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23rd Single Molecule Workshop

23rd International Workshop on
“Single Molecule Spectroscopy and Super-resolution Microscopy in the Life Sciences”

September 13-15, 2017 in Berlin, Germany

Image 23rd Single Molecule Workshop 2017

Join our workshop by giving a talk, presenting a poster, or without any presentation. We especially encourage young scientists to present their work with an oral presentation. A special prize will be awarded for the “Best Student Talk”.

Aim and Purpose

Poster session at the 18th workshop

The workshop aims to provide an interdisciplinary platform for the exchange of experience and information as well as sharing recent findings in the field of ultrasensitive optical detection down to the single molecule level and beyond the classical diffraction limit. The target audience are researchers and developers from Physics, Chemistry, Biology as well as from Life or Materials Science.

Ultrasensitive microscopy and spectroscopy techniques have become standard tools for fundamental biological and biomedical research, allowing studying the function and structure of individual biomolecules as well as interactions between them. The range of techniques and methods has steadily grown since the first report of single molecule detection in 1976.

Today, single molecules can be detected using either wide field or confocal fluorescence microscopy, Scanning Nearfield Optical Microscopy (SNOM), Atomic Force Microscopy (AFM), or Raman scattering. Time-resolved methods such as Fluorescence Lifetime Imaging (FLIM), Fluorescence Correlation Spectroscopy (FCS) or even multidimensional fluorescence methods are used on a daily basis in imaging facilities. Measurements beyond Abbe's diffraction limit are possible thanks to techniques such as Stimulated Emission Depletion Microscopy (STED), localization microscopy (PALM, STORM, dSTORM, GSDIM), or fluctuation microscopy (SOFI). Interest in super-resolution microscopy techniques has increased in recent years and their importance was recognized by awarding the Nobel Price in Chemisty 2014 to Eric Betzig, Stefan W. Hell, and W.E. Moerner.

The current focus in ultrasensitive optical detection lies not only on improving and extending the range of single molecule and super-resolution techniques, but also to use them for challenging applications ranging from chemical analysis to biophysics, biological and biomedical research, medical diagnostics, and materials research.

Combining Conferences: MAF 2017

The conference on Methods and Applications in Fluorescence (MAF) has a long standing tradition of bringing together experts working on (super-resolution) fluorescence microscopy, lifetime spectroscopy, imaging and probe design, as well as on nanomaterials, opto-electronics, or data acquisition and processing.

The 15th MAF will take place in Bruges (Belgium) from September 10th to 13th, 2017, just a few days before PicoQuant's 23rd Single Molecule Workshop. Since both events cover similar topics, why not combine them by taking a short trip from Bruges to Berlin?

Abstract submission

Abstract submission for both oral and poster presentations is now closed.

The deadline for abstract submission is May 31, 2017. Post deadline abstracts may not be considered.

The deadline for abstract submission has been extended to June 6, 2017. Post deadline abstracts may not be considered.

Abstract submission for oral presentations is closed. Abstracts for post deadline poster presentations can still be submitted until August 15, 2017.

Abstract submission for oral presentations is closed. Abstracts for post deadline poster presentations can still be submitted until August 15, 2016.

Please contact us via email if you are interested in presenting a poster at the workshop.

  • Abstracts can be submitted for oral or poster presentation. Depending on the number of received abstracts, some oral presentations may be changed to a poster presentation.
  • Abstracts can only be submitted along with the registration for the workshop.
  • Abstracts must be submitted in English containing not more than 200 words and no graphics.

Student award

PicoQuant especially wants to encourage young scientists to present their work. Therefore, the “Best Student Talk”  will be awarded with a special prize of 750 Euro. Undergraduate and graduate students are invited to submit their contributions until May 31, 2017. Please indicate during the registration/abstract submission if you wish to participate in the contest.

Important dates

  • Deadline for submission of abstracts: (May 31, 2017) extended to June 6, 2017
  • Deadline for early bird registration: (May 31, 2017) extended to June 6, 2017
  • Deadline for a fee waiver application: May 31, 2017
  • Deadline for submission of abstracts for post-deadline posters: August 15, 2017
  • Final deadline for workshop registration: August 15, 2017
  • Notification on acceptance of abstracts: July 2017
  • Program available: July 2017

SymPhoTime Training Day

One day before the workshop, on September 12, PicoQuant will host the “SymPhoTime Training Day” for users of the SymPhoTime and SymPhoTime 64 software. Participation is free of charge.

For details visit the event website.

Conference on Single Molecule Spectroscopy at BiOS 2018

Within the framework of the Biomedical Optics Symposium BiOS, PicoQuant is co-organizing the special conference "Single Molecule Spectroscopy and Superresolution Imaging XI" (BO503)." The call for papers is now open and abstracts are due July 17, 2017. If you would like to submit a paper, please visit the SPIE's abstract submission page. As a special motivation for young researchers, PicoQuant is presenting the "Young Investigator Award" as part of this session. Young scientists (age 32 or below and not yet full faculty members) are encouraged to participate in this best paper competition which offers a cash award worth 1000 USD.

Future workshops

  • September 5-7 or 12-14, 2018
  • September 3-6 or 10-13, 2019


Workshop coordinator: Jana Bülter

Tel: +49-30-1208820-0
Fax: +49-30-1208820-90
Email: workshop@picoquant.com

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

Invited speakers and their tentative titles

The list of speakers will be announced at a later date.

Workshop location

The workshop will be held in Berlin-Adlershof. Details will be announced at a later date.

The workshop will be held in the "Max-Born-Saal" in Berlin-Adlershof, located in the south-east of Berlin.

12489 Berlin

Local area map showing the workshop location (red marker)


The program consists of invited and contributed oral presentations, as well as poster presentations.

The schedule will be published online in July 2017.

We have received an overwhelming large amount of abstracts for talks and posters. We thank all particpants for their contribution.

12:00 - 13:15Registration and collection of workshop material
13:15 - 13:30Rainer Erdmann, Berlin, Germany
Opening Remarks
Session: FRET/FCS/FLIM 1Chair: Thorben Cordes
13:30 - 14:00
Paul W. Wiseman, Montreal, Canada (Invited Talk)

ICS with a new focus…pairing image correlation spectroscopy with super-resolution imaging

Paul W. Wiseman

Departments of Chemistry and Physics McGill University, paul.wiseman@mcgill.ca

Image correlation spectroscopy (ICS) methods allow analysis of protein-protein interactions and macromolecular transport properties from fluorescence microscopy images of living cells. These techniques are based on space and time correlation analysis of fluctuations in fluorescence intensity within the image time series. Previously applications of spatio-temporal image correlation spectroscopy (STICS) have measured vectors of protein flux in cells based on the calculation of a spatial correlation function as a function of time from an image time series. As well the two color extension, spatio-temporal image cross-correlation spectroscopy (STICCS), was used to measure co-transport different proteins labeled with fluorophores of different emission wavelengths, however, these previous applications all involved analysis of diffraction limited optical microscopy data. Here I will present new directions for the image correlation methods involving extensions to super-resolution microscopy. Application of STICS and STICCS paired with structured illumination microscopy (SIM) imaging of actin and membrane component dynamics in migrating Jurkat cells will be shown. Also the combination of k-space image correlation spectroscopy (kICS) with super-resolution optical fluctuation imaging (SOFI) to obtain both probe transport dynamics and a super-resolved image of blinking but static probes will be introduced with application to Dronpa-actin expressed in cultured cells.

14:00 - 14:20
Ali Ibrahim, Orsay, France (Student Award)

Spectral and fluorescence lifetime measurement of brain tumor tissues using a customized double-clad optical fiber

Ali. Ibrahim1, Fatima. Melouki2, Fanny. Poulon3, Marc Zanello4, Darine. Abi Haidar5

1IMNC Laboratory, UMR 8165-CNRS, Orsay, France
2IMNC Laboratory, UMR 8165-CNRS, Orsay, France
3IMNC Laboratory, UMR 8165-CNRS, Orsay, France
4Neuropathology Department, Sainte-Anne Hospital, France
5IMNC Laboratory, UMR 8165-CNRS, Orsay, France

Minimal invasive surgery is becoming the gold standard in oncology surgery. The 21th surgery requires new tools designed to slide into small surgical approaches and able to give fast and precise information on the tissue met by the surgeon. To address this need, we develop a multimodal nonlinear endomicroscope[1][2]. The endoscopic system enables imaging fluorescence and second harmonic generation as well as spectral and fluorescence lifetime.

In this work, we present a new customized microstructure Double Clade Optical Fiber (DCF) dedicated to in vivo multimodal imaging. This fiber insures short pulse duration thanks to a compensating grism line and to its small core diameter. We characterized this DCF using visible and near infrared excitation wavelength. A spectral and lifetime measurement was accomplished on fresh and fixed tumoral brain sample (mouse and human).  Results were compared to those obtained with other fibered system. 

 [1]C. Lefort, H. Hamzeh, F. Louradour, and D. A. Haidar, J. Biomed. Opt. 19, 076005–076005 (2014)

 [2]H. Hamzeh, C. Lefort, F. Pain, and D. A. Haidar, Opt. Lett. 40, 808–811 (2015)

14:20 - 14:40
Jonas Mücksch, Martinsried, Germany (Student Award)

FCS on lipid membranes in sugar solutions: Disentangling the effects of viscosity and refractive index mismatch

Jonas Mücksch, Petra Schwille, Eugene P. Petrov

Max Planck Insitute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

Fluorescence Correlation Spectroscopy (FCS) is known to be a powerful tool to assess molecular mobilities in various settings ranging from bulk solutions to model lipid membranes, to living cells. In case of membrane studies, FCS is frequently performed on giant unilamellar vesicles (GUVs) serving as a model of a freestanding lipid bilayer. In a number of published FCS studies, GUVs are prepared in sugar solutions, rather than in pure water. As a result, the medium surrounding the lipid membrane has not only a higher viscosity compared to water, but also a higher refractive index. Similarly, all FCS measurements in cells and dense macromolecular solutions are inherently performed at a refractive index different from water. At the same time, it has previously been shown that the refractive index mismatch can result in a strong systematic bias in the results of FCS measurements. We explore whether it is possible to disentangle the effects of the solution viscosity and refractive index mismatch on FCS measurements carried out using a standard confocal microscope-based setup.

14:40 - 15:00
Narain Karedla, Göttingen, Germany (Student Award)

Measurement of Thickness and Leaflet-Dependent Diffusion of Lipids in Lipid Bilayers using MIET and 2f-FLCS

Narain Karedla1, Sebastian Isbaner1, Falk Schneider2, Alexey I. Chizhik1, Ingo Gregor1, Jörg Enderlein1

1III. Institute of Physics, Georg-August-Universität, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany
2Weatherall Institute of Molecular Medicine, John Raddcliffe Hospital, University of Oxford, United Kingdom

Diffusion plays a key role for passive transport and signaling in cell membranes. A lipid bilayer is a simple model system for these membranes that has been used extensively for diffusion studies.  As the thickness of a bilayer is just a few nanometers, the diffusion coefficient is usually an average over both leaflets. We report here the first simultaneous measurement of the thickness and lipid diffusion within the bilayers in a leaflet-dependent manner. We use the recently developed method of Metal-Induced Energy Transfer (MIET) [1], which is based on the distance dependent metal-induced fluorescence quenching, in combination with dual-focus Fluorescence Lifetime Correlation Spectroscopy (2f-FLCS) [2,3], which yields temporal intensity correlations of each lifetime species and cross-correlations between them. This allows us to determine the location and diffusion coefficients of lipids in a leaflet-dependent manner, interleaflet coupling and flip-flop dynamics.

[1] Karedla, N.; Chizhik, A. I.; Gregor, I.; Chizhik, A. M.; Schulz, O.; Enderlein, J., ChemPhysChem, 15, 705-711 (2014).

[2] Weiß, K.; Enderlein, J., ChemPhysChem, 13, 990-1000 (2012).

[3] Benda A.; Fagulova V.; Deyneka A.; Enderlein J.; Hof M., Langmuir, 22, 9580-9585 (2006).

