)

F. Dituri, M. Centonze, E. J. W. Berenschot, N. R. Tas, A. Susarrey-Arce, and S. Krol, Nanomaterials, 11 (12), 3233 (2021)

Anindita Dasgupta, Jena, Germany

Effect of spectral shift of organic dyes in STED and STED-FCS microscopy

Flash talk
Felix Erichson, Mittweida, Germany

FRET-assisted RNA modeling

Felix Erichson1, Fabio D. Steffen2, Richard Börner1

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

Long-range tertiary interactions between RNA tetraloops (TL) and their receptors, such as kissing loops (KL), stabilize the tertiary fold of ribosomal RNA (rRNA) during the maturation process of the ribosome [1]. A minimal construct of a recently encountered KL-TLGAAA tertiary contact located in the rRNA of baker’s yeast was used in vitro to mimic the interaction during the maturation process [2]. Therein, polyA/U linker are necessary to link secondary structure elements (KL, TL) which are distant within the original sequence. Here, we use an integrative modeling approach that simplifies the linking of these building blocks in silico as PyMOL-based RNA Lego. Further we build the docked and the undocked state of the KL-TLGAAA construct, label this RNA models in silico and compute FRET histograms based on MD simulations and multiple-accessible contact volumes (mACV) [3, 4]. The latter allows us to compare our modeling results with single-molecule FRET experiments. This hybrid approach of experiment and simulation will promote RNA integrative modeling and elucidate our understanding of dynamic RNA tertiary contacts. Together, our approach will help to accelerate the discovery of novel RNA-RNA/DNA contacts and RNA-protein interactions as potential drug targets [5].

[1] P. Nissen et al., PNAS., 98, 4899–4903 (2001).
[2] S. Gerhardy, M. Oborská-Oplová, L. Gillet, R. Börner et al., Nat. Commun., 12, 4696 (2021).
[3] F. Erichson, F. D. Steffen, and R. Börner, Proceedings IWKM, 230–233 (2021). https://doi.org/10.48446/opus-12283 
[4] F. D. Steffen, R. K. O. Sigel, and R. Börner, Bioinformatics, 37, 3953–3955 (2021).
[5] M. Hoefling and H. Grubmüller, Comput. Phys. Commun., 184, 841–852 (2013).

Gregory-Neal Gomes, New Haven, United States

Structural Characterization of Pathological and Protective Isoforms of ApoE in the Presence of Lipids

Gregory-Neal Gomes1,2, Zachary Levine1,2

1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
2Department of Pathology, Yale School of Medicine, New Haven, CT 06520, USA

A major risk determinant for late-onset Alzheimer’s disease (AD) is polymorphism in apolipoprotein E (ApoE), a protein which plays a critical role in redistributing cholesterol and other lipids to neurons. Of the three isoforms, ApoE4(R112,R158) is associated with an increased risk and ApoE2(C112,C158) is associated with a decreased risk of AD, relative to the common ApoE3(C112,R158) isoform. How single amino-acid changes can substantially alter AD pathology remains unclear, though several lines of evidence suggest that isoforms differ in lipid binding and subsequent binding to cell surfaces receptors. However, a molecular level characterization of ApoE isoforms and their interactions with diverse lipids and receptors is challenging due to the presence of disordered regions in ApoE and its propensity to self-associate. To circumvent these challenges, we used single-molecule Förster Resonance Energy Transfer (smFRET) and replica-exchange molecular dynamics (REMD) to structurally characterize the conformational ensembles of full-length ApoE2/3/4 free in solution and bound to phospholipid vesicles of varying composition and curvature. We identify compact and extended conformations of ApoE that are modulated by mutations and lipid binding. These observations are critical for explaining the precise pathological mechanism(s) by which the ApoE polymorphism mediates AD risk.

Zhongying Han, Munich, Germany

Conformational Changes in the Glutamine Binding Protein are Driven by Protein-Ligand Interactions

Zhongying Han1, Sabrina Panhans1, Marija Ram1, Anna Herr1, Alessandra Narducci1, Michael Isselstein1, Paul D. Harris2, Eitan Lerner2,3, Douglas Griffith1, Niels Zijlstra*1, Thorben Cordes*1

1Physical and Synthetic Biology, Faculty of Biology, Großhadernerstr. 2-4, Ludwig-Maximilians-Universität München, 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
3The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel

Conformational changes in biomacromolecules underlie various biochemical pathways. Structural dynamics in periplasmic substrate binding proteins (SBPs) have been studied extensively over the past decades. The available structures of SBPs show open (often ligand-free, apo) and closed conformations (often liganded, holo). This suggests a ligand binding mechanism, where the open SBP recruits the ligand to form the closed state. There is, however, a growing body of evidence for the existence of intrinsic conformational changes in SBPs, which is ligand independent. In this work, we characterize structural dynamics of the glutamine binding protein (GlnBP) from Escherichia coli on timescales ranging from 100 µs to seconds. Our results suggest that the freely-diffusing GlnBP has two conformational states in solution, an open and a closed state, which are both static in the absence and the presence of saturating ligand. Furthermore, the exchange between the open and the closed state of GlnBP occurs slowly at low ligand concentrations. Once GlnBP becomes surface-immobilized, we show that a large fraction of the protein becomes non-functional. In summary, we find that GlnBP recognizes L-glutamine through a venus-fly-trap mechanism with a single unique conformation under both apo and holo conditions.

