22nd Single Molecule Workshop
22nd International Workshop on
“Single Molecule Spectroscopy and Super-resolution Microscopy in the Life Sciences”
September 14-16, 2016 in Berlin, Germany
We invite you to 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”. A welcome reception and a workshop dinner give you the opportunity to meet the invited speakers and the workshop attendees.
Aim and Purpose
The aim of this workshop is to provide an interdisciplinary platform for users as well as developers from Physics, Chemistry, and Biology to share their experience, exchange information, and report their recent findings and developments in the field of ultrasensitive optical detection down to the single molecule level and below the classical diffraction limit.
Ultrasensitive spectroscopic techniques have evolved into standard tools in fundamental biological and biomedical research, as they allow the study of function, structure, and interaction of individual single biomolecules. Since the first report of the detection of a single molecule in solution in 1976, the range of techniques and methods have constantly grown. Today, single molecules can be detected using widefield and confocal fluorescence microscopy, Scanning Nearfield Optical Microscopy (SNOM), Atomic Force Microscopy (AFM) or Raman scattering. Time-resolved methods such as Fluorescence Lifetime Imaging (FLIM) or Fluorescence Correlation Spectroscopy (FCS) and even multidimensional fluorescence methods are used on a daily basis in imaging facilities. Measurements below Abbe's classical diffraction limit have become possible with techniques such as Stimulated Emission Depletion Microscopy (STED) and techniques based on single molecule detection capabilities such as localization microscopy (PALM, STORM, dSTORM, GSDIM), or fluctuation microscopy (SOFI). These super-resolution microscopy techniques have gained much interest in the recent years and have especially been recognized by the award of the Nobel Price in Chemisty 2014 to Eric Betzig, Stefan W. Hell, W.E. Moerner.
Nowadays, not only improving and extending the existing arsenal of single molecule and super-resolution techniques and methods is still of paramount interest, but also the beneficial usage of the existing and already established techniques is a major challenge for applications ranging from chemical analysis to biophysics, biological and biomedical research, medical diagnostics, and materials research.
The deadline for abstract submission is May 31, 2016. 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, 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.
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, 2016. Please indicate during the registration/abstract submission if you wish to participate in the contest.
- Deadline for submission of abstracts for post-deadline posters: August 15, 2016
- Deadline for workshop registration: August 15, 2016
- Notification on acceptance of abstracts: July 2016
- Program available: July 2016
- Deadline for submission of abstracts: May 31, 2016
- Deadline for early bird registration: May 31, 2016
SymPhoTime Training Day
One day before the workshop, on September 13, PicoQuant will host the “SymPhoTime Training Day” for users of the SymPhoTime and SymPhoTime 64 software.
For details visit the event website.
Conference on Single Molecule Spectroscopy at BiOS 2017
Within the framework of the Biomedical Optics Symposium BiOS, PicoQuant is co-organizing the special conference "Single Molecule Spectroscopy and Superresolution Imaging X" (BO503)." The call for papers is now open and abstracts are due July 18, 2016. 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.
Workshop coordinator: Jana Bülter
The list of speakers will be announced at a later date.
- Thorben Cordes (University of Groningen, The Netherlands)
“Dynamic structural biology of membrane transporters: mechanisms and tool development”
- Yuval Ebenstein (Tel Aviv University, Israel)
“Beyond sequencing: Single-molecule genomics”
- Philipp Kukura (University of Oxford, UK)
“Towards label-free single molecule microscopy with interferometric scattering”
- Melike Lakadamyali ( Institut de Ciències Fotòniques, Spain)
“Decoding chromatin organization with super-resolution”
- Viola Vogel (ETH Zurich, Switzerland)
“Mechanobiology: from single molecules to disease”
- Katrin Willig (Max Planck Institute of Experimental Medicine, Germany)
“STED microscopy of the living mouse brain”
- Paul W. Wiseman (McGill University, USA)
“ICS with a new focus…pairing image correlation spectroscopy with super-resolution imaging”
- Haw Yang (Princeton University, USA)
“Real-Time 3D Single-Particle Tracking Spectroscopy and Its Applications”
The workshop will be held in Berlin-Adlershof.
Local area map showing the workshop location (red marker)
Time schedule (as of July 20)
We have received an overwhelming large amount of abstracts for talks and posters. We thank all particpants for their contribution.
|12:00 - 13:15||Registration and collection of workshop material|
|13:15 - 13:30||Rainer Erdmann, Berlin, Germany|
|Session: FRET/FCS/FLIM 1Chair: Thorben Cordes|
|13:30 - 14:00|
ICS with a new focus…pairing image correlation spectroscopy with super-resolution imaging
Paul W. Wiseman
Departments of Chemistry and Physics McGill University, firstname.lastname@example.org
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|
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
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. 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.