15:00 - 15:35COFFEE BREAK
Session: FRET/FCS/FLIM 2Chair: Paul Wiseman
15:35 - 16:05
Thorben Cordes, Groningen, Netherlands (Invited Talk)

Dynamic structural biology of membrane transporters: mechanisms and tool development

Thorben Cordes

University of Groningen

Membrane transporters are vital to any living system and are involved in the translocation of a wide variety of substrates. Despite their importance, mechanistic models for transport are only based on indirect evidence. My group has recently started to use single-molecule fluorescence microscopy to characterize conformational states and changes in active transporters and with that to directly observe how different steps in transport are coordinated.[1,2] In my talk I describe mechanistic studies of substrate binding and release in ATP binding cassette importers using single-molecule Förster resonance energy transfer (FRET). I further describe our latest developments of “enabling technology”, i.e., a simple two-color FRET assay that allows to simultaneously monitor multiple distances within biochemical complexes.[3] I finally summarize our contributions towards the development of """self-healing""" organic fluorophores and their applications in single-molecule FRET and super-resolution microscopy.[2]

[1] G. Gouridis et al., Nature Structural & Molecular Biology 22 (2015) 57-64

[2] J.H.M. van der Velde et al., Nature Communications 7:10144 (2016)

[3] E. Ploetz and E. Lerner et al., bioRxiv doi: 10.1101/047779 (2016)

16:05 - 16:25
Antonino Ingargiola, Los Angeles, United States

Multi-spot approach for high-throughput freely-diffusing single-molecule FRET

Antonino Ingargiola1, Eitan Lerner1, SangYoon Chung1, Angelo Gulinatti2, Ivan Rech2, Massimo Ghioni2, Shimon Weiss1, Xavier Michalet1

1Dept. Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA, USA.
2Dip. di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milano, Italy.

Freely-diffusing single-molecule FRET (smFRET) allows detecting conformational changes or binding interactions of biomolecules in a heterogeneous sample. As opposed to surface-immobilized techniques, freely-diffusing measurements have the advantage of a simpler sample preparation and of being immune from surface interactions which can affect conformation or kinetics of molecules under study. One of the main limitations of freely-diffusing experiments is the long acquisition time needed to characterize the different subpopulations. This is due to the high number of photon-bursts from single-molecules crossing the excitation volume needed to build a representative distribution of subpopulations. To overcome this limitation, the multi-spot system[1] parallelizes both the excitation and the detection of the fluorescent signal by using an extended excitation pattern (series of confocal spots) coupled to a new generation of high photon detection efficiency SPAD arrays[2]. In this configuration, the acquisition time is divided by the number of pixels allowing to perform high-throughput smFRET experiments (i.e. testing several experimental conditions in a short time span) or to follows kinetic activity on faster timescales than possible with single-spot systems (seconds versus several minutes). We report the progress and challenges toward building the first 48-spot smFRET system with dual-color laser excitation and detection using 48-pixels SPAD arrays.

[1] Antonino Ingargiola; Francesco Panzeri; Niusha Sarkosh; Angelo Gulinatti; Ivan Rech; Massimo Ghioni; Shimon Weiss; Xavier Michalet, Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 85900E (2013)

[2] Xavier Michalet; Antonino Ingargiola; Ryan A. Colyer; Giuseppe Scalia; Shimon Weiss; Piera Maccagnani; Angelo Gulinatti; Ivan Rech; Massimo Ghioni, IEEE Journal of Selected Topics in Quantum Electronics, vol 20-6, 3804420 (2014).

16:25 - 16:45
Iwo König, Zürich, Switzerland (Student Award)

Single-molecule spectroscopy of intrinsically disordered proteins in live eukaryotic cells

Iwo König, Arash Zarrine-Afsar, Mikayel Aznauryan, Andrea Soranno, Jakob Stüber, Daniel Nettels, Ben Schuler

University of Zurich, Department of Biochemistry, Winterthurerstr. 190, 8057 Zürich, Switzerland

Single-molecule methods have become a powerful tool for quantifying the conformational heterogeneity and structural dynamics of intrinsically disordered proteins (IDPs) in vitro, e.g. the effects of crowding on the dimensions of IDPs [1]. The application of those experiments in vivo, however, has remained challenging due to shortcomings concerning the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing IDP dimensions and dynamics down to the nanosecond regime in live eukaryotic cells with confocal single-molecule FRET spectroscopy [2]. We illustrate the versatility of the approach by determining the dimensions and sub-microsecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens new possibilities for assessing the effect of the cellular environment on IDP conformation, dynamics, and function.

[1] A. Soranno, I. König, M. B. Borgia, H. Hofmann, F. Zosel, D. Nettels, B. Schuler, Proc. Natl. Acad. Sci. U.S.A., 111, 4874–4879 (2014).

[2] I. König, A. Zarrine-Afsar, M. Aznauryan, A. Soranno, B. Wunderlich, F. Dingfelder, J. C. Stüber, A. Plückthun, D. Nettels, B. Schuler, Nat Meth, 12, 773–779 (2015).

16:45 - 17:05
Piau Siong Tan, Heidelberg, Germany

Single molecule studies reveal conformational features and binding mechanisms of phenylalanine-glycine rich nucleoporins

Piau Siong Tan, Swati Tyagi, Iker Valle Aramburu, Edward A. Lemke

Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany

FG-nucleoporins (FG-Nups) are intrinsically disordered proteins (IPDs) located in the nuclear pore complex (NPC) where they form a selective barrier that can be crossed by nuclear transport receptors (NTRs).  How interaction of NTRs affects the conformational features of these FG-Nups in a way that allows them to control nucleocytoplasmic transport is still unclear. We previously showed that the folded NTRs can interact with the FG-Nups with remarkably fast kinetics and that the conformational ensemble of Nups are largely unaffected by the multivalent interaction with NTRs, which ensures both rapid yet specific transport [1]. In this work, we revealed another type of FG-Nup•NTR interaction by using multi-parameter fluorescence detection (MFD) to perform single-molecule FRET studies. We demonstrate that the binding of the NTR CRM1, an export receptor, can induce a conformational change in the Nup214. The affinity of this complex can be further modulated by Ran-GTP, which can bind to CRM1. The conformational change suggests a stable bound complex which is different from the ultrafast dynamic multivalent-fuzzy complex that previously showed for different FG-Nup•NTR interactions. These findings bring us one step forward to understand the mechanistic functions and regulatory role of different FG-Nups as well as the molecular details of transport complex passing through NPC.

[1] Sigrid Milles, Davide Mercadante, Iker Valle Aramburu, Malene Ringkjøbing Jensen, Niccolò Banterle, Christine Koehler, Swati Tyagi, Jane Clarke, Sarah L. Shammas, Martin Blackledge, Frauke Gräter, Edward A. Lemke, Cell, 163, 734-745, 2015.

17:05 - 17:25
Niels Zijlstra, Zürich, Switzerland

Rapid microfluidic dilution device for probing low‐affinity biomolecular complexes with single-molecule spectroscopy

Niels Zijlstra, Fabian Dingfelder, Franziska Zosel, Stephan Benke, Daniel Nettels, Benjamin Schuler

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

Single‐molecule spectroscopy has developed into a powerful strategy for investigating biomolecular structure, dynamics, and interactions, especially in combination with Förster resonance energy transfer (FRET). However, a major challenge in studying biomolecular complexes by single-molecule spectroscopy is that their affinity is often low, resulting in rapid dissociation at the exceedingly low concentrations required for single-molecule detection.

Here, we demonstrate a microfluidic mixing device capable of diluting a concentrated sample more than 10’000‐fold within ~5 ms, which can be easily combined with confocal single‐molecule detection. The microfluidic device combines diffusion-limited dilution with multiple, long observation channels offering a wide range of dilution factors for precise tuning of the sample concentrations and observation times ranging from milliseconds to minutes after dilution.

To test the capabilities of our microfluidic device, we used single‐molecule two-color and three-color FRET to study a protein complex consisting of the two intrinsically disordered protein domains ACTR and NCBD. We show that we can transiently populate and study the structural properties of these low-affinity complexes with single-molecule spectroscopy and quantify the dynamics of the dissociation process over a wide range of timescales.

Session: Biological applications Chair: Melike Lakadamyali
9:00 - 09:35
Viola Vogel, Zurich, Switzerland (Invited Talk)

Mechanobiology: from single molecules to disease

Viola Vogel

Laboratory of Applied Mechanobiology, , Department of Health Sciences and Technology, ETH Zurich, Switzerland. , E-mail: viola.vogel@hest.ethz.ch

Proteins can be stretched into intermediate states by tensile forces, thus altering their structure-function relationships, for example by altering their dissociation rates or the accessibility of binding or cleavage sites. As proteins have unique structural motifs, the mechanisms by which they act as mechano-chemical switches are quite specific for each protein. Microbes as well as eukaryotic cells evolved complex nanoarchitectures of bonds that act as mechano-chemical switches to sense and respond to their environments. While much has been learned in the last decade at the single molecule and single cell levels, the next challenge is to translate some of this knowledge into an improved understanding of diseases. Examples will be discussed that illustrate how the discovery of mechanical principles of functional regulation are indeed exploited by bacteria, immune cells, and at the tissue level, and how such knowledge can be utilized to improve on the diagnosis and therapy of disease.


09:35 - 09:55
Falk Schneider, Oxford, United Kingdom (Student Award)

Lipid and protein diffusion in actin-free plasma membrane vesicles

Falk Schneider, Erdinc Sezgin, Christian Eggeling

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS, Oxford, United Kingdom

Stimulated emission depletion (STED) nanoscopy is one of the tools to gain resolution beyond the diffraction limit of about 200 nm. By combining it with fluorescence correlation spectroscopy (FCS), diffusive processes on the nanoscale can be studied.  With STED-FCS so called the diffusion law is obtained by measuring the diffusion coefficient at different sizes of the observation spot [1].

Determination of the FCS diffusion law of lipids and proteins in the cellular membrane revealed different diffusion modes namely trapped diffusion, domain diffusion and hop diffusion [2]. These diffusion characteristics have been suggested to be associated with plasma membrane organisation and its interactions with the underlying actin cytoskeleton [3].

In this study plasma membrane derived vesicles, giant plasma membrane vesicles (GPMVs), are used as a model system for cellular plasma membrane which retain the compositional complexity of the native cellular membrane but lack of organized actin cytoskeleton. Thus, remaining heterogeneity resulting in obstructed diffusion would solely be associated with membrane components. Here, we will present the diffusion law of different types of lipids as well as GPI-anchored proteins in GPMVs and compare it with the diffusion law in live cell membrane.

[1] Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, et al., Nature, 457(7233), 1159–62 (2009)

[2] Wawrezinieck L, Rigneault H, Marguet D, Lenne P-F., Biophys J, 89(6), 4029–42 (2005)         

[3] Fujiwara T, Ritchie K, Murakoshi H, Jacobson K, Kusumi A, J Cell Biol, 157(6), 1071–81 (2002)

09:55 - 10:15
Piotr Trochimczyk, Warsaw, Poland (Student Award)

How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles.

Piotr Trochimczyk, Sen Hou, Lili Sun, Robert Hołyst

Institute of Physical Chemistry PAS, Kasprzaka 44/52, 01-224 Warsaw, Poland

Previous studies indicate that macromolecular crowding thought to be always capable of increasing reaction rate of most biological reactions. However our study show that in some case the biological reaction can be inhibited. As an example of reaction inhibition caused by macromolecular crowding we have used cleavage of the DNA by restriction enzyme. The impede of enzyme activity was caused by formation of DNA nanoparticles, which have created the steric obstruction for enzyme active site, thus the attachment of enzyme to DNA was hindered. In order to clarify what happens during the reaction flow we have used fluorescence correlation spectroscopy. In normal autocorrelation curve the number of particles in focal volume is inversely proportional to the amplitude of the curve, this peculiarity allows to determine when single fluorescently labeled DNA molecules ensemble into nanoparticles. To overcome the repulsive interactions of DNA both macromolecular crowder and divalent cationic ions had to be present in reaction solution. As a confirmation to FCS results of nanoparticles creation we have used FAUC and DLS, while EMSA confirmed the shielding aspect of nanoparticles. All methods mentioned above have proven that FCS results were legitimate and that macromolecular crowding can inhibit biological reactions.