Flash talk
Ignacio Izeddin, Paris, France

Event-based sensor for fast and dense single-molecule localization microscopy

Clément Cabriel1, Christian Specht2, Ignacio Izeddin1

1Institut Langevin, ESPCI Paris, Université PSL, CNRS, 75005 Paris, France
2DHNS, Inserm U1195, Université Paris-Saclay, 94276 Le Kremlin-Bicêtre, France

Although single-molecule localization microscopy (SMLM) gives access to spatial resolutions down to the scale of protein size, its main advantage compared to electron microscopy is its viability for imaging dynamic processes in living samples. In that regard, a number of applications remain challenging due to the tradeoff between temporal and spatial samplings. This is particularly problematic whenever the system studied displays heterogenous protein densities and dynamic processes at different temporal scales.

Here, we propose a new approach to SMLM using an event-based sensor in place of conventional scientific cameras. Event-based sensors are commercially-available matrices of independent pixels that are sensitive to intensity variations rather than to absolute intensity values. In addition, their response time is very fast and a given frame rate need not be chosen before the experiment. This allows us to easily detect blinking molecules among overlapping fluorophores that stay in a bright state.

After describing the working principle and implementation of such event-based sensor for single-molecule microscopy, we provide details about data acquisition, treatment, and display in an SMLM workflow. We then demonstrate the capability of such an event-based sensor to provide super-resolution images with spatial resolution similar to that of EMCCD cameras on biological samples labelled with dSTORM organic dyes. Finally, we demonstrate the performance of event-based SMLM to work on a high-density regime. In such a situation, the event-based detection allows detecting only the moment when a molecule turns on or off, making it distinguishable from other molecules emitting in its diffraction-limited vicinity. All in all, we demonstrate a novel approach to SMLM with an event-based sensor, compare its performance with that of classical scientific cameras, and highlight the added value of event-based SMLM to perform experiments in the regime of high density overlapping PSFs.

Flash talk
Sankar Jana, Aachen, Germany

Interaction between linear pNIPAM and Nile Red studied with FCS and TCSPC

Sankar Jana, Dominik Wöll

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

The biocompatible homopolymer, poly(N-isopropylacrylamide) (pNIPAM) has been extensively studied microscopically and spectroscopically due to its applications in the fields of drug delivery, bioimaging, and polymer physics. For further progress for these applications, a detailed understanding of the properties of pNIPAM is essential. Studies with the solvatochromic dye Nile Red open new possibilities to understand the structure of pNIPAM gels and the solvent conditions in them. Also, Nile Red has been found to be an ideal dye for PAINT studies in pNIPAM microgels. We studied the interaction between Nile Red and two linear pNIPAM polymers with different chain lengths of molecular mass 2.5 and 1600 kg/mol, respectively, and different concentrations in aqueous solution. The diffusion coefficient of Nile Red was determined by FCS and the Nile Red fluorescence lifetime measured with TCSPC revealed its surrounding. Surprisingly, the diffusion coefficient of Nile Red does not reflect the one of the polymer chains which means that it does not permanently bind to pNIPAM. That binding occurs, however, can be shown with the fluorescence lifetimes which can be fitted with two main components, one for Nile Red in surrounding of (dense) pNIPAM and the other one rather resembling Nile Red in aqueous solution.

[1] A. Purohit, S. P. Centeno, S. K. Wypysek, W. Richtering, D. Wöll, Chem. Sci., 10, 10336 (2019).

[2] M. Sutter, S. Oliveira, N. N. Sanders, B. Lucas, A. van Hoek, M. A. Hink, A. J. W. G. Visser, S. C. De Smedt, W. E.  Hennink, W. Jiskoot, J. Fluoresc.,  17, 181 (2007).

[3] A. S. Klymchenko, Acc. Chem. Res., 50, 366 (2017).

Flash talk
Jiří Junek, Liberec, Czech Republic

Random Temporal Signals (RATS) Method for Fluorescence Lifetime Imaging

Jiří Junek1, Karel Žídek2

1Technical University in Liberec, Faculty of Mechatronics, Informatics and Interdisciplinary Studies, Studentská 1402/2, 461 17 Liberec, Czech Republic
2Regional Center for Special Optics and Optoelectronic Systems TOPTEC, Institute of Plasma Physics of the Czech Academy of Sciences v.v.i., Za Slovankou 1782/3, 182 00 Prague 8, Czech Republic

We present a novel RATS (RAndom Temporal Signals) method for fluorescence lifetime imaging (FLIM). The method is based on a random excitation of the measured sample. Therefore, PL dynamics is examined on a wide range of frequencies within the same dataset, and thus an arbitrary photoluminescence (PL) decay could be reconstructed from a single dataset via deconvolution. By using a suitable deconvolution regularization, we can highly limit the effect of noise. The RATS concept was implemented in a microscope setup based on a single-pixel camera configuration to reach two-dimensional PL mapping and FLIM, which can be attained even for the noise level of 3% of the PL amplitude. Moreover, we present an approach for the direct reconstruction of amplitude maps corresponding to lifetimes present in multi-exponential PL decay. Hence, in the case of N-exponential PL decay, it is possible to evaluate the FLIM spectrogram using just N reconstruction, which is not demanding on postprocessing time. Using our current optical setup, it is possible to map PL dynamics in the nanosecond or microsecond time scale with impulse response function (IRF) in units of a nanosecond.  

[1] J. Junek, L. Ondič, and K. Žídek, Opt. Express, 28(8), 12363–12372 (2020).

[2] J. Junek and K. Žídek, Opt. Express, 29(4), 5538–5551 (2021).

[3] J. Junek and K. Žídek, Opt. Express, 30(8), 12654-12669 (2022).