C. Lefort, H. Hamzeh, F. Louradour, and D. A. Haidar, J. Biomed. Opt. 19, 076005–076005 (2014)
H. Hamzeh, C. Lefort, F. Pain, and D. A. Haidar, Opt. Lett. 40, 808–811 (2015)
|14:20 - 14:40|
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|
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
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) , 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.
 Karedla, N.; Chizhik, A. I.; Gregor, I.; Chizhik, A. M.; Schulz, O.; Enderlein, J., ChemPhysChem, 15, 705-711 (2014).
 Weiß, K.; Enderlein, J., ChemPhysChem, 13, 990-1000 (2012).
 Benda A.; Fagulova V.; Deyneka A.; Enderlein J.; Hof M., Langmuir, 22, 9580-9585 (2006).
|15:00 - 15:35||COFFEE BREAK|
|Session: FRET/FCS/FLIM 2Chair: Paul Wiseman|
|15:35 - 16:05|
Dynamic structural biology of membrane transporters: mechanisms and tool development
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. I finally summarize our contributions towards the development of """self-healing""" organic fluorophores and their applications in single-molecule FRET and super-resolution microscopy.
 G. Gouridis et al., Nature Structural & Molecular Biology 22 (2015) 57-64
 J.H.M. van der Velde et al., Nature Communications 7:10144 (2016)
 E. Ploetz and E. Lerner et al., bioRxiv doi: 10.1101/047779 (2016)
|16:05 - 16:25|
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.
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 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. 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.
 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)
 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|
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 . 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 . 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.
 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).
 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|
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 . 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.
 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|
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.
|18:00 - …||WELCOME RECEPTION|
|Session: Biological applications Chair: Melike Lakadamyali|
|9:00 - 09:35|
Mechanobiology: from single molecules to disease
Laboratory of Applied Mechanobiology, , Department of Health Sciences and Technology, ETH Zurich, Switzerland. , E-mail: email@example.com
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|
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 .
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 . These diffusion characteristics have been suggested to be associated with plasma membrane organisation and its interactions with the underlying actin cytoskeleton .
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.
 Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, et al., Nature, 457(7233), 1159–62 (2009)
 Wawrezinieck L, Rigneault H, Marguet D, Lenne P-F., Biophys J, 89(6), 4029–42 (2005)
 Fujiwara T, Ritchie K, Murakoshi H, Jacobson K, Kusumi A, J Cell Biol, 157(6), 1071–81 (2002)
|09:55 - 10:15|
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.
 Ellis, R. John. Trends in biochemical sciences 26.10 Page: 597-604 (2001)
 Zimmerman, Steven B., and Barbara H. Pheiffer. Proceedings of the National Academy of Sciences 80.19 Page: 5852-5856 (1983)
 Sasaki, Yoshiharu, Daisuke Miyoshi, and Naoki Sugimoto. Nucleic acids research 35.12 Page: 4086-4093 (2007)
 Hou, Sen, et al. Soft Matter 7.7 Page: 3092-3099 (2011)
|10:15 - 10:35|
A paradigm model of induced-fit recognition by the lysine riboswitch aptamer unveiled using a combined single molecule FRET and Optical Force approach
J. Carlos Penedo1, Kaley McCluskey1, Julien Boudreault2, Patrick St-Pierre2, Yann Chemla3, Taekjip Ha3, Daniel A Lafontaine2
1SUPA School of Physics and Astronomy, University of St. Andrews, Scotland, UK KY16 9SS
Riboswitches are 5’ untranslated mRNA elements that regulate gene expression through the allosteric interaction of a ligand-responsive aptamer domain and a downstream expression platform. Among them, the 5-way lysine-binding riboswitch, that is present in very pathogenic bacteria, has attracted interest as a target for antibiotic development because lysine biosynthesis and transport could be suppressed by compounds that mimic the ligand structure. Here, we have applied a combination of single-molecule fluorescence resonance energy transfer (sm-FRET) and single-molecule force-induced mechanical manipulation using optical tweezers (sm-OT) to determine the interplay between structural folding and ligand-binding kinetics on the lysC riboswitch. Using a detailed single-molecule analysis of the kinetics of ligand binding, we demonstrate that the lysC aptamer regulates gene expression on a Mg2+-tuneable scale between kinetic and thermodynamic control. Our work also demonstrates that the lysC riboswitch has been evolutionary optimized not only to specifically recognize the lysine ligand using a ‘pure’ induced-fit model, but also fine-tuned to do so by adapting its folding landscape accordingly to small variations within the physiological bracket of Mg2+ concentrations. Our data provide useful insights for a rational design of ligand analogs and demonstrate that the lysC can be considered as the first unambiguous example of ‘induced-fit’ recognition by a natural aptamer regulating bacterial gene expression.