[1] Ellis, R. John. Trends in biochemical sciences 26.10 Page: 597-604 (2001)

[2] Zimmerman, Steven B., and Barbara H. Pheiffer. Proceedings of the National Academy of Sciences 80.19 Page: 5852-5856 (1983)

[3] Sasaki, Yoshiharu, Daisuke Miyoshi, and Naoki Sugimoto. Nucleic acids research 35.12 Page: 4086-4093 (2007)

[4] Hou, Sen, et al. Soft Matter 7.7 Page: 3092-3099 (2011)

10:15 - 10:35
Roman Tsukanov, Göttingen, Germany

Investigating conformational dynamics of DNA hairpin and Holliday junction using single-molecule fluorescence techniques

Roman Tsukanov1,2, Menahem Pirchi3, Toma E. Tomov2, Yaron Berger2, Miran Liber2, Dinesh Khara2, Eyal Nir2, Gilad Haran3

1III. Institute of Physics – Biophysics, Department of Physics, Georg August University, Friedrich-Hund-Platz 1, D-37077 Göttingen, Germany
2Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer Sheva, 84105, Israel
3Chemical Physics Department, Weizmann Institute of Science, Herzl St 234, Rehovot 76100, Israel

DNA is a highly-designable, easily modified and cost-efficient biological molecule. DNA hairpin and Holliday junction are responsible for genetic recombination and other important biological processes. The interconversion rates of synthetic DNA hairpin and Holliday junction molecules can be programmed by designing its sequence and changing the environment of the molecule (ionic strength, temperature, viscosity). These properties make DNA hairpin and Holliday junction perfect dynamic model molecules for development and validation of single-molecule fluorescence techniques and approaches. We will discuss the implementations of Probability Distribution Analysis and photon-by-photon Hidden Markov Model for DNA hairpin and Holliday junction conformational dynamics study on a broad time-scale.

10:35 - 11:10COFFEE BREAK
Session: Super-resolution 1Chair: Philipp Kukura
11:10 - 11:40
Melike Lakadamyali (presented by Anna Oddone), Castelldefels, Barcelona, Spain (Invited Talk)

Decoding chromatin organization with super-resolution

Melike Lakadamyali

ICFO – Institut de Ciències Fotòniques, Advanced Fluorescence Imaging and Biophysics Group, Av. Carl Friedrich Gauss, 3, 08860 Castelldefels, Barcelona, Spain, Melike.Lakadamyali@icfo.es

Nucleosomes help structure chromosomes by compacting DNA into fibers. Chromatin organization plays an important role for regulating gene expression; however, due to the nanometer length scales involved, it has been very difficult to visualize chromatin fibers in vivo. Using super-resolution microscopy, quantitative analysis and simulations, we have been gaining new insights into chromatin organization at nanometer length scales in intact nuclei. For example, we found that nucleosomes assemble into heterogeneous groups of varying sizes, which we named “clutches,” in analogy with “egg clutches”. Clutch organization is highly cell specific and I will give various examples of this specificity in the context of stem cells as well as immune cells. Overall, our results reveal how the chromatin fiber is formed at nanoscale level and link chromatin fiber architecture to cell state.

11:40 - 12:00
Alex von Diezmann, Stanford, United States (Student Award)

Correcting nanoscale aberrations over the field of view in three-dimensional localization microscopy

Alex von Diezmann1, Maurice Y. Lee1,3, Thomas H. Mann2, Keren Lasker2, Lucy Shapiro2, W. E. Moerner1

1Department of Chemistry, Stanford University, Stanford, CA 94305
2Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305
3Biophysics Program, Stanford University, Stanford, CA 94305

Single-molecule tracking and super-resolution imaging rely on the ultraprecise localization of fluorescent or scattering point emitters and can be extended to three dimensions by engineering a microscope’s point spread function (PSF). However, the nanoscale accuracy of 3D PSF localization over a single-molecule microscope’s field of view cannot be taken for granted. By finely sampling the 3D PSF with an array of regularly-spaced subdiffraction “nanoholes” filled with fluorescent dye, we reveal field-dependent errors as large as 50-100 nm, and show they can be corrected to less than 25 nm over an extended 3D volume. We show the applicability of this calibration approach for two engineered PSFs, the double-helix PSF and astigmatic PSF (1). A related error results from imperfect registration of spectrally separated imaging channels. By generating control points with a nanohole array filled with dyes spectrally matched to each channel, we achieve 3D multicolor registration with < 10 nm accuracy. The power of this approach is exemplified by multicolor super-resolution imaging of the ≤ 200 nm polar nanodomain in live cells of Caulobacter crescentus, a model organism for the study of cell polarity and differentiation (cf. 2).

(1) Alex von Diezmann, Maurice Y. Lee, Matthew D. Lew, and W. E. Moerner, Optica 2, 985 (2015)
(2) Jerod L. Ptacin, Andreas Gahlmann, Grant R. Bowman, Adam M. Perez, Alexander R. S. von Diezmann, Michael R. Eckart, W. E. Moerner, and Lucy Shapiro, PNAS 111, E2046 (2014)

12:00 - 12:20
Christian Franke, Wuerzburg, Germany (Student Award)

Three-dimensional localization microscopy based on accurate intensity estimation

12:20 - 12:40
Jörg Enderlein, Göttingen, Germany

Image Scanning Microscopy

Jörg Enderlein

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

Classical fluorescence microscopy is limited in resolution by the wavelength of light (diffraction limit) restricting lateral resolution to ca. 200 nm, and axial resolution to ca. 500 nm (at typical excitation and emission wavelengths around 500 nm). However, recent years have seen a tremendous development in high- and super-resolution techniques of fluorescence microscopy, pushing spatial resolution to its diffraction-dictated limits and much beyond. One of these techniques is Image Scanning Microscopy (ISM). In ISM, the focus of a conventional laser-scanning confocal microscope (LCSM) is scanned over the sample, but instead of recording only the total fluorescence intensity for each scan position, as done in conventional operation of an LCSM, one records a small image of the illuminated region. The result is a four-dimensional stack of data: two dimensions refer to the lateral scan position, and two dimensions to the pixel position on the chip of the image-recording camera. This set of data can then be used to obtain a super-resolved image with doubled resolution, completely analogously to what is achieved with Structured Illumination Microscopy. However, ISM is conceptually and technically much simpler, suffers less from sample imperfections like refractive index variations, and can easily be implemented into any existing LSCM. I will also present recent results of combining ISM with two-photon excitation, which is important for deep-tissue imaging of e.g. neuronal tissue.

[1] Müller, C.B.; Enderlein J. "Image scanning microscopy" Phys. Rev. Lett. 104, 2010, 198101.

[2] Schulz, O.; Pieper, C.; Clever, M.; Pfaff, J.; Ruhlandt, A.; Kehlenbach, R.H.; Wouters, F.S.; Großhans, J.; Bunt, G.; Enderlein, J.
"Resolution doubling in fluorescence microscopy with Confocal Spinning-Disk Image Scanning Microscopy" PNAS, 110, 2013, 21000-21005.

12:40 - 12:50GROUP PICTURE
12:50 - 14:20LUNCH BREAK
Session: Methods and techniques 1Chair: Viola Vogel
14:20 - 14:50
Philipp Kukura, Oxford, United Kingdom (Invited Talk)

Towards label-free single molecule microscopy with interferometric scattering

Philipp Kukura

Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK, philipp.kukura@chem.ox.ac.uk

The fundamental goal of optical microscopy is to visualise and thereby enable the study of dynamics on the microscopic or even nanoscopic scale. The past decades have been dominated by fluorescence-based approaches, in particular in super-resolution methodologies. Despite the advantages of fluorescence imaging, the requirement of introducing labels can be both complex and perturbative, while photophysics and photochemistry limits imaging speed, precision and duration. I will highlight the capabilities of an alternative approach to optical microscopy based on light scattering called interferometric scattering microscopy (iSCAT). Contrary to intuition, I will show that iSCAT can achieve sensitivities approaching and possibly rivalling those of a fluorescence microscope including label-free imaging of single molecules. Importantly, this sensitivity has wide-ranging applications for studies of nanoscale phenomena in general, such as phase separation, dynamics at interfaces, bilayers or biological filaments.

14:50 - 15:10
Felix Koberling, Berlin, Germany

Fast TCSPC Based Confocal Microscopy Optimised for Hz Image Frame Rates with High Photon Throughput

Benedikt Kraemer1, Paja Reisch1, Marcus Sackrow1, Sandra Orthaus-Mueller1, Sebastian Tannert1, Felix Koberling1, Piau Siong Tan2, Edward Lemke2, Rainer Erdmann1

1icoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany, info@picoquant.com
2EMBL, Cell Biology and Biophysics Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany

Fluorescence lifetime imaging (FLIM) as a tool for monitoring fast, dynamic processes is becoming more important in fields such as live cell imaging. New imaging approaches like rapidFLIM are increasingly demanding in terms of data throughput rates and short frame acquisition times. In order to meet these requirements, we combined fast confocal imaging capabilities including STED super-resolution with a superior single photon counting throughput concept in a single system, while maintaining key features needed for single molecule sensitivity.

We expanded the MicroTime 200 time-resolved microscopy platform with a fast 3 mirror galvo scanner allowing both fast imaging with up to several FLIM frames per second and stable point positioning using the same beam path and within an experimental sequence. Furthermore, by exploiting recent hardware developments such as TCSPC modules with ultra short dead times and hybrid photomultiplier detector assemblies, significantly higher detection count rates (> 10 Mcps) can be used for imaging while maintaining good temporal accuracy.
We took special care to ensure synchronisation between pixel dwell time and laser repetition rate. This enables generation of an equal number of laser pulses per pixel, which is especially important for fast imaging with high pixel numbers per line and e.g., phosphorescence imaging requiring low laser repetion rates.

In this presentation, we will demonstrate the speed, accuracy, and versatility of our prototype by means of multispecies STED imaging as well as single molecule detection.

15:10 - 15:30
Sebastian Isbaner, Göttingen, Germany (Student Award)

Dead-time correction of fluorescence lifetime measurements and fluorescence lifetime imaging

Sebastian Isbaner1, Narain Karedla1,2, Daja Ruhlandt1, Simon Christoph Stein1, Anna Chizhik1, Ingo Gregor1, Jörg Enderlein1,2

1III. Institute of Physics, Georg August University, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany
2DFG Research Center """Nanoscale Microscopy and Molecular Physiology of the Brain""" (CNMPB), Göttingen, Germany

Dead-time artifacts can dramatically influence the shape of Time-Correlated Single Photon Counting (TCSPC) histograms such as fluorescence lifetime curves [1]. These artifacts occur at high count rates, which limit the acquisition speed in Fluorescence Lifetime Imaging Microscopy (FLIM). We present an algorithm that corrects the distortions of TCSPC histograms which are caused by constant electronics and/or detector dead-times [2]. We verified the algorithm with Monte-Carlo simulations and fluorescence lifetime measurements. Furthermore, we performed FLIM measurements on densely labeled cells at various excitation powers and corrected the lifetime and intensity values for each pixel.

Our correction method is not restricted to TCSPC measurements only, but can be applied to any periodic single-event counting or timing measurement. Since it corrects dead-time artifacts for both lifetime and intensity, the algorithm could be beneficial for example for lidar or time-resolved fluorescence anisotropy measurements.

[1] W. Becker, Advanced Time-Correlated Single Photon Counting Techniques (Springer, 2005).

[2] S. Isbaner, N. Karedla, D. Ruhlandt, S.C. Stein, A. Chizhik, I. Gregor, and J. Enderlein, Opt. Express 24, 9429-9445 (2016)

15:30 - 15:45COFFEE BREAK
15:45 - 18:15POSTER SESSION
19:30 - 22:30DINNER
Session: Super-resolution 2 & Material sciencesChair: Yuval Ebenstein
9:00 - 09:35
Katrin I. Willig, Göttingen, Germany (Invited Talk)

STED microscopy of the living mouse brain

Katrin I. Willig1,2, Waja Wegner1,2, Carola Gregor3, Heinz Steffens1,3

1Center for Nanoscale Microscopy and Molecular Physiology of the Brain, , Göttingen, Germany
2Max Planck Institute of Experimental Medicine, Göttingen, Germany
3Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, E-mail: kwillig@em.mpg.de

Far-field light microscopy is a powerful technique for imaging structures inside living cells, tissue or living animals. From all novel light microscopy or super-resolution microscopy techniques available presently, STED microscopy stands out for its imaging capabilities in tissue: It is live-cell compatible, able to record 3D images from inside transparent tissue and the imaging speed is fast.