Flash talk
Agnes Koerfer, Jena, Germany

A new Analysis Approach to study non-Brownian diffusion in Biomembranes with temporal Image Correlation Spectroscopy

Agnes Koerfer1, Bela Vogler1, Jacopo Abramo2, Francesco Reina2, Christian Eggeling1,2

1Institute of Applied Optics and Biophysics, Friedrich-Schiller University, Jena, Germany
2Leibniz Institute of Photonic Technologies, Jena, Germany

Studying the diffusion of single molecules reveals important insights into the complex organization and function of the cellular plasma membrane. For example, the difference between molecules that undergo trapping in their diffusion, and those that diffuse freely on the plasma membrane is a consequence of their biophysical properties, and often relates to their physiological relevance.

Typically, the methods used to study diffusion in the cellular plasma membrane are Fluorescent Fluctuation Spectroscopy-techniques (FFS) and Single Particle Tracking (SPT). While FFS-techniques provide averaged results of a system of diffusing particles, SPT enables to study individual tracks of particles. In my project I am developing a new analysis routine for temporal Image Correlation Spectroscopy (tICS - part of the FFS-techniques [1]) to study non-Brownian lipid diffusion in the cellular plasma membrane. Further I want to combine tICS with STED-super resolution microscopy to get an enhanced insight into the dynamics of trapping events. In addition to test the capabilities and the limitations of this new analysis approach we set up a simulation toolbox for different particle diffusion modes. Furthermore, the data acquisition with the specific experimental conditions of our microscopy set up and the analysis routine were implemented in the toolbox.

[1] Wiseman, P. W., Cold Spring Harbor Protocols, 4, 2015

Maria Loidolt-Krüger, Berlin, Germany

An easy and reliable way to perform single molecule FRET measurements

Maria Loidolt-Krüger

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.

Flash talk
Maria Loidolt-Krüger, Berlin, Germany

Fast analisis with minimal user interaction in Fluorescence Lifetime Imaging

Maria Loidolt-Krüger

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 bining format and GPU-based algorithms FLIM images of 1024x1024 can be anaylsised 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.

Flash talk
Sarojini Mahajan, Eindhoven, Netherlands

Single-molecule localization microscopy on plasmonic nanoparticles

Sarojini Mahajan1, Teun Huijben2, Vincenzo Lamberti3, Swayandipta Dey4, Kim Mortensen6, Rodolphe Marie5, Peter Zijlstra7


Functionalized nanoparticles such as gold and silica are widely used for bio-sensing and drug-delivery applications. The sensitivity and specificity of these sensors depend on the number and the distribution of functional groups on the nanoparticle’s surface [1]. These properties are traditionally characterized using ensemble-averaged techniques which do not capture inter-and intra-particle heterogeneity.

Single-Molecule Localization Microscopy can provide insight into these heterogeneities at the single-particle and single-molecule level. However, the emitter and the nanoparticle couple to each other, resulting in a displaced and/or distorted point-spread function (PSF) causing mislocalization [2]. This is particularly the case for metallic particles that exhibit plasmon resonances, hampering the use of localization-microscopy for biosensing applications [3].

We use an approach based on DNA-PAINT [4] wherein the transient binding of DNA-labelled fluorophores to a DNA-functionalized particle is used to collect hundreds of PSFs from many individual particles. A wide range of exotic PSFs including donut, bi-lobed, and elliptical shapes are observed from gold nanorods, nanospheres, and nanocubes due to plasmon-fluorophore coupling. The experimental results are compared to the numerically calculated PSFs to predict the position of the fluorophore based on PSF shape.

In the future, machine learning algorithms may enable the fitting of distorted PSFs to rectify mislocalization.

[1] Horáček, M., Engels, D.J., & Zijlstra, P, Nanoscale,12, 4128-4136 ( 2020)

[2] Harrison J. Goldwyn, Kevin C. Smith, Jacob A. Busche, and David J. Masiello, ACS Photonics, 5, pp. 3141-3151 (2018).

[3] Frank Bloksma and Peter Zijlstra, J. Phys. Chem. C, 125, 40, 22084–22092 (2021).

[4] Adam Taylor, René Verhoef, Michael Beuwer, Yuyang Wang, and Peter Zijlstra, The Journal of Physical Chemistry. C, Nanomaterials and Interfaces 122 , 2336 - 2342 (2018).

Flash talk
Samuel Naudi-Fabra, Berlin, Germany

Quantitative description of intrinsically disordered proteins using single-molecule FRET, NMR and SAXS

Samuel Naudi-Fabra1,2, Maud Tengo1, Malene Ringkjøbing Jensen1, Martin Blackledge1, Sigrid Milles1,2

1Institute for Structural Biology. 71 Av. des Martyrs, 38000 Grenoble, France
2Current address : Leibniz-Forschungsinstitut für Molekulare Pharmakologie. Robert-Rössle-Straße 10, 13125 Berlin, Germany

Intrinsically Disordered Proteins (IDPs) are a broad family of molecules often undertaking essential cellular processes, and are notorious for eluding classical structural biology investigation strategies. Their highly dynamic behavior and their spanning of manifold conformations makes them suitable for vastly different arrays of functions, which cannot be linked to singular defined structures, but rather to ensembles of conformers. Building such ensembles requires extracting parameters from experimental data only accessible to solution state techniques ; such as small-angle scattering techniques, nuclear magnetic resonance (NMR), and single-molecule Förster resonance energy transfer (smFRET). When combined, these techniques are sensitive to local structural propensities, specific intermediate- to long-range contacts, as well as the overall dimension of the system. We set out to integrate large sets of smFRET efficiencies and fluorescence lifetimes with NMR paramagnetic relaxation enhancements (PREs), chemical shifts, and small-angle X-ray scattering (SAXS) data, measured on measles virus phosphoprotein as a model system. Cross-validation against experimental data not used for ensemble calculation, showed that our integrative approach allows us to derive quantitative and accurate conformational ensembles of predictive nature and carries potential for integrative structural biology studies of IDPs.