|10:35 - 11:10||COFFEE BREAK|
|Session: Super-resolution 1Chair: Katrin Willig|
|11:10 - 11:40|
Decoding chromatin organization with super-resolution
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|
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
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)
|12:00 - 12:20|
Christian Franke, Wuerzburg, Germany (Student Award)
Three-dimensional localization microscopy based on accurate intensity estimation
|12:20 - 12:40|
High-Definition Biological Imaging using DNA PAINT
Mael Manesse, Xi Chen, Stephanie Walter, Michael Natan
Ultivue, Inc. 763D Concord Ave., Cambridge, MA 02138, USA
Ultivue Inc. has developed a series of powerful fluorescence microscopy reagents for high-definition biological imaging of fixed cell and tissue samples. Based on DNA-PAINT technology, these reagents enable both single molecule, super-resolution imaging using oblique illumination on conventional microscopes, as well as high-multiplex imaging at standard resolution using epifluorescence instruments. Our approach relies on the finely-tuned transient binding of fluorescent DNA barcodes to label targets of interest.
This technology enables a series of very important user benefits: (i) super-resolution imaging without the need for any specialized instrumentation; (ii) unlimited multiplexing of protein and nucleic acid targets using a single fluorescence channel; (iii) photobleaching-independent image capture; (iv) single molecule data collection with high signal-to-noise ratio; (v) quantitative analysis; and (vi) repetitive imaging of archived samples.
Super-resolution and multiplexed images of key organelle markers will be presented to highlight the application of Ultivue’s reagents for single molecule detection and co-localization of protein targets in cells and tissue, and to illustrate the potential application in life science research in general.
|12:40 - 12:50||GROUP PICTURE|
|12:50 - 14:20||LUNCH BREAK|
|Session: Super-resolution 2 & Material scienceChair: Viola Vogel|
|14:20 - 14:50|
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
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 . 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.
 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)
|14:50 - 15:10|
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.
The discovery of Förster resonance energy transfer (FRET)  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  to accurately deduce the distance between the molecules and metal . 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 .
 Förster, Th. Ann. Phys. 437, 55-75 (1948).
 Drexhage, K. H. Prog. Opt. 12, 163-232 (1974).
 Chizhik, A. I., Rother, J. Gregor, I., Janshoff, A., Enderlein, J. Nature Photon. 8, 124-127 (2014).
 Chizhik, A. I., Baronsky, T., Ruhlandt, D., Karedla, N., Haehnel, D., Gregor, I., Janshoff, A., Enderlein, J. submitted (2016)
|15:10 - 15:30|
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. 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.
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.
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.
 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).
 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).
 Steiner F., Bange S., Vogelsang J., Lupton J.M., Journal of Physical Chemistry Letters, Vol. 6, p. 999 (2015).
|15:30 - 15:45||COFFEE BREAK|
|15:45 - 18:15||POSTER SESSION|
|19:30 - 22:30||DINNER|
|Session: Methods and techniques 1Chair: Yuval Ebenstein|
|9:00 - 09:35|
Towards label-free single molecule microscopy with interferometric scattering
Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK, firstname.lastname@example.org
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.
|09:35 - 09:55|
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, email@example.com
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.
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.
|09:55 - 10:15|
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
Dead-time artifacts can dramatically influence the shape of Time-Correlated Single Photon Counting (TCSPC) histograms such as fluorescence lifetime curves . 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 . 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.
 W. Becker, Advanced Time-Correlated Single Photon Counting Techniques (Springer, 2005).
 S. Isbaner, N. Karedla, D. Ruhlandt, S.C. Stein, A. Chizhik, I. Gregor, and J. Enderlein, Opt. Express 24, 9429-9445 (2016)
|10:15 - 10:35|
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
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. 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. 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.