Here we present an application of STED microscopy to image dendritic spines in the brain of a living mouse. It was shown that STED microscopy is capable to image dendritic spines up to 120 µm deep inside living organotypic brain slices and to resolve distinct distributions of actin inside dendrites and spines. We use STED microscopy to image the cerebral cortex of a living mouse through a glass window, so that we can observe the dynamics of dendritic spines in the molecular layer of the visual cortex. We reveal filamentous actin in dendrites down to a depth of 40 µm [1]. Time-lapse recordings disclose dynamic changes at a resolution of ~ 60 nm. Very recently, we have extended in vivo STED microscopy to the imaging of other synaptic protein structures such as the post-synaptic density protein PSD95.

[1] Willig, K. I., H. Steffens, C. Gregor, A. Herholt, M. J. Rossner, S. W. Hell """Nanoscopy of Filamentous Actin in Cortical Dendrites of a Living Mouse""" Biophys. J. 106, L01 - L03 (2014)

09:35 - 09:55
Alexey Chizhik, Göttingen, Germany

Metal-induced energy transfer for live cell nanoscopy

Alexey Chizhik1, Daja Ruhlandt1, Thilo Baronsky2, Anna Chizhik1, Narain Karedla1, Jan Rother2, Ingo Gregor1, Andreas Janshoff2, Jörg Enderlein1

1III. Institute of Physics, Georg August University, 37077 Göttingen, Germany.
2Institute of Physical Chemistry, University of Göttingen, Tammannstrasse 6, 37077 Göttingen, Germany.

The discovery of Förster resonance energy transfer (FRET) [1] has revolutionized our ability to measure inter- and intramolecular distances on the nanometre scale using fluorescence imaging. The phenomenon is based on electromagnetic-field-mediated energy transfer from an optically excited donor to an acceptor. We replace the acceptor molecule with a metallic film and use the measured energy transfer efficiency from donor molecules to metal surface plasmons [2] to accurately deduce the distance between the molecules and metal [3]. Like FRET, this makes it possible to localize emitters with nanometre accuracy, but the distance range over which efficient energy transfer takes place is an order of magnitude larger than for conventional FRET. This creates a new way to localize fluorescent entities on a molecular scale, over a distance range of nearly 200 nm. We demonstrate the power of metal-induced energy transfer (MIET) by profiling the basal lipid membrane of living cells. Using MIET, we gain a new insight into mechanics of epithelial-to-mesenchymal cellular transition. This process allows epithelial cells to enhance their migratory and invasive behaviour and plays a key role in embryogenesis, fibrosis, wound healing and metastasis [4].

[1] Förster, Th. Ann. Phys. 437, 55-75 (1948).

[2] Drexhage, K. H. Prog. Opt. 12, 163-232 (1974).

[3] Chizhik, A. I., Rother, J. Gregor, I., Janshoff, A., Enderlein, J. Nature Photon. 8, 124-127 (2014).

[4] Chizhik, A. I., Baronsky, T., Ruhlandt, D., Karedla, N., Haehnel, D., Gregor, I., Janshoff, A., Enderlein, J. submitted (2016)

09:55 - 10:15
Florian Steiner, Regensburg, Germany (Student Award)

Spontaneous fluctuations of transition dipole orientation in OLED triplet emitters revealed by single-molecule spectroscopy

Florian Steiner, Jan Vogelsang, John M. Lupton

Department of Experimental and Applied Physics, University of Regensburg, Regensburg, Germany

The efficiency of organic light-emitting diodes (OLEDs) strongly depends on the microscopic orientation of transition dipole moments of the molecular emitters. In conventional measurement techniques, however, it is not possible to determine the polarization anisotropy of the individual emitter due to ensemble averaging in solution or bulk film[1]. In contrast, single-molecule spectroscopy is capable of acquiring microscopic insight into the spatial and temporal arrangement of transition dipole moments from single luminescent particles. Recent measurements showed that the dipole moment orientation can fluctuate spontaneously in planar carbazole macrocycles[2].

Similar fluctuations also exist for phosphorescent molecules as used in OLEDs. We investigated the prototypical triplet emitter tris(1-phenylisoquinoline)iridium(III) (Ir(piq)3) with regards to its emission polarization properties one molecule at a time[3].

Here, spontaneous symmetry breaking occurs in the excited state after every excitation, leading to a random selection of one of the three ligands to form a charge-transfer state with the metal in the center of the molecule. This nondeterministic localization is revealed in switching of the linear polarization of phosphorescence. Such polarization scrambling will raise out-coupling efficiency and should be taken into account when deriving molecular orientation of the guest emitter within the OLED host from ensemble angular emission profiles[1].

[1] Jurow M. J., Mayr C., Schmidt T. D., Lampe T., Djurovich P. I., Brütting W., Thompson M. E., Nature Materials, Vol. 15, p. 85 (2015).

[2] Aggarval V., Thiessen A., Idelson A., Kalle D., Würsch D., Stangl T,. Steiner F., Jester S.-S., Vogelsang J., Höger S., Lupton J.M., Nature Chemistry, Vol. 5, p. 964 (2013).

[3] Steiner F., Bange S., Vogelsang J., Lupton J.M., Journal of Physical Chemistry Letters, Vol. 6, p. 999 (2015).

10:15 - 10:35
Izabela Kaminska, Braunschweig, Germany

DNA origami-based antennas for a broadband fluorescence enhancement

Izabela Kaminska1,2, Anastasiya Puchkova1, Maria Sanz Paz1, Carolin Vietz1, Enrico Pibiri1, Bettina Wünsch1, Guillermo P. Acuna1, Philip Tinnefeld1

1Braunschweig Integrated Centre of Systems Biology, Institute of Physical and Theoretical Chemistry, TU Braunschweig, Rebenring 56, 38106 Braunschweig, Germany
2Institute of Physics, Department of Physics, Astronomy and Informatics, Nicolaus Copernicus University, Grudziadzka 5/7, 87-100 Torun, Poland

DNA origami technology is employed to fabricate self-assembled nanoantennas composed of two metallic nanoparticles and a fluorophore positioned at the hotspot. Our initial experiments with a DNA origami pillar and gold nanoparticles provided 117-fold fluorescence enhancement factor for a dye molecule incorporated at the 23-nanometer gap.[1] The performance of this structure was improved by: development of a new DNA origami template design, reduction of the interparticle distance, alignment of the light polarization and quenching of the fluorophore’s quantum yield.[2,3] These factors allowed us to obtain fluorescence enhancement above 5000 and single molecule detection at 25 µM concentration.[2] Additionally in this contribution we show how the fluorescence enhancement can be extended to a broader range, in particular for applications in the field of biosensing/imaging and single molecule detection at high concentrations where more than one wavelength is needed. In this case, gold nanoparticles are replaced with silver nanoparticles which have stronger localized surface plasmon resonance in the visible region, and can be used to achieve higher fluorescence enhancement factor in that range. We investigate fluorophores emitting at different frequencies. While both gold and silver nanoantennas give similar results in red, dimers based on silver are advantageous in the range from orange to blue.

[1] G. P. Acuna, F. M. Möller, P. Holzmeister, S. Beater, B. Lalkens, P. Tinnefeld, ACS Nano, 6, 3189 (2012).

[2] A. Puchkova, C. Vietz, E. Pibiri, B. Wuensch, M. Sanz Paz, G. P. Acuna, P. Tinnefeld, Nano Letters, 15, 8354 (2015).

[3] Carolin Vietz, Birka Lalkens, Guillermo P Acuna, Philip Tinnefeld, New Journal of Physics, 18, 045012 (2016)

10:35 - 11:10COFFEE BREAK
Session: Methods and techniques 2Chair: Katrin Willig
11:10 - 11:40
Haw Yang, Princeton, United States (Invited Talk)

Real-Time 3D Single-Particle Tracking Spectroscopy and Its Applications

Haw Yang

Department of Chemistry, Princeton University

Real-time 3D single-particle tracking spectroscopy is an experimental technique that allows one to follow a nanoprobe as its moves in three-dimensional space, either by diffusion or by active locomotion. The technique keeps the nanoprobe at the center of the focus at all times by moving the sample stage to counter any movements that the probe exhibits. Effectively, the apparatus transforms the experimental coordinate from lab-based from to probe-based frame. This way, it would become possible to do time-dependent experiments on the probe or the molecule tethered to the probe as if the particle or molecules are immobilized. This presentation will discuss its principles, capabilities, and applications—including (1) 3D multi-resolution imaging of virus-like particle interacting with a live cell, (2) single-particle temperature-jump experiment testing the hot Brownian motion theory, and (3) 3D steering of self-propelled micro-swimmers and its implications in bacterial locomotion.

11:40 - 12:00
Dmitry Torchinsky, Tel Aviv, Israel (Student Award)

Single-molecule counting highlights the efficiency and specificity of DNA repair enzymes

Dmitry Torchinsky, Yuval Ebenstein

Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 6997801, Israel.

DNA, under exposure to various chemicals such as oxidizing agents as well as various types of radiation, can undergo physical breakage of one or both DNA strands. However, the most common yet least studied form of DNA damage are chemical modification known as DNA damage adducts[1]. These damage lesions can influence transcription and replication, and eventually can lead to DNA breaks. DNA adducts are repaired by specialized enzymes that specifically recognize and remove them but difficulties in the detection of these damage types have hampered their research. In this project we introduce a single molecule approach which takes advantage of natural repair enzymes to label damage sites in vitro by incorporating fluorescent nucleotides as a part of the repair process[2]. The labeled DNA is extended on positively activated glass coverslips and damage sites are visualized as fluorescent spots along the DNA contour. Using model DNA we establish the efficiency of the labeling process as well as it’s specificity towards particular damage type, opening up new opportunities for DNA damage studies. 

[1] RP Sinha, DP Häder - Photochemical & Photobiological Sciences, 1, 225-236, 2002

[2] Shahar Zirkin, Sivan Fishman, Hila Sharim, Yael Michaeli, Jeremy Don, and Yuval Ebenstein, J. Am. Chem. Soc., 136 (21), pp 7771–7776, 2014

12:00 - 12:20
Aquiles Carattino, Leiden, Netherlands (Student Award)

Background-Suppression in the Detection of Gold Nanoparticles in Cells through Anti-Stokes Photoluminescence

Aquiles Carattino, Veer Keizer, Marcel Schaaf, Michel Orrit

Niels Bohrweg 2, 2333CA, Leiden, The Netherlands

In microscopy one of the fundamental limitations for particle imaging, localization and tracking is the signal-to-background ratio. Many techniques have been proposed to lower the background, including two-photon excitation, lifetime measurements, photothermal signal, etc., but they are usually hard to implement and require a dedicated setup. We present a simple solution that is readily achievable in any confocal microscope by detecting the anti-Stokes photoluminescence of single gold particles. We apply this technique to the imaging of gold nanorods in living cells. We show a high background rejection, lowering the effect of the cell self-fluorescence. This proves to have a great impact in the case of stained cells, where the background arises also from dye molecules. This new technique provides a simple detection scheme of smaller nano objects with standard setups, and will be an ideal approach for cellular biology.

12:20 - 12:40
Hao Cheng, Göttingen, Germany (Student Award)

Single-Molecule Brightness Analysis by Stroboscopic Imaging in Nanofluidic-Channels

Hao Cheng, Simon Stein, Jan Thiart, Ingo Gregor, Jörg Enderlein

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

Fluorescence brightness is one of the fundamental parameters of a fluorescing molecule which is determined by its absorption cross-section and its quantum yield.  A method which could accurately and quantitatively determine the brightness of single molecules in solution could be used for analyzing complex mixtures of molecular species, studying molecular aggregation, or determining stoichiometry of molecular complexes. One of techniques which has been used in the past for molecular brightness distribution analysis is fluorescence-fluctuation-spectroscopy, but it employs complicated statistical models and has very limiting resolving power when it comes to mixtures of molecular species. Here, we present a different approach which is based on direct imaging of individual molecules during their diffusion and directed transport through nanofluidic-channels. For this purpose, we employ full-glass-chips with channel heights of less than 200nm, which confine molecular motion to the focal plane of the observing microscope. In combination with high-speed stroboscopic imaging and active flow control, we obtain high-quality images of freely diffusing molecules and measure their brightness with high accuracy.  The method is high-throughput, allowing for analyzing thousands of individual molecules within only few minutes. We demonstrate the capability of our method by determining the labeling stoichiometry of multiply-labeled DNA fragments. Our combination of cutting-edge nanofluidics with advanced single-molecule imaging opens up fascinating opportunities for numerous biomedical applications. 