S. Naudi-Fabra, M. Tengo, M.R. Jensen, M. Blackledge, S. Miles, J Am Chem Soc. 2021 Dec 8;143(48):20109-20121. (2021)

Valentin Pitzen, Potsdam, Germany

Elucidating the Nanoscale: Clustering of Influenza A Viral Proteins at the Plasma Membrane

Valentin Pitzen, Annett Petrich, Salvatore Chiantia

University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, 14476 Potsdam, Germany

Understanding the molecular processes involved in the assembly of the influenza A virus (IAV) in host cells is an important step towards the design of targeted therapeutic approaches against this respiratory pathogen, which can cause illnesses with a significant social impact and mortality. Here, we present highly resolved microscopy datasets, based on PALM approach, investigating the interaction of the viral proteins HA, M1 and M2 at the plasma membrane with different lipid markers, such as KrΦ, Lact-C2, PLCδ-PH and GPI. We use transfected as well as infected cell models in our study to shed light on the clustering behavior of IAV structural proteins. Our results present new insights regarding the molecular interactions involved in IAV assembly, as a function of viral strain and host type.

Niels Radmacher, Göttingen, Germany

Fluorescence Lifetime Image Scanning Microscopy

Niels Radmacher

Friedrich-Hund-Platz 1, 37077 Göttingen, Germany

The spatial arrangement and relative composition of proteins in cells can be an essential clue for the health of an organism. Thus capturing high-resolution images of living cells in native environment is a vital tool for diagnostics in medicine and biology.
Image Scanning Microscopy (ISM) provides twice the resolution of a confocal microscope by replacing the single-point detector with a CCD chip.
Another excellent tool for bio-imaging is Fluorescence Lifetime Imaging Microscopy (FLIM), which allows for distinguishing different markers by their specific lifetime.
Combining these two techniques into a Fluorescence Lifetime Image Scanning Microscope (FL-ISM) enables superresolution microscopy with fluorescence lifetime multiplexing for live cell and tissue imaging. This requires an array of single-photon detectors such as SPADs (Single Photon Avalanche Diodes) or MPMTs (Multianod Photomultiplier Tube).
Our results show simultaneous fluorescence lifetime multiplexing for up to three different structures in two spectral regions. At the same time, we are keeping acquisition times comparable to other Fluorescence Lifetime Imaging techniques.

Flash talk
Elizabete Ruppeka Rupeika, Leuven, Belgium

Optical mapping: detecting kilo-basepair range elements in prokaryotic genomes

Elizabete Ruppeka Rupeika, Sergey Abakumov, Laurens D'Huys, Xiong Chen, Johan Hofkens

KU Leuven, Department of Chemistry, , Research group of Molecular Imaging and Photonics, , Celestijnenlaan 200F, B 3001 Leuven

Global metagenomic sequencing efforts have resulted in databases with thousands of entries. One way to make use of these databases is by using a shorthand approach to compare “fingerprints” of sequences to the database. One such shorthand is amplicon sequencing. We present optical mapping as an alternative, amplification-free cross-taxonomic shorthand for precise microbial identification from environmental samples. Enzymatically labelled single molecules are deposited on a microscopy cover-slide in a linear fashion. The fluorescent labels appear in a site-specific pattern upon imaging with wide-field fluorescence microscope systems, resulting in a barcode  or rather - a ’fluorocode’. The acquired experimental fluorocodes are compared to in silico generated fluorocodes of database entries to determine matches.

Genomes of bacteria, fungi, archaea and even phages/viruses (with sufficient dsDNA length) can be detected and identified from pure and complex samples (Bouwens et al, 2020). 

Finally, the method can also be used to detect unique elements in genomes starting at ~40 kbp in length (D’Huys et al., 2021).

D’Huys, L., Vitale, R., Ruppeka-Rupeika, E., Goyvaerts, V., Ruckebusch, C., & Hofkens, J. (2021). Assessing the Resolution of Methyltransferase-Mediated DNA Optical Mapping. ACS Omega, 6(33), 21276–21283. https://doi.org/10.1021/acsomega.1c01381

Bouwens, A., Deen, J., Vitale, R., D’Huys, L., Goyvaerts, V., Descloux, A., Borrenberghs, D., Grussmayer, K., Lukes, T., Camacho, R., Su, J., Ruckebusch, C., Lasser, T., Van De Ville, D., Hofkens, J., Radenovic, A., & Frans Janssen, K. P. (2020). Identifying microbial species by single-molecule DNA optical mapping and resampling statistics. NAR Genomics and Bioinformatics, 2(1), lqz007. https://doi.org/10.1093/nargab/lqz007

Vanessa Schumann, Mittweida, Germany

From in vitro transcription to biophotonic assays: a complete RNA production line including RNA labeling

Vanessa Schumann, Richard Börner

Laserinstitut Hochschule Mittweida, Mittweida, University of Applied Sciences Mittweida, Germany

Elucidating the function of RNA regarding structure and dynamics is imperative to gain a complete understanding of life at molecular level. Our investigations concentrate on a ribosomal RNA tertiary contact consisting of a kissing loop and a GAAA tetraloop. Only recently it was shown that the formation of this tertiary contact in presence of the protein Puf6[1] is mandatory for the ribosomal maturation process in Saccharomyces cerevisiae at lower temperatures. Our current research aims to establish a complete RNA production line for biophotonic assays and beyond. It includes RNA synthesis by in vitro transcription, subsequent purification steps and its 3’/5’-end fluorescence labeling for use in biophotonic assays[2]. The large scale in vitro synthesis allows further investigations in NMR, UV-VIS absorption measurements along with thermodynamic melting studies, both demanding high amounts of RNA. Furthermore, we present a fluorescence labeling technique to be used for biophotonic assays: the coupling of N-hydroxysuccinimide ester with a primary amino group as a useful strategy for postsynthetic fluorescent modifications of our RNA construct[3,4,5]. The labeling will enable fluorescence assays such as FRET on the ensemble and single molecule level[6]. The latter will be used to receive kinetic data of the RNA folding process and distance constraints for a dynamic view of the three-dimensional structure of this important ribosomal RNA tertiary contact.