 G. P. Acuna, F. M. Möller, P. Holzmeister, S. Beater, B. Lalkens, P. Tinnefeld, ACS Nano, 6, 3189 (2012).
 A. Puchkova, C. Vietz, E. Pibiri, B. Wuensch, M. Sanz Paz, G. P. Acuna, P. Tinnefeld, Nano Letters, 15, 8354 (2015).
 Carolin Vietz, Birka Lalkens, Guillermo P Acuna, Philip Tinnefeld, New Journal of Physics, 18, 045012 (2016)
|10:35 - 11:10||COFFEE BREAK|
|Session: Methods and techniques 2Chair: Philipp Kukura|
|11:10 - 11:40|
Real-Time 3D Single-Particle Tracking Spectroscopy and Its Applications
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|
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. 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.
 RP Sinha, DP Häder - Photochemical & Photobiological Sciences, 1, 225-236, 2002
 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|
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|
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 - 14:10||LUNCH BREAK|
|Session: Methods and techniques 3Chair: Haw Yang|
|14:10 - 14:40|
Beyond sequencing: Single-molecule genomics
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||tba|
|15:00 - 15:20|
Sebastian Kruss, Göttingen, Germany
Chemical imaging of cell communication using near infrared fluorescent carbon nanotube sensors
|15:20 - 15:40|
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.
 He Z.; Zhong, C.; Su, S.; Xu, M.; Wu, H.; Cao, Y., Nature Photonics, 6, 591–595, (2012)
 Ouyang, X.; Peng, R.; Ai, L.; Zhang, X.; Ge, Z., Nature Photonics, 9, 520, (2015)
|15:40 - 15:50||STUDENT AWARD PRESENTATION|
|15:50 - 16:00||CONCLUDING REMARKS|
|16:00 -||END OF WORKSHOP|
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
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.
Estimating the emitter number on diffusive complexes by means of photon statistics
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
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.
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
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 .
We demonstrate super-resolution optical fluctuation imaging (SOFI) of cells labeled with dual-color carbon nanodots . 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.
 Ghosh, S. et al. Photoluminescence of Carbon Nanodots: Dipole Emission Centers and Electron-Phonon Coupling. Nano Letters 14, 5656-5661 (2014).
 Chizhik, A.M. et al. Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots. Nano Letters 16, 237-242 (2016).
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
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 .
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.
 Tarpani, et al., Nano Letters, in press (2016).
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
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.
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
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. 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.
 Turin L., Chem Senses, 21, 773–791(1996).
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
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, and single color multiplexing. 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.
 A. Kiel et al., Angew. Chem. Int. Ed., 119, 3427( 2007).
 M. Schwering et al., Angew. Chem. Int. Ed., 50, 2940(2011).
 D. Brox et al., PLoS ONE, 8, e58049( 2013).
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
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)  towards its use in fixed cells.
 Kristin S. Grußmayer, Florian Steiner, John M. Lupton, Dirk-Peter Herten and Jan Vogelsang, ChemPhysChem, 16, 3578-3583 (2015)
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.
 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.
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
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 . 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.
 J. Ma and W. Yang, PNAS, vol. 107, no. 16, pp. 7305–7310 (2010).
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
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.
Enhanced SOFI algorithm with deconvolution pre-processing
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, firstname.lastname@example.org
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.
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 . 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 . 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.
 S Mahajan, J Richardson, T Brown, P.N Bartlett, Journal of the American Chemical Society, 130(46), 15589-15601 (2008).
 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)
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.
 G. P. Acuna, F. M. Möller, P. Holzmeister, S. Beater, B. Lalkens, P. Tinnefeld, Science, 338, 506–510 (2012)
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, email@example.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.
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,
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 firstname.lastname@example.org (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
Comparative iSCAT and STED-FCS measurements on diffusing gold-tagged phospholipids
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
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.
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
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.
The program consists of invited and contributed oral presentations, as well as poster presentations.
The schedule will be published online in July 2016.
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
The fee structure as well as terms and conditions for payment will be released at a later date.
|Until May 31, 2016||June 1, 2016 until August 15, 2016|
|Academic/University||290 €||340 €|
|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, three lunches, and an abstract book. Attendees will be responsible for their own travel, lodging, and meals.
Please note the terms and conditions for payment.
- 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.
- After online registration, you will receive an email notification including a PDF file that includes information on the payment procedure.
- In order to take advantage of the early bird rate (registration deadline: May 31, 2016), payments have to be received by June 7, 2016.
- All other payments have to be received within 14 days after date of registration.
- 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.
- A receipt of payment will be included in our email confirmation of participation.
- 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.
- No visa letters will be issued until payment of the registration fee is received and confirmed.
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.
Details on the fee waiver application