12:40 - 14:10LUNCH BREAK
Session: Methods and techniques 3Chair: Haw Yang
14:10 - 14:40
Yuval Ebenstein, Tel Aviv, Israel (Invited Talk)

Beyond sequencing: Single-molecule genomics

Yuval Ebenstein

Department of Chemical Physics, Tel-Aviv University, Israel

Next generation sequencing (NGS) is revolutionizing all fields of biological research but it fails to extract the full range of information associated with genetic material and is lacking in its ability to resolve variations between genomes. As a consequence, many genomic features remain poorly characterized in the human genome reference. Chromosomes contain a plethora of variable regions that include single point mutations (SNP), structural variations (SV), copy number variations (CNV) and DNA repeats. In addition, the information content of the genome extends beyond the base sequence in the form of chemical modifications such as DNA methylation or DNA damage lesions. By applying experimental principles of single molecule detection we gain access to the structural variation and long range patterns of genetic and epigenetic information. We show how physical extension of long DNA molecules on surfaces and in nanofluidic channels reveals such information in the form of a linear, optical “barcode”, like beads threaded on a string, where each bead represents a distinct type of observable. Recent results from our lab demonstrate our ability to detect epigenetic marks and various forms of DNA damage on individual genomic DNA molecules and use this information for medical diagnostics.

14:40 - 15:00
Sebastian Kruss, Göttingen, Germany

Chemical imaging of cell communication using near infrared fluorescent carbon nanotube sensors

15:00 - 15:20
Gordon J. Hedley, Regensburg, Germany

Exploring Molecular Aggregation at the Single-Molecule Level in a High-Performance Organic Photovoltaic Polymer

Gordon J. Hedley, Florian Steiner, Jan Vogelsang, John M. Lupton

Institut für Experimentelle und Angewandte Physik, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany

The nature of the absorption and emission of light from chromophores is important to understand if one wants to tailor or control it. Altering these properties by changing the conformation of the molecule or by aggregating the molecules themselves may also enable powerful probes of the local molecular environment. Using single molecule spectroscopy we have investigated the process of aggregation in a light absorbing polymer used in high performance organic solar cells.[1-2]  In ensemble measurements the polymer absorbs in the red and emits in the near-infrared. We have found that this is actually due to chromophore aggregation, and if one separates the polymer chains sufficiently, they absorb at a wavelength of 488 nm and have red PL.  Single molecule lifetimes, spectra, emission polarization measurements and FCS have been measured in both the “aggregated” and “isolated” cases. Clear differences were observed linked to electronic aggregation of the chromophores during the self-assembly into mesoscopic objects. This work contributes to the understanding of how dramatically interactions between chromophores dictate the final photophysical properties and not only the chemical structure itself, implying routes towards control by following the aggregation process.

[1] He Z.; Zhong, C.; Su, S.; Xu, M.; Wu, H.; Cao, Y., Nature Photonics, 6, 591–595, (2012)

[2] Ouyang, X.; Peng, R.; Ai, L.; Zhang, X.; Ge, Z., Nature Photonics, 9, 520, (2015)

Iman Abdollahzadeh, Jülich, Germany

Single Molecule Localization Microscopy (SMLM) of autophagy-relevant proteins in mammalian cells.

Iman Abdollahzadeh1,2, Alexandra Boeske2, Johnny Hendriks1, Oliver H. Weiergräber2, Christoph Fahlke1, Dieter Willbold2, Silke Hoffmann2, Thomas Gensch1

1ICS-4 (Cellular Biophysics); Institute of Complex System, Forschungszentrum Jülich, Germany
2ICS-6 (Structural Biochemistry); Institute of Complex System, Forschungszentrum Jülich, Germany

Autophagy is a homeostatic process by which cells can survive under stress and nutrition shortage. It denotes a group of distinct pathways, the most prominent being macroautophagy, which converge to the lysosomal compartment. The morphological hallmark of macroautophagy is the formation of a double membrane structure (the phagophore), which expands to engulf its cargo and finally closes to form an autophagosome. Autophagosome formation and maturation critically depend on two sub-families of ATG8 proteins (GABARAP and LC3). Whereas our knowledge regarding the individual protein components involved in macroautophagy has advanced dramatically, their dynamic localization behavior and the resulting spatio-temporal patterning of interactions are still poorly understood. Given the size of the structures in question (50 nm to 1 µm), the distribution of proteins cannot be resolved by conventional fluorescence microscopy. The situation has changed with the development of super-resolution microscopy techniques. One of the new methods – Single Molecule Localization Microscopy (SMLM) – relies on the precise localization (10 to 30 nm accuracy) of single fluorescent molecules (organic dyes or fluorescent proteins) decorating the autophagy-relevant and other cellular structures. Here we used HEK293 cells stably expressing EYFP-ATG8 fusion proteins to investigate the distribution of the proteins under fed and starvation condition.

Subhasis Adhikari, Leiden, Netherlands

Simultaneous detection of absorption and fluorescence of single conjugated polymer chains

Subhasis Adhikari, Lei Hou, Michel Orrit

Niels Bohrweg 2, 2333 CA Leiden, The Netherlands

Optical properties of conjugated polymers strongly depend on their conformations. Single molecule fluorescence studies have found that the whole polymer chain acts as a single emitter in its collapsed state. This result is very surprising as a single polymer chain consists of hundreds of chromophores. An intra- or inter-molecular energy transfer from high energy sites to the lowest energy site (i.e., red site) can explain the photo-blinking and single step photo-bleaching of such a multichromophoric system .

We aim to understand detailed insights of energy transfer processes by simultaneous detection of absorption and fluorescence of single conjugated polymer chains. We use a combined scanning confocal fluorescence and photothermal setup to measure temporal response of absorption and fluorescence of single polymer chains.

For the first time, we will present photothermal measurements of conjugated polymer (MEHPPV and P3HT) thin films and single MEHPPV polymer chains in PMMA. We will report quantum yields of single MEHPPV chains and their relation to polymeric properties.

Alexander Block, Castelldefels (Barcelona), Spain

Tracking nanoscale energy transport in photosynthetic light-harvesting complexes

Alexander Block1, Pu Qian2, Cvetelin Vassilev2, Matz Liebel1, C. Neil Hunter2, Niek van Hulst1

1ICFO – Institut de Ciences Fotoniques, Mediterranean Technology Park, 08860 Castelldefels (Barcelona), Spain
2Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom

Light-harvesting complexes play a crucial role in photosynthesis as they capture solar energy and transport it to the reaction center where charge separation takes place. Recent experimental results suggest that this highly efficient and fast transfer process makes use of quantum coherences even under biologically relevant conditions (Science 340, 1448 (2013)). Yet, many questions regarding the transport mechanism between adjacent light-harvesting complexes remain open. To address this, we investigate tubular crystal sheets of light-harvesting antenna complexes (LH2) with scanning confocal fluorescence microscopy and time-correlated single photon counting detection with few picosecond temporal resolution.


We observe rapid, <20ps, energy transport over hundreds of nanometers out of the excitation volume which is directed along the tubular axis alongside a significant lifetime reduction of the crystallized light-harvesting complexes. Comparison to Monte Carlo simulations allows us to directly characterize and quantify the highly efficient excitation energy transfer mechanism governing light harvesting in LH2.

Jan-Hendrik Budde, Düsseldorf, Germany

Monitoring the dimerization of a large GTPase from sub-microseconds to minutes

Jan-Hendrik Budde1, Thomas-Otavio Peulen1, Carola S. Hengstenberg2, Ralf Biehl3, Mykola Dimura1, Alessandro Valeri1, Semra Ince2, Tobias Vöpel2, Bela Farago4, Holger Gohlke5, Christian Herrmann2, Johann Klare6, Andreas Stadler3, Claus A.M. Seidel1.

1Chair for Molecular Physical Chemistry, Heinrich Heine University, Düsseldorf, Germany,
2Physical Chemistry I, Ruhr-University Bochum, Bochum, Germany,
3Institute of Complex System, Forschungszentrum Jülich, Jülich, Germany,
4Institut Laue-Langevin, Grenoble, France,
5Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine University, Düsseldorf, Germany,
6Macromolecular Structure Group, University of Osnabrück, Osnabrück, Germany.

In context of immunology large GTPase such as the human guanylate binding protein 1 (hGBP1) take an important role in intracellular pathogen defense. In hGBP1 we resolved two distinct conformational changes by combining double electron-electron resonance (EPR), small angle x-ray scattering (SAXS) and time-resolved fluorescence-measurements (TCSPC). hGBP1 is composed out of three domains, a GTPase-, a middle- and a helical-domain. We find that the C-terminal helix of the helical domain, which is important for oligomerization [Vöpel, Tobias, et al.], has two distinct major conformations with respect to the GTPase domain. We monitored its dynamics by neutron spin echo (NSE) and filtered FCS (fFCS). NSE observed no mayor dynamics upto 100 nanoseconds while fFCS found conformational exchanges from sub-micro to milli seconds. By multiple FRET-fFCS measurements we determined the internal flexibility of the protein and find that the C-terminal helices a12/a13 are highly flexible relatively to the middle domain. We previously showed that the GTPase- and the C-terminal helices of two hGBP1s associate in presence of non-hydrolysable GTP analogue (GTPγS) If the protein remains in its major state at room temperature an association of the helices a13 is sterically impossible. Hence, we conclude that the minor state at room temperature or a transient state populated during the domain flip is relevant for association of the C-terminal helices a13. To understand potential importance of this dynamics for oligomerization we combine time resolved ensemble FRET and stopped flow measurements. This allows us to investigate conformational changes from milliseconds to seconds. We correlate internal conformational changes with dimerization by intra- and inter protein multiparametric stopped flow fluorescence decay measurements of FRET-samples in presence of GTPγS. This unique combination of experiments may shed new light on the importance of molecular promiscuity for oligomerization.

Vöpel, Tobias, et al. "Triphosphate induced dimerization of human guanylate binding protein 1 involves association of the c-terminal helices: a joint double electron–electron resonance and FRET study." Biochemistry 53.28 (2014): 4590-4600.

Alexey I. Chizhik, Göttingen, Germany

Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots

Anna M. Chizhik1, Simon Stein1, Mariia O. Dekaliuk2, Christopher Battle1, Weixing Li1, Anja Huss1, Mitja Platen1, Iwan A. T. Schaap3, Ingo Gregor1, Alexander P. Demchenko2, Christoph F. Schmidt1, Jörg Enderlein1, Alexey I. Chizhik1

1Third Institute of Physics, Georg August University, 37077 Göttingen, Germany
2A. V. Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Leontovicha Street 9, Kiev 01601, Ukraine
3Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot Watt University, Edinburgh EH14 4A, United Kingdom

The rapid development of far-field super-resolution microscopy techniques has created an increasing demand for novel types of fluorophores with unique photophysical properties. The most essential among these properties are high photostability, biocompatibility, and small size. Carbon nanodots are a novel type of fluorescent probe which, in addition to having the above properties, are attractive for their simple, environmentally friendly, and inexpensive one-step synthesis [1].

We demonstrate super-resolution optical fluctuation imaging (SOFI) of cells labeled with dual-color carbon nanodots [2]. The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

[1] Ghosh, S. et al. Photoluminescence of Carbon Nanodots: Dipole Emission Centers and Electron-Phonon Coupling. Nano Letters 14, 5656-5661 (2014).

[2] Chizhik, A.M. et al. Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots. Nano Letters 16, 237-242 (2016).

Alexey I. Chizhik, Göttingen, Germany

Is there Fluorescence after Photo-Bleaching?

Luigi Tarpani1, Daja Ruhlandt2, Loredana Latterini1, Dirk Haehnel2, Ingo Gregor2, Jörg Enderlein2, Alexey I. Chizhik2

1Dipartimento di Chimica, Biologia e Biotecnologie, Università di Perugia and Centro di Eccellenza sui Materiali Innovativi Nanostrutturati, Via Elce di Sotto 8, 06123 Perugia, Italy
2III. Institute of Physics, Georg August University, 37077 Göttingen, Germany

Photo-bleaching of fluorophores is one of the key problems in fluorescence microscopy. So far, the only approach for diminishing the effect of photo-bleaching has been to enhance the photostability of an emitter. In this work, we present a fundamentally new solution for increasing the number of photons emitted by a fluorophore. We show that by exposing a single SiO2 nanoparticle to UV illumination one can create new luminescent centers within this particle after its photo-bleaching [1].