[1] S. Gerhardy et al., Nat. Commun.,12:4696, 1-16 (2021)

[2] S. Gallo, M, Fuler, R. K. O. Sigel, Chimia, 59:11, 812-816 (2005)

[3] M. Li, Bioengineered, 11:1, 599-606 (2020)

[4] F. D. Steffen et al., Chimia, 73:4, 257-261 (2019)

[5] E. Ahunbay et al.,  G. Steger et al. (eds.), DNAzymes: Methods and Protocols, Methods in Molecular Biology, 2439, 173-190 (2022)

[6] G. Hanspach et al., RNA Biol., 16:9, 1119–1132 (2019)

Flash talk
Chiara Schirripa Spagnolo, Pisa, Italy

Two-colour single-particle tracking from its experimental to computational aspects to unveil biomolecules interactions

Chiara Schirripa Spagnolo, Aldo Moscardini, Rosy Amodeo, Stefano Luin


Single-particle tracking (SPT) has become a powerful technique to investigate the spatio-temporal organization of living systems at the single-molecule level. Multicolour SPT extensions can provide a quantitative description of biological interactions, but they are still limited. We identified several experimental and computational challenges for their implementation.

The first crucial point concerns the measurement of fluorescent labelling efficiency, an essential requirement for studying biomolecule interactions. The procedures found in the literature are limited or unconvincing. We developed a robust method based on a ratiometric multichannel procedure that allows an efficient determination of labelling degree.

Then I will discuss the experimental challenges arising from minimally invasive probes, which create limitations in signal-to-noise ratio and time resolution. We characterized different sources of undesired background, finding the best TIRF microscopy configuration with the proposal of novel strategies.

Finally, I will focus on the computational aspect. While different software are available for single-colour SPT, there are none specific for multicolour applications. We designed an approach based on parallel computing and a message-passing procedure. We developed algorithms for simulations of two-colour SPT to perform testing by varying different parameters. I show the greater robustness of our tracking method to challenging conditions in comparison to existing approaches.

Flash talk
Tobias Starling, Fleet, United Kingdom

Live-Cell Fluorescence Lifetime Imaging Microscopy Multiplexing Using Genetically Encodable Fluorescent Proteins to Characterize the HIV-1 Virological Synapse and Cell-Cell fusion in live cells.

Tobias Starling1, Irene Carlon-Andres1, David Williamson1, Sergi Padilla-Parra1,2

1Department of Infectious Diseases, King's College London, Faculty of Life Sciences & Medicine, London, United Kingdom.
2Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.

Fluorescence Lifetime Imaging Microscopy (FLIM) visualises the unique fluorescence lifetimes of fluorophores or fluorescent proteins; (FP) acting as a spectral fingerprint. This allows multiple spectrally similar fluorophores to be imaged simultaneously, as previously shown with inorganic fluorophores both in fixed and live cells. FLIM multiplexing with genetically encodable fluorescent proteins (FP) provides an opportunity to understand complex; multi-protein biological problems in live cells such as the HIV-1 virological synapse formation and cell-cell fusion.

There is currently no comprehensive list of fluorescent protein lifetimes under different experimental conditions. From a panel of 43 fluorescent proteins, we identified 21 pairwise to hexawise combinations in three spectral channels suitable for multiplexing. Indeed, employing either a single excitation or pulsed interleaved excitation (PIE), in excess of 8 colour simultaneous imaging has been demonstrated in a single acquisition using each FPs unique fluorescence lifetime.

Importantly, current lifetime multiplexing methods have avoided unmixing of spectrally similar fluorophores in the same pixel, irrespective of their lifetime. Here; we have unmixed spectrally similar fluorophores (mTFP1 and mTQ2) in a pixel by pixel manner and used this to determine the ratio of CD4 (64%) and CXCR4 (45%) at the HIV-1 virological synapse.

Flash talk
Matthew Steinsaltz, New Haven, United States

Spectroscopic Observations of p53 DNA-Binding Domain Aggregation

Matthew Steinsaltz1, Zachary Levine2

1Yale University, 266 Whitney Ave. New Haven, CT 06511
2Altos Labs, 5510 Morehouse Dr. San Diego, CA 92121

WT tumor suppressor protein 53 (p53) is a transcription factor that forms a homo-tetrameric complex; however, the DNA Binding Domain (DBD) can assemble into cross beta-sheet stacked fibrils and non-fibrilar aggregates with unclear functions1-4. Here, non-fibrillar aggregates encompass all non-native p53-p53 complexes currently off-pathway to a highly structured low-energy endpoint fibril. Notably, patient-derived tumor samples containing pathological hotspot mutants in the DBD can result in p53 aggregates and a loss of apoptosis1,3-5. Moreover, while aggregation-inhibiting peptide aptamers targeting p53 have mainly focused on a single DBD interface4,6, several solvent-exposed surfaces likely coordinate the aggregation phenotype3. Typically, thioflavin assays and electron microscopy demonstrate insoluble p53 aggregates; however, these methods often fail to clearly distinguish structural information of non-fibrillar aggregates. To address these concerns and deduce the properties of soluble non-fibrillar p53 aggregates, we employ fluorescence correlation spectroscopy to characterize the diffusion of non-fibrillar p53-DBD aggregates with and without a pathological hotspot mutation (R248Q or R175H). Additionally, we introduce therapeutic peptides to deduce the structural modularity of WT vs. mutant p53 aggregates. Together, these experiments help elucidate biophysical mechanisms of p53-DBD aggregation and suggest multiple p53 pharmacophores may exist that can be concurrently targeted to treat tumor growth in cancer.