By analogy with nanodiamonds where the luminescent centers can be created by exposing a particle to a high-energy source, creation of luminescent defects in SiO2 nanostructure occurs upon breaking a chemical bond. However, because of sufficiently weaker bonds, generation of defects in SiO2 requires much lower energies, which allows for a reactivation of the nanoparticle’s fluorescence within the same sample. This opens new perspectives for a drastic enhancement of the number of photons emitted by a particle. Moreover, a new approach to the photo-induced transition between the on- and off-states may potentially become a new tool for enhancing the spatial resolution in imaging by exploiting the stochastic photoswitching-based super-resolution microscopy techniques (such as STORM, SOFI, or PALM) or RESOLFT microscopy.

[1] Tarpani, et al., Nano Letters, in press (2016).

Nader Danaf, München, Germany

Image Correlation Spectroscopy Based Assay to Investigate G-Protein Coupled Receptors

Nader Danaf1, Stefan Hannus2, Don C. Lamb1

1Department Chemie, Ludwig¬Maximilians¬Universität München, Butenandtstr. 5-13, 81377 München, Germany
2Intana Bioscience GmbH, Lochhamerstrasse 29A, D-82152 Martinsried

G-Protein coupled receptors (GPCRs) coordinate and regulate several cellular mechanisms and vital human activities such as behavior and sensing. Hence, GPCRs constitute a superfamily of membrane proteins that encode approximately 4 - 5 % of the entire human genome.1 Interestingly, few GPCRs represent a huge pharmacological interest making them the target of ~ 50% of the prescribed drugs on the market. GPCRs, being ubiquitos and diverse, made these membrane proteins an interesting system to study and understand. Most current assays lack the ability to provide the essential physiological experimental conditions, which enables in vivo studies addressing the GPCR-ligand interactions. Here, we provide an image correlation spectroscopy based assay that monitors and investigates GPCRs in live cells on a single molecule basis. The presently developed assay utilizes fluorescence and image correlation methods2,3 to investigate several aspects of the GPCR membrane proteins. Raster image correlation spectroscopy, allows to scan and determine diffusion coefficients of the receptors, was implemented to study the behavior of different GPCRs before and after the binding of a certain ligand. Furthermore, to study the associaton of GPCRs in the membrane, number and brightness would be a feasible analysis to perform in order to question the degree of oligomerization the GPCRs would show in the membranes.

1 a) Rosenbaum, D. M; Søren, S. G. F.; Kobilka, B. K. Nature 2009, 459, 356-363; b) Bjarndóttir, T. K; Gloriam, D. E.;

  Hellstrand, S. H.; Kristiansson, H.; Fredriksson, R.; Schiöth, H. B Genomics 2006, 88, 263-273.

2 Foo, Y. H.; Naredi-Rainer, N; Lamb, D. C.; Ahmed, S.; Wohland, T. Biophys. J. 2012, 102, 1174-1183.

3 a) Müller, B. K.; Zaychikov, E.; Bräuchle, C.; Lamb, D. C. Biophys. J. 2005, 89, 3508-3522; b) Hendrix, J.; Schrimpf,

  W.; Höller, M.; Lamb, D. C. Biophys. J. 2013, 105, 848-861.

M. Alejandrina Martínez Gámez, leon Guanajuato, Mexico

Can an Electromagnetic Pulse Evoke a Neural Olfactory Response?

M. Alejandrina Martínez Gámez1, Paola Segoviano-Arias2

1Centro de Investigaciones en Óptica, Loma del Bosque 115, Col. Lomas del Campestre, , León 37150, Guanajuato, México
2División de Ciencias e Ingenierías del Campus León de la Universidad de Guanajuato. , Loma del Bosque 103, Col. Lomas del Campestre 37150, León Guanajuato, México

The olfactory transduction process begins in the receptor neuron cilia where receptor proteins bind odorants. It has been proposed that besides the structural motifs olfactory receptors might sense vibrational energy levels of a given molecule[1]. Odorants have different vibrational frequencies. Our aim is be able to stimulate olfactory neuron receptors replacing odorant and evoke a neural response by using an electromagnetic pulse given the odorant’s spectrum and measure current kinetics. By placing the odorant near a receptor we can measure the resonant frequency and current through the channels. Given the resonant frequencies of odorants we can replace them with the electromagnetic pulse stimulating exactly with the same resonant frequency as the odorant does. We are eliciting the olfactory response with amines, thiols, and alcohols mixtures due to the fact that sensitivity and amplitude response changes with concentration and bonding between functional groups. For future work we aim to measure the interaction between receptor proteins and odorant binding to understand better how the mechanism of vibration theory of olfaction works. 

[1] Turin L., Chem Senses, 21, 773–791(1996).

Andreas Haderspeck, Heidelberg, Germany

Chemically switchable Fluorescent Probes:A versatile tool for Super-resolution Microscopy

Andreas Haderspeck1, Thorben Cordes2, Paja Reisch3, Ben Krämer3, Felix Koberling3, Dirk-Peter Herten1

1Institute for Physical Chemistry, Heidelberg University, Germany
2Zernike Institute for Advanced Materials, University of Groningen, Netherlands
3PicoQuant GMBH, Berlin, Germany

Super-resolution microscopy is a well-established tool for investigating cellular structures below the Abbe limit. However, the development of new fluorescent probes with specifically tailored properties offers still space for further improvements and also helps expanding their range of applications. Currently, we work on modular systems allowing facile combination of different dye and receptor moieties to establish highly versatile fluorescent probes that can be switched between a bright and a dark state by chemical reactions, e.g. reversible coordination of metal ions to a ligand. Previously we reported the utilization of this stochastic process in localization microscopy[1],[2] and single color multiplexing.[3] Here, we present new fluorescent probes based on a compact organic scaffold, which we recently synthesized and characterized in respect of their photophysical properties and demonstrate their potential for localization microscopy imaging. Furthermore, we highlight the application of our multiplexing technique in combination with STED microscopy making it a powerful tool to image multiple structures with a resolution of up to 70 nm by employing single color excitation.

[1] A. Kiel et al., Angew. Chem. Int. Ed., 119, 3427( 2007).

[2] M. Schwering et al., Angew. Chem. Int. Ed., 50, 2940(2011).

[3] D. Brox et al., PLoS ONE, 8, e58049( 2013).

Siegfried Hänselmann, Heidelberg, Germany

Measuring the kinetics and stoichiometry of protein complex formation on a single-molecule level

Siegfried Hänselmann1, Florian Salopiata3, Yu Qiang2, Karl Rohr2, Ursula Klingmüller3, Dirk-Peter Herten1

1Institute for Physical Chemistry, Heidelberg University, Im Neuenheimer Feld 267, 69210 Heidelberg, Germany
2Biomedical Computer Vision Group, Heidelberg University, Im Neuenheimer Feld 267, 69210 Heidelberg, Germany
3Systems Biology of Signal Transduction,German Cancer Research Center,Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

An initial event in many cellular signaling cascades is the assembly of receptors and associated downstream molecules in the plasma membrane. We have developed a two-color single-molecule tracking routine based on SNAP-tag and HaloTag fusion proteins that allows us to follow receptor heterodimerization and dissociation in the plasma membrane of live cells. This enables us to determine interaction kinetics even for weakly interacting receptors. To quantify the stoichiometry of diffraction-limited protein complexes, we are extending the reference-free counting method Counting by Photon Statistics (CoPS) [1] towards its use in fixed cells.

[1] Kristin S. Grußmayer, Florian Steiner, John M. Lupton, Dirk-Peter Herten and Jan Vogelsang, ChemPhysChem, 16, 3578-3583 (2015)

Dirk-Peter Herten, Heidelberg, Germany

Estimating the emitter number on diffusive complexes by means of photon statistics

Kristýna Holanová, Praha 8 - Kobylisy, Czech Republic

Optical imaging of sub-protein sized scattering labels

Kristýna Holanová, Marek Piliarik

Institute of Photonics and Electronics of the CAS, Chaberská 57, 18251, Praha 8 - Kobylisy, Czech Republic

Labeling is the key technique in real-time tracking of processes in biological matter. The major challenge of conventional microscopy methods is the spatial and the temporal resolution of observed biological event. Fluorescent labeling have been in the forefront of the super-resolution and nanoscopic tracking techniques for years. However, the saturation of fluorescent signal puts an intrinsic limit on the imaging resolution which has been already reached. Elastic scattering has none of these limitation and indeed scattering labels such as gold nanoparticles have recently proven much faster and more accurate localization and tracking of biological analytes. The obvious handicap of scattering labels is their size which is often way much larger than the investigated biomolecule (e.g. a lipid or a protein).

In this contribution we demonstrate that scattering labels smaller than 2 nm can be imaged and localized using interferometric detection of scattering (iSCAT). Our iSCAT microscope enables imaging and tracking extremely small nanoparticles or even unlabeled protein. Here for the first time we demonstrate imaging of metallic nanoparticles considerably smaller than the size of a single protein molecule.

[1] S. Spindler, J. Ehrig, K. Koenig, T. Nowak, M. Piliarik, H. Stein, R. Taylor, E. Garanger, S. Lecommandoux, I. Alves, V. Sandoghdar, Journal of Physics D: Applied Physics, in print.
[2] M. Piliarik, V. Sandoghdar, Nature Communications 5, 4495 (2014).

Sen Hou, Warsaw, Poland

Enhanced DNA nanoparticle formation in macromolecular crowding environment studied by fluorescence correlation spectroscopy

Sen Hou, Piotr Trochimczyk, Lili Sun

Institute of Physical Chemistry, Polish Academy of Sciences Kasprzaka 44/52 Warsaw Poland

Macromolecular crowding is defined as the presence of high concentration of macromolecules in a solution. Macromolecules occupy an overwhelmingly large fraction of the solution volume and herein increases the effective concentration of the rest reaction molecules. Therefore, previous researches usually report that macromolecular crowding enhanced the biological reactions inside. However, our recent research shows the macromolecular crowding inhibits DNA cleavage by restriction enzyme, which leads to the unearthing of the enhanced formation of DNA nanoparticles in macromolecular crowding environment. The formation of DNA nanoparticles as well as the prerequisite of the process are studied with fluorescence correlation spectroscopy technique by labeling DNA with fluorescent dyes. The formation of DNA nanoparticles is observed by the autocorrelation curves. Results show that macromolecular crowding alone is insufficient to induce DNA nanoparticle formation. Cationic ions (magnesium ions) are necessary to assist this process. However cationic ions cannot cause DNA nanoparticle formation alone either. Herein, in this sense, the macromolecular crowding enhances the formation of DNA nanoparticles in cationic ion solutions. The conclusion is confirmed by electrophoresis assay, dynamic light scattering assay and analytical ultracentrifugation assay, etc. The enhanced DNA nanoparticle formation is meaningful for the controllable DNA compaction, a first step to efficient gene delivery.

Sen Hou, Piotr Trochimczyk, Lili Sun, Agnieszka Wisniewska, Tomasz Kalwarczyk, Xuzhu Zhang, Beata Wielgus-Kutrowska, Agnieszka Bzowska, Robert Holyst.  Scientific Reports 6 (2016) DOI: 10.1038/srep22033

Maximiliaan Huisman, Worcester, United States

Reconstructing discrete features of nucleocytoplasmic transport using projected density maps.

Maximiliaan Huisman1, Yu-Chieh Chung4, Li-Chun Tu2, Carlas Smith3, David Grünwald5


Time-resolved single molecule experiments have provided tremendous information pertaining to molecular dynamics and localization over the last decade. Biological processes take place in a light sensitive environment, on time scales from sub-millisecond (ms) to hours and length scales from nanometer to millimeter, presenting a number of experimental challenges. One such challenge is the need for speed in image acquisition to correctly follow single molecule movements during translocation through the nuclear pore complex (NPC). Translocation through the central channel has repeatedly been reported to be faster than 20 ms.

Biochemical and structural data of the components that make up the NPC have led to the question if specific spatial transport routes exist within the NPC in vivo. Millisecond time resolutions and three-dimensional spatial resolution in the range of only a few nm are needed in order to resolve the path traveled by transport receptors and cargos by single molecule real-time microscopy. These imaging requirements are challenging and are not met by current technology; however, it was suggested that highly time-resolved 2D tracking data can be interpreted as projected cargo densities and subsequently transformed into a 3D cargo density [1]. Such density maps would provide valuable insights into the function of NPC mediate transport in cells. Here we present a thorough analysis of the conditions needed for this method to work for the nuclear pore complex and the limits to which data can be interpreted.

[1] J. Ma and W. Yang, PNAS, vol. 107, no. 16, pp. 7305–7310 (2010).

Ali Ibrahim, Orsay, France

FLIM and spectral non-linear imaging of human brain tumors samples 

Ali Ibrahim1, Fanny Poulon2, Fatima Melouki3, Marc Zanello4, Pascale Varlet5, Bertrand Devaux6, Darine Abi Haidar7

1IMNC Laboratory, UMR 8165-CNRS, Orsay, France
2IMNC Laboratory, UMR 8165-CNRS, Orsay, France
3IMNC Laboratory, UMR 8165-CNRS, Orsay, France
4Neuropathology Department, Sainte-Anne Hospital, France
5Neuropathology Department, Sainte-Anne Hospital, France
6Neurosurgery Department, Sainte-Anne Hospital, France
7IMNC Laboratory, UMR 8165-CNRS, Orsay, France

We will investigate autofluorescence of normal brain, different tumor types and infiltrated boundaries, in order to build a database of human tissue optical signature. This series of specific multimodal “optical signatures” linked to different types of tissues will help to (i) define the better excitation and collection parameters, (ii) obtain data allowing differentiation of healthy and tumor tissues, and (iii) give new insights into brain morphology. These results confirmed the clinical relevance of this real-time multimodal optical analysis, which can be easily applied to neurosurgical purpose for a better definition of surgical margins for gliomas, metastases and meningiomas.

We work at the excitation wavelength of 890nm, measuring the two-photon emission spectra and fluorescence lifetime. By combining these two responses we were able to discriminate the tumorous tissue from the healthy one, even more precisely, we were able to discriminate between the two different types of tumors, mainly by looking at the shape of the emission spcectra.

Shan Jiang, Suzhou, China

Enhanced SOFI algorithm with deconvolution pre-processing

André Klauss, Potsdam, Germany

Light modulator based sensorless aberration correction for improved STED nanoscopy

André Klauss, Florian Conrad, Carsten Hille

Physical Chemistry / ALS ComBi, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany

In STED nanoscopy diffraction-unlimited fluorescence imaging is achieved by superimposing a stimulated emission inducing laser beam featuring a zero-intensity point on the excitation beam in the focal plane of a confocal microscope. STED intensity distributions like the ‘doughnut’ for lateral resolution improvement and the ‘optical bottle’ for axial squeezing of the effective fluorescence spot are most commonly produced by phase modulation, imprinting a helical phase ramp or a circular π phase into the STED wavefront, respectively.

Due to their free programmability, spatial phase only light modulators (SLM) used for this task bring some advantages, comprising fast switching between different STED modes, automatized holographic fine adjustment, and, most importantly, adaptive aberration correction by addressing compensation phasemasks on the SLM. Especially for (3D-)STED imaging several micrometers deep inside the specimen aberration correction becomes an issue. To quantify distortions in the wavefront, partly introduced by the microscopic specimen itself, we propose and demonstrate the use of special light pattern generated by the SLM in a sensorless scheme. This allows to determine many parameters for aberration correction and fine alignment by simple intensity measurements at one single point in the specimen and without the need for an additional wavefront sensor.

Ben Kraemer, Berlin, Germany

Expanding the Capabilities of Single Molecule STED with Advanced Pulsed Interleaved Excitation

Marcelle Koenig, Paja Reisch, Rhys Dowler, Benedikt Kraemer, Sebastian Tannert, Matthias Patting, Felix Koberling, Rainer Erdmann

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

Stimulated Emission Depletion (STED) microscopy has evolved into an established imaging method offering super-resolution well beyond 50 nm. Whereas STED is now available in many laboratories, it is still in the focus of research to push the boundaries of its capabilities and applications. Time-resolved STED microscopy using time correlated single photon counting (TCSPC) is advantageous for many applications and promises further development for increased resolution and less photo-damage.

Here, we show the application of established methods (e.g., gSTED) as well as emerging applications of time-resolved STED. We employ pulsed interleaved excitation (PIE), where the STED laser is pulsed at half the frequency of the excitation laser, such that STED and confocal data is taken practically at the same time. By using this approach, single molecule STED experiments can be carried out while the confocal control-experiment is performed simultaneously, allowing to account for measurement artifacts due to the high power of the STED laser. We will show examples from single molecule imaging, where blinking and bleaching are monitored using the confocal data. Furthermore, we will present STED-FCS data, where the confocal data allows insight into changes of the sample due to the STED laser. Since the control experiment for the influence of the STED laser is performed at the same time as the STED measurement, experimental parameters can be adjusted online to give highest resolution while ascertaining that the relevant information drawn from the experiment is not affected.

Luis Mateos, Southampton, United Kingdom

Gold porous microcavities for plasmon-enhanced DNA sequence analysis

Luis Mateos, Tracy Melvin

Optoelectronics Research Centre, University of Southampton, Higfield, SO17 1BJ, U.K.

Current methods for DNA sequence analysis are now fast and highly-accurate over long-read lengths for single-stranded DNA. Even so there is a growing need for sequence analysis of low copy numbers of double-stranded DNA, notably for epigenetics.

The use of metallic nanostructures with different sizes and geometries has shown much potential for optical based biosensing applications [1]. Here, we apply a gold film consisting of a hexagonal lattice of 3D gold semi-spherical microcavities where a nanosized pore is milled at its base [2]. The design of the cavity is such that the plasmon enhancement is maximized at the nanopore, our ongoing research including the design, fabrication and optical interrogation will be presented.

[1] S Mahajan, J Richardson, T Brown, P.N Bartlett, Journal of the American Chemical Society, 130(46), 15589-15601 (2008).

[2] SZ Oo, G Silva, F Carpignano, A Noual, K Pechstedt, L Mateos, J.A Grant-Jacob, B Brocklesby, P Horak, M Charlton, S.A Boden, T Melvin, Sensing and Bio-Sensing Research, 7, 133-140 (2016)

Sarah Ochmann, Braunschweig, Germany

Diagnostics based on Fluorescence Enhancement with Nanoantennas

Sarah Ochmann, Carolin Vietz, Birka Lalkens, Philip Tinnefeld

TU Braunschweig, BRICS, NanoBioSciences, Rebenring 56, 38106 Braunschweig

Usually, the detection of pathogens with low abundance is limited by the obtainable signal. For the specific detection of DNA sequences, often a molecular multiplication of these sequences is necessary. In our lab we use an amplification method based on a purely physical enhancement of the fluorescence signal. 
Based on a DNA-nanostructure which is capable of enhancing fluorescence in a plasmonic hotspot by several orders of magnitude [1] we developed a universal signal-enhancement method which is compatible to existing assay technologies, lowering the detection limit and/or reducing the lead time of the assay. Additionally, low-tech detection systems can be used, enabling point-of-care diagnostics.
Target molecules can be nucleic acids or other biomarkers like small molecules or proteins.

[1] G. P. Acuna, F. M. Möller, P. Holzmeister, S. Beater, B. Lalkens, P. Tinnefeld, Science, 338, 506–510 (2012)

Sandra Orthaus-Mueller, Berlin, Germany

Rapid FLIM: the new and innovative method for ultra-fast imaging of biological processes

Sandra Orthaus-Mueller, Ben Kraemer, Astrid Tannert, Tino Roehlicke, Michael Wahl, Hans-Juergen Rahn, Felix Koberling, Rainer Erdmann

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

Over the last two decades, time-resolved fluorescence microscopy has become an essential tool in Life Sciences thanks to measurement procedures such as Fluorescence Lifetime Imaging (FLIM), lifetime based Foerster Resonance Energy Transfer (FRET), and Fluorescence (Lifetime) Correlation Spectroscopy (F(L)CS) down to the single molecule level. Today, complete turn-key systems are available either as stand-alone units or as upgrades for confocal laser scanning microscopes (CLSM). Data acquisition on such systems is typically based on Time-Correlated Single Photon Counting (TCSPC) electronics along with picosecond pulsed diode lasers as excitation sources and highly sensitive, single photon counting detectors.

Up to now, TCSPC data acquisition is considered a somewhat slow process as a large number of photons per pixel is required for reliable data analysis, making it difficult to use FLIM for following fast FRET processes, such as signal transduction pathways in cells or fast moving sub-cellular structures. We present here a novel and elegant solution to tackle this challenge.

Our approach, named rapidFLIM, exploits recent hardware developments such as TCSPC modules with ultra short dead times and hybrid photomultiplier detector assemblies enabling significantly higher detection count rates. Thanks to these improved components, it is possible to achieve much better photon statistics in significantly shorter time spans while being able to perform FLIM imaging for fast processes in a qualitative manner and with high optical resolution. FLIM imaging can now be performed with up to several frames per second making it possible to study fast processes such as protein interactions involved in endosome trafficking.

Dina Petrova, Amsterdam, Netherlands

Microscopic vizualization of contacts and friction

Dina Petrova, Tomislav Suhina, Michiel Hilbers, Daniel Bonn, Fred Brouwer

Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands

New viscosity sensitive-probes, which were synthesized in our lab(1), are extremely sensitive to mechanical confinement. Due to their sensitivity, they present ideal means of imaging contacts between the solid surfaces both in static and dynamic regime. In order to carefully interpret the fluorescence intensity changes due to the load and the friction force variation one has to understand the behavior of the molecules on the surface. The spectroscopic properties of the immobilized molecules on the coverslip with and without solvent presence are investigated and compared to the spectra obtained within the measured contact zone. Our results indicate that the real contact area is smaller than the one which is possible to resolve with the conventional microscopy techniques. In order to investigate the finer structures within the contact area, the next step of the project is using TIRF microscopy and photoswitchable fluorophores for the super-resolution contact area imaging.

1. Tomislav Suhina, Bart Weber, Chantal E. Carpentier, Kinga Lorincz, Peter Schall, Daniel Bonn,
and Albert M. Brouwer, Angewandte Chemie 2015, 127, 3759-3762

Elena Polo, Göttingen, Germany

Impact of Redox-Active Molecules on the Fluorescence of Polymer-Wrapped Carbon Nanotubes

Elena Polo, Sebastian Kruss

Institute of Physical Chemistry, Department of Chemistry, Göttingen University Germany, Tammannstrasse 6, 37077 Göttingen, Germany, Phone +49 551 39 10633; e-mail skruss@gwdg.de (S.K.).

The near-infrared (nIR) fluorescence of polymer-wrapped single-walled carbon nanotubes (SWCNTs) is very sensitive to the local chemical environment. It has been shown that certain small reducing molecules can increase the fluorescence of SWCNTs. However, so far the role of the polymer around the SWCNT as well as the mechanism is not understood. 

We investigated how reducing and oxidizing small molecules affect the nIR fluorescence of polymer-wrapped SWCNTs. Our results show that the polymer plays an essential role. Reducing molecules such as ascorbic acid, epinephrine, and trolox increased the nIR fluorescence up to 250% but only if SWCNTs were suspended in negatively charged polymers such as DNA or poly(acrylic acid) (PAA). In comparison, phospholipid–poly(ethylene glycol) wrapped SWCNTs did not respond at all while positively charged polyallylamine-wrapped SWCNTs were quenched. Oxidized equivalents such as dehydroascorbic acid did not show a clear tendency to quench or increase fluorescence. Only riboflavin with an intermediate oxidation potential and light absorption in the visible range quenched all polymer-wrapped SWCNTs. In general, polymer-wrapped SWCNTs that responded to reducing molecules (e.g., +141%, ascorbic acid) also responded to oxidizing molecules (e.g., −81%, riboflavin). Nevertheless, several reducing molecules showed only a small fluorescence increase (NADH, +21%) or even a decrease (glutathione, −14%), which highlights that the redox potential alone cannot explain fluorescence changes.

Furthermore, we show that neither changes of absorption cross sections, scavenging of reactive oxygen species (ROS), nor free surface areas on SWCNTs explain the observed patterns. However, results are in agreement either with a redox reaction of the polymer or conformational changes of the polymer that change fluorescence decay routes. In summary, we show that the polymer around SWCNTs governs how redox-active molecules change nIR fluorescence (quantum yield) of SWCNTs. Molecules with a low redox potential (<−0.4 V) are more likely to increase SWCNT fluorescence, but a low redox-potential alone is not sufficient.