[1] Xu J., Reumers J., Couceiro J., De Smet F., Gallardo Rodrigo., Rudyak S., Cornelis A., Rozenski J., Zwolinska       A., Marine JC., Lambrechts D., Suh YA., Rousseau F., Schymkowitz J., Nat Chem Biol., 7, 285–295 (2011)

[2] Ano Bom A.P., Rangel L.P., Costa DC., de Oliveira G.A., Sanches D., Braga C.A., Gava L.M., Ramos C.H., Cepeda A.O., Stumbo A.C., De Moura Gallo C.V., Cordeiro Y., Silva J.L., J Biol Chem., 287(33), 28152-62, (2012)

[3] Wang, G., Fersht, A.R, Proc Natl Acad Sci., 114(13), E2634-E2643 (2017)

[4] Palanikumar L., Karpauskaite L., Al-Sayegh, M., Chehade I., Alam M., Hassan S., Maity D., Ali L., Kalmouni M., Hunashal Y., Ahmed J., Houhou T., Karapetyan S., Falls Z., Samudrala R., Pasricha R., Esposito G., Afzal A.J., Hamilton A.D., Kumar S., Magzoub M., Nat Commun., 12, 3962, (2021)

[5] Ghosh S., Salot S., Sengupta S., Navalkar A., Ghosh D., Jacob R., Das S., Kumar R., Nath Jha N., Sahay S., Mehra S., Mohite G.M., Ghosh S.K., Kombrabail M., Krishnamoorthy G., Chaudhari P., Maji S.K., Cell Death Differ., 24, 1784–1798, (2017)

[6] Soragni A., Janzen D.M., Johnson L.M., Lindgren A.G., Nguyen, A.TQ., Tiourin E., Soriaga A.B., Lu J., Jiang L., Faull KF., Pellegrini M., Memarzadeh S., Eisenberg DS., Cancer Cell., 29(1), 90-103, (2016)

Flash talk
Liangxuan Wang, Tübingen, Germany

Monitoring and controlling tautomerization of single hypericin molecules in a tunable optical λ/2 microcavity

Liangxuan Wang1,2, Quan Liu1, Frank Wackenhut1, Johannes Giershcner2, Alfred J. Meixner1

1University of Tübingen, Institute of Physical and Theoretical Chemistry, Auf der Morgenstelle 18, D-72076 Tübingen, Germany
2IMDEA Nanoscience , C/ Faraday 9, Ciudad Universitaria de Cantoblanco, 28049, Madrid, Spain

Fluorescent organic bio-reporters are of great importance in bio- and biomedical applications. The advance of spectroscopic and computational techniques permits a unique, detailed nanoscopic insight into the interactions of the chromophore with its surrounding by combining single-molecule spectroscopy with quantum chemistry calculations1,2,3. The computational results validate the experimental findings by imaging the absolute orientation of the single molecule transition dipole moments (TDMs) in the three-dimensional manner.2,3 The current study is an important first step to develop a joint experimental-computational tool to probe nano-environmental effects at a single molecule level.

Tautomerization in hypericin is accompanied by reorientation of the TDM, which can be directly observed using confocal microscopy combined with higher-order laser modes. Quantitative tautomerization residence times can be obtained from the autocorrelation of the temporal emission behaviour revealing that hypericin stays in the same tautomeric state for several seconds, which can be influenced by the embedding matrix.3 Moreover, the study can be extended to a λ/2 Fabry–Pérot microcavity, where the local photonic environment is modified, leading to an alteration of the spontaneous emission rate.4 Our approach paves the way to monitor and even control reactions for a wider range of molecules at the single molecule level.

[1] Liu, Quan and Wackenhut, Frank and Hauler, Otto and Scholz, Miriam and zur Oven-Krockhaus, Sven and Ritz, Rainer and Adam, Pierre-Michel and Brecht, Marc and Meixner, Alfred J., The Journal of Physical Chemistry A, 124, 2497-2504 (2020).

[2] Liu, Quan and Wackenhut, Frank and Wang, Liangxuan and Hauler, Otto and Roldao, Juan Carlos and Adam, Pierre-Michel and Brecht, Marc and Gierschner, Johannes and Meixner, Alfred J., The Journal of Physical Chemistry Letters, 12,1025-1031 (2021).

[3] Liu, Quan and Wang, Liangxuan and Roldao, Juan Carlos and Adam, Pierre-Michel and Brecht, Marc and Gierschner, Johannes and Wackenhut, Frank and Meixner, Alfred J., Advanced Photonics Research, 2, 2000170 (2021).

[4] Wang, Liangxuan and Liu, Quan and Wackenhut, Frank and Brecht, Marc and Adam, Pierre-Michel and Gierschner, Johannes and Meixner, Alfred J., The Journal of Chemical Physics, 56, 014203 (2022).

Aditya Yadav, Mandi, India

Bovine Serum Albumin-Conjugated Red Emissive Gold Nanocluster as a Fluorescent Nanoprobe for Super-resolution Microscopy

Aditya Yadav1, Navneet C. Verma1, Chethana Rao1, Pushpendra M. Mishra1,2, Amit Jaiswal2, Chayan K. Nandi1

1School of Basic Sciences, Indian Institute of, Technology Mandi, Mandi 175075, H.P., India.
2School of Basic Sciences and BioX Centre, Indian, Institute of Technology Mandi, Mandi 175075, H.P., India.