Polo et al, J. Phys. Chem. C, 2016, 120 (5), pp 3061–3070

Francesco Reina, Oxford, United Kingdom

Comparative iSCAT and STED-FCS measurements on diffusing gold-tagged phospholipids

Frank Schreiber, Berlin, Germany

Correlative imaging of gene expression and metabolic activity by combining single-molecule mRNA FISH and nanometer-scale secondary ion mass spectrometry

Frank Schreiber1,2,3, Sten Littmann4, Stephane Escrig5, Gaute Lavik4, Marcel Kuypers4, Anders Meibom5,6, Martin Ackermann2,3

1Department of Materials and Environment, BAM- Federal Institute for Materials Research and Testing (BAM),Unter den Eichen 87, 12205 Berlin, Germany
2Department of Environmental Systems Science, ETH Zurich, Swiss Federal Institute of Technology, Universitätsstrasse 16, 8092 Zurich, Switzerland
3Department of Environmental Microbiology, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse 133, 8600 Dübendorf, Switzerland
4Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany
5Laboratory for Biological Geochemistry, School of Architecture, Civil and Environmental Engineering (ENAC), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
6Center for Advanced Surface Analysis, Institute of Earth Sciences, University of Lausanne, Lausanne, Switzerland

Populations of genetically identical cells that share the same environment can differ markedly in their phenotypes. This phenomenon has been termed phenotypic heterogeneity. While a few molecular mechanisms that lead to heterogeneity in gene expression have been elucidated, it remains unclear how heterogeneity in gene expression is transmitted to heterogeneity in activity; especially in metabolism. Metabolic activity of single bacterial cells can be quantified by labeling the substrate with stable isotopes and by measuring label uptake with nanometer-scale secondary ion mass spectrometry (NanoSIMS). Here we combined NanoSIMS with single-molecule mRNA fluorescence in situ hybridization (smFISH) to link heterogeneity in gene expression and metabolism in nitrogen fixing bacteria. We find that gene expression and metabolic activity are decoupled in single cells. However, heterogeneity in gene expression is correlated with heterogeneity in metabolic activity on the population level. Gene expression kinetics can provide insights into the molecular mechanisms that lead to heterogeneity in metabolism.

Maryam Hashemi Shabestari, Amsterdam, Netherlands

Phosphorylation and acetylation of TFAM have contrasting mechanisms for regulating non-specific TFAM-DNA interactions 

Maryam Hashemi Shabestari1, Graeme A. King1, Wouter H. Roos2, Carolyn K. Suzuki3, Gijs J.L. Wuite1

1Department of Physics and Astronomy and LaserLaB, Vrije Universiteit, De Boelelaan 1081, Amsterdam, The Netherlands
2Moleculaire Biofysica, Zernike Institute, Rijksuniversiteit Groningen, Nijenborgh 4, Groningen, The Netherlands
3Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA

Mitochondrial transcription factor A (TFAM) is a multifunctional protein that orchestrates compaction, and helps initiate transcription, of the mitochondrial genome. While post-translational modifications of TFAM, including phosphorylation and acetylation, are thought to regulate its activity, the mechanisms by which this can occur are poorly understood. Here, using biochemical, spectroscopic and single-molecule methods, we probe the non-specific binding of relevant phosphomimics and acetylmimics of TFAM to DNA. We demonstrate that these modifications do not alter the protein’s ability to compact DNA, but rather lower its DNA binding affinity. We determine that this arises primarily from a decrease in on-rate in the case of the acetylmimic. In contrast, the phosphomimic is distinct in that it shows a faster (1.5-fold) off-rate and a significantly (7-fold) higher rate of diffusion relative to both the acetylmimic and wild-type. This indicates that the phosphomimic specifically lowers the stability of TFAM when bound to DNA. This work provides a mechanistic basis for how phosphorylation and acetylation can regulate TFAM binding to DNA and, in turn, its function in vivo.

Dmitry Torchinsky, Tel Aviv, Israel

Single-molecule counting highlights the efficiency and specificity of DNA repair enzymes

Dmitry Torchinsky

Tel Aviv University, Chemical Physics, Haim Lebanon, Tel Aviv, Israel

DNA, under exposure to various chemicals such as oxidizing agents as well as various types of radiation, can undergo physical breakage of one or both DNA strands. However, the most common yet least studied form of DNA damage are chemical modification known as DNA damage adducts. These damage lesions can influence transcription and replication, and eventually can lead to DNA breaks. DNA adducts are repaired by specialized enzymes that specifically recognize and remove them but difficulties in the detection of these damage types have hampered their research. In this project we introduce a single molecule approach which takes advantage of natural repair enzymes to label damage sites in vitro by incorporating fluorescent nucleotides as a part of the repair process. The labeled DNA is extended on positively activated glass coverslips and damage sites are visualized as fluorescent spots along the DNA contour. Using model DNA we establish the efficiency of the labeling process as well as it’s specificity towards particular damage type, opening up new opportunities for DNA damage studies.

Dongfang Wang, Braunschweig, Germany

Title: A DNA Walker in the plasmonic hotspot for fluorescence enhancement

Florian Weigert, Berlin, Germany

Photoluminescence Properties of core-shell CdSe Nanocrystals with Different Shells and Surface Chemistries Derived from Ensemble and Single Particle Measurements

Florian Weigert1, Christian Strelow2, Chris Guhrenz3, Chistopher Wolter2, Pavel Samokhvalov4, Christian Würth1, Alf Mews2, Nikolai Gaponik3, Alexander Eychmüller3, Igor Nabiev4, Ute Resch-Genger1

1Federal Institute for Materials Research and Testing, Division Biophotonics, D-12489 Berlin, Germany
2University of Hamburg, D- 20146 Hamburg, Germany
3Technical University Dresden, D-01062 Dresden, Germany
4National Research Nuclear University MEPHI, 115409 Moscow, Russia

The optical properties of semiconductor nanocrystals (SCNCs) depend on constituent material, particle size, and surface chemistry, with the size of the photoluminescence (PL) quantum yield (QY) and the PL decay kinetics being largely controlled by the number of dangling bonds, which have to be properly passivated for high quality materials. Hence, PL measurements can provide insight not only in SCNC photophysics, yet can be also used for quality control of SCNC synthesis and surface modification. In this respect, steady state and time-resolved fluorometry and confocal microscopy with time correlated single photon counting were used to study the PL properties of core-shell CdSe SCNCs with different shells and surface chemistries on ensemble and single particle level, thereby focusing on a correlation of ensemble PL QY and PL decay kinetics with particle brightness, PL time traces, and the On-time fraction of single SCNCs. Additionally, confocal PL images were correlated with AFM measurements in order to derive the amount of absorbing, yet non-emissive ”dark” SCNCs, the presence of which resulting in  an underestimation of ensemble PL quantum yields. The results of this study can help to identify synthetic routes and surface modifications, which minimize the fraction of dark SCNCs.

We have received an overwhelming large amount of abstracts for talks and posters. We thank all particpants for their contribution.

The originally planned schedule did unfortunately not allow to accept all submitted abstracts for talks. We therefore included four "flash talk" sessions into the program. A flash talk offers with a maximum of 4-5 transparencies and 4 minutes a way to highlight a poster. There will also be no questions during the flash talks as there will be plenty of time for questioning and discussions at the poster session that follows the flash talks on the same day.

Registration and abstract submission (Post-deadline poster only!)

Workshop fees*
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. A few days after online registration, you will receive an email notification including a PDF file that gives detailed information on the payment procedure.
  3. In order to take advantage of the early bird rate (deadline: May 31, 2016), payments have to be received by June 7, 2016.
  4. All other payments have to be received within 14 days after date of registration.
  5. 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.
  6. A receipt of payment will be included in our email confirmation of participation.
  7. 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 15, 2016, 75 % of the received registration fee will be reimbursed. In case of cancellations after August 15, 2016, 25 % of the registration fee will be reimbursed.
    • It is possible to name and send a substitute participant.
  8. No visa letters will be issued until payment of the registration fee is received and confirmed.
  9. Schedule and content of this event is subject to change without notice.
  10. PicoQuant records photographs and video material of participants at the events. By registering for a PicoQuant event, you agree that we may include images of yourself (either as a full image or in parts) for promotional purposes (press releases, event website, etc.). If you do not want us to use pictures in which you appear, please inform us in written form.
Additional information

Workshop fees

The fee structure as well as terms and conditions for payment will be released at a later date.

  Until June 6, 2017 June 7, 2017 until August 15, 2017
Academic/University 300 € 350 €
Industry and Private Sector 750 € 900 €

Besides full workshop attendance, the fee includes all coffee breaks, a reception with free food and drinks, one dinner, two lunches, and an abstract book. Attendees will be responsible for their own travel, lodging, and 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. In order to take advantage of the early bird rate (registration deadline: June 6, 2017), payments have to be received by June 13, 2017.
  4. All other payments have to be received within 14 days after date of registration.
  5. 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.
  6. A receipt of payment will be included in our email confirmation of participation.
  7. 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 15, 2017, 75 % of the received registration fee will be reimbursed. In case of cancellations after August 15, 2017, 25 % of the registration fee will be reimbursed.
    • It is possible to name and send a substitute participant.
  8. No visa letters will be issued until payment of the registration fee is received and confirmed.
  9. Schedule and content of this event is subject to change without notice.
  10. PicoQuant records photographs and video material of participants at the events. By registering for a PicoQuant event, you agree that we may include images of yourself (either as a full image or in parts) for promotional purposes (press releases, event website, etc.). If you do not want us to use pictures in which you appear, please inform us in written form.

Financial support

As in the previous years, PicoQuant will grant a fee waiver to a few participants from the university and academic sector of economically less privileged countries. 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.

To apply for a fee waiver, please send us your application:

a letter of application and
a formal letter of recommendation from your department/institute

Deadline for a fee waiver application is May 31, 2017.

Please note that only one person per research group can be considered for a fee waiver.

Details on the fee waiver application process will be published at a later date.



Details about booking accommodations will be announced at a later date.

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.

City Tax

Please note that since the beginning of the year 2014, tourists staying overnight in Berlin are subject to paying an accommodation tax, the so-called City Tax. It amounts to five percent of the room rate (net price), excluding VAT and fees for amenities and services such as mini-bar, sauna, or spa area. The City Tax does only affect private overnight stays and NOT business travellers. The business purpose of a trip can be verified by a bill that is paid by or issued to the employer, or a letter from the company. If the accommodation is booked by the employer in the first place, there is no further proof necessary.

Also see the information at www.berlin.de.

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
  • single room: 63 € (excl. breakfast)
  • double room: 78 € (excl. breakfast)
  • breakfast: 13 € per day and person
Airporthotel Berlin-Adlershof

Booking code: PicoQuant 23 Workshop.

Please use the booking form to reserve a room.

The rooms are bookable at this rate until August 23, 2017. We can not guarantee any reservations to these prices or any reservation at all after this date.

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: 69 € (excl. breakfast)
  • double room: 90 € (excl. breakfast)

Guests can join the breakfast buffet at the ADAPT Hotel or in nearby hotels for a special price of 10-12.50 € per person and day.

Wireless LAN is included in the room price.

ADAPT Apartments Berlin-Adlershof

Please book your room via e-mail, using the booking code: WORKSHOPPICO

The rooms are bookable at this rate until July 13, 2017 on a first come, first served basis. We cannot guarantee reservations at these prices or any reservations at all after this date.

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

Room prices per night
  • single room: 82 € (incl. breakfast)
  • double room: 110 € (incl. breakfast)

Wireless LAN is included in the room price.

Dorint Hotel Berlin Adlershof

Booking code: PicoQuant Workshop.

Please contact the Dorint Adlershof Berlin via phone, fax, or e-mail to book a room. 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 August 16, 2017 on a first come, first served basis. We cannot guarantee reservations at these prices or any reservations at all after this date.



The workshop on "Single Molecule Spectroscopy and Ultra Sensitive Analysis in the Life Sciences" is an annual event since 1995. For a summary of each year's event, please select the year from the list below.


Thank you for registering for the 23rd Single Molecule Workshop!

An email with the supplied information has been sent to the provided address.