The gold nanocluster (GNC), because of its interesting photoluminescence properties and easy renal clearance from the body, has tremendous biomedical applications. Unfortunately, it has never been explored for super-resolution microscopy (SRM). Here, we present a protein-conjugated red emissive GNC for super-resolution radial fluctuation (SRRF) of the lysosome in HeLa cells. The diameter of the lysosome obtained in SRRF is ∼59 nm, which is very close to the original diameter of the smallest lysosome in HeLa cells. Conjugation of protein to GNC aided in the specific labeling of the lysosome. We hope that GNC not only will replace some of the common dyes used in SRM but due to its electron beam contrast could also be used as a multimodal probe for several other correlative bioimaging techniques.

(1) Zheng, J.; Zhang, C.; Dickson, R. M. Phys. Rev. Lett. 2004, 93 (7), 77402.

(2) Loynachan, C. N.; Soleimany, A. P.; Dudani, J. S.; Lin, Y.; Najer, A.; Bekdemir, A.; Chen, Q.; Bhatia, S. N.; Stevens, M. M. Nat. Nanotechnol. 2019, 14 (9), 883-890.

(3) Yadav, A.; Verma, N. C.; Rao, C.; Mishra, P. M.; Jaiswal, A.; Nandi, C. K. J. Phys. Chem. Lett. 2020, 11 (14), 5741-5748.

Flash talk
Mehrta Shirzadian Yazd, Vienna, Austria

Designing efficient MINFLUX schemes

Mehrta Shirzadian Yazd, Maximilian Korbinian GEISMANN, Alessandro PASSERA, Alba GOMEZ SEGALAS, Francisco Balzarotti

IMP, Vienna

MINFLUX [1,2] is a novel single-molecule localization strategy based on sequential excitation with tailored illumination patterns featuring a zero of intensity. Such schemes make individual photons more informative, leading to sub 3 nm isotropic 3D localization precision for imaging and ~100-fold increase in temporal resolution, in comparison to classical camera localization.

In search for even higher photon efficiencies, here we present a mathematical model that describes MINFLUX localization performance, including the generation of background light. A numerical implementation of the model is used to characterize the performance of different MINFLUX schemes. We studied the effect of mixing different beam shapes and adding information sources such as the fluorescence emission spectra and lifetime as well as detection arrangements with access to spatial information. We present several optimized configurations and implementations that surpass previous reports by 2–3 fold and extend the usable MINFLUX range in all three dimensions.

[1]                   F. Balzarotti et al., “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science, vol. 355, no. 6325, pp. 606–612, Feb. 2017, doi: 10.1126/science.aak9913.

[2]                   Y. Eilers, H. Ta, K. C. Gwosch, F. Balzarotti, and S. W. Hell, “MINFLUX monitors rapid molecular jumps with superior spatiotemporal resolution,” PNAS, vol. 115, no. 24, pp. 6117–6122, Jun. 2018, doi: 10.1073/pnas.1801672115.

Simon Zhamkochyan, Yerevan, Armenia

Advanced Radio Frequency Timing Apparatus: Technique and Applications

Amur Margaryan Simon Zhamkochyan

Alikhanyan National Science Laboratory, 2 Alikhanyan Bros., 0036, Yerevan

 We presentt a new RF timing apparatus, where the position sensor, consisting of microchannel plates and a delay-line anode, produces ~ns duration pulses with small dead time. Measurements made with sub-ps duration laser pulses, synchronized to the radio frequency power, produced a timing resolution of ~10 ps. The time stability of the technique over a period of ~1 hour is within the range of the statistical uncertainty which is about 0.5 ps, FWHM. This ultra-high precision and ultra-stable timing technique has potential applications in a large variety of scientific devices. A possible application of this new timing technique is in the single photon sensitive detector, namely in the Radio Frequency Photo Multiplier Tube (RFPMT). The RFPMT has potential applications in many fields of science and industry, which include fundamental physics, high energy and nuclear physics, chemistry, medical and biomedical imaging and material science.

A. Margaryan et al., An RF timer of electrons and photons with the potential to reach picosecond precision. Nucl. Instr. and Methods A1038 (2022) 166926, https://doi.org/10.1016/j.nima.2022.166926

Workshop fees

  until June 24, 2022 June 25, 2022 until August 7, 2022
Academic/University 340 € 390 €
Industry and Private Sector 750 € 950 €

Besides full workshop attendance, the fee includes coffee breaks, a reception with food and drinks, and two lunches. Attendees will be responsible for their own travel, lodging, and additional meals.

Please note the terms and conditions

  1. For payment you can choose between credit card (Visa, Master Card) and bank transfer. Possible bank charges have to be paid by the participant. Please note, that we do not accept checks.
  2. After online registration, you will receive an email notification including a PDF file that includes information on the payment procedure.
  3. All payments have to be received within 14 days after date of registration.
  4. We will send an email confirming your participation once we have received your payment. If payment is overdue, your registration will not be processed and considered invalid.
  5. A receipt of payment will be included in our email confirmation of participation.
  6. Cancellation of registration must be submitted in writing or via email and is valid only with acknowledgment of receipt by PicoQuant GmbH. A refund of registration fees is dependent on the notice given:
    • For cancellations made until August 7, 2022, 75 % of the registration fee will be reimbursed. In case of cancellations after August 7, 2022, 25 % of the registration fee will be reimbursed.
    • It is possible to name and send a substitute participant.
  7. No visa letters will be issued until payment of the registration fee is received and confirmed.
  8. Schedule and content of this event is subject to change without notice.
  9. PicoQuant records photographs and video material (including audio) of participants at the events. By registering for a PicoQuant event, you agree that we may include pictures or video material of yourself (either as a full image or in parts) for promotional purposes in various publications (press releases, promotional activities, event website, social media, etc.). If you do not want us to use pictures or video material in which you appear, please inform us in written form.

Financial support

PicoQuant has a fee waiver program for a small number of participants from universities or academic sector. Accommodation, travel, and personal expenses still need to be paid by the participants themselves. The selection of sponsored people is completely the sole decision of PicoQuant and there is no right or guarantee to receive a fee waiver.

The deadline to apply for a fee waiver has passed. We can no longer accept any fee waiver applications.



Registration is closed. Please contact us via workshop@picoquant.com in case you want to be placed on the waiting list. 

Workshop location

The workshop will be held in the science and technology park of Berlin-Adlershof, located in the south-eastern part of Berlin, not far from the Schönefeld Airport.

Carl-Scheele-Straße 6 / Max-Born-Straße
12489 Berlin

Local area map showing the symposium location (red marker)


We have negotiated special rates for a limited number of rooms in several hotels/appartment block, located close to the workshop location. The number of rooms as well as booking time are limited and we therefore advise to reserve your room as soon as possible.

Hotels within walking distance to the workshop location


ADAPT Apartments
Erich-Thilo-Straße 3, 12489 Berlin
Phone: +49-30-678-929-80
Fax: +49-30-678-929-82
Website of the apartment house

Room prices per night
  • single room: 78.40 € (excl. breakfast)

Breakfast is available for 12 € per day and person at the hotel‘s catering partners in walking distance. It can be booked directly through the hotel.

Wireless LAN is included in the room price.

ADAPT Apartments Berlin-Adlershof

Booking code: WS27

Please book your room via e-mail or phone using the booking code.

The rooms are bookable at this rate until July 28, 2022 on a first come, first served basis. We cannot guarantee reservations at these prices or any reservations at all after this date.

Airporthotel Berlin-Adlershof
Rudower Chaussee 14, 12489 Berlin
Phone: +49-30-720-2222-000
Fax: +49-30-720-2222-100
Website of the hotel

Room prices per night
  • studio apartment for single use: 104 € (incl. breakfast)
  • studio apartment for double use: 117 € (incl. breakfast)

breakfast is included in the room price

Airporthotel Berlin-Adlershof

Booking code: PicoQuant WS 27

Please book your room by e-mail, phone or fax using the booking code PicoQuant WS 27

The rooms are bookable at this rate until July 26, 2022. We can not guarantee any reservations at these prices or any reservation at all after this date.

Essential by Dorint Berlin Adlershof
Rudower Chaussee 15, 12489 Berlin
Phone: +49-30-67822-0
Fax: +49-30-67822-1000
Website of the hotel

Room prices per night
  • single room: 109 € (incl. breakfast)
  • double room: 128 € (incl. breakfast)

Wireless LAN is included in the room price.

Dorint Hotel Berlin Adlershof

Booking code: Workshop

Please book your room via e-mail using the booking code.

If you do not wish to have breakfast included, please inform the hotel when making your reservation.

The rooms are bookable at this rate until July 15, 2022 on a first come, first served basis. We cannot guarantee reservations at these prices or any reservations at all after this date.


CoVID-19 information

Currently there are no CoVID-19 specific regulations for hosting the workshop in September. However, we all know that this might change quickly, depending on local spread of infections. We will therefore make sure that this place always contain the latest information. The currently valid regulation regarding CoVID-19 for the city of Berlin can be found at https://www.berlin.de/corona/en/measures/directive/

Note that is currently still mandatory to wear FFP2 or medical face masks in public transport.

Although there are no regulations, the safety of participants is important for us. We will therefore handout FFP2 masks as well as self-test kits to all participants during the workshop.


Information on travel to Germany

There are no travelling restrictions at the moment. For updated information please check the Information on entry restrictions, testing and quarantine regulations by the German Federal Foreign Office

Be aware that when travelling from Germany back to your country, other rules, restrictions, and regulations will apply. Make sure to also regularly read up on the rules governing travel back to your country.

Remember: when travelling, stay informed and (above all) stay safe!


Conditions for in-person participation

Based on the currently valid SARS-CoV-2 Infection Protection Measures of the city of Berlin, workshop partcipation is only possible for persons that can provide proof of either having:

  • been fully vaccinated against Covid-19 with a vaccine approved by the European Union (refer to the link for a list of currently approved vaccines), or
  • completely recovered from a SARS-CoV-2 infection, or
  • tested negative by either a point-of-care (PoC) antigen test not older than 24 hours or a current PCR test not older than 48 hours

Please prove your status by showing an appropriate document (either in digital or paper form) during the registration. Note: if you are not fully vaccinated or recovered you need to provide an offical negative test result every day.

Please contact us early in advance if you have any questions regarding the proof of your status. Please understand that during the registration process there will be no time to discuss this matter any further.

More details on the hygiene measures implemented at the venue will follow in September.

Information on travel to Germany

Due to the pandemic, international travel has become quite a bit more complicated. You can find the currently applicable rules for travelling to Germany by following the links below. One thing that COVID-19 taught us is that rules can (and do) change quite quickly.

Information on entry restrictions, testing and quarantine regulations by the German Federal Foreign Office

Current information for travellers by the German Federal Minsitery of Health

Be aware that when travelling from Germany back to your country, other rules, restrictions, and regulations will apply. Make sure to also regularly read up on the rules governing travel back to your country.

Remember: when travelling, stay informed and (above all) stay safe!



The workshop on "Single Molecule Spectroscopy and Ultra Sensitive Analysis in the Life Sciences" is an annual event since 1995. To get an impression of our Single Molecule Workshops have a look at the video below.

For a summary of each year's event, please select the year from the list below.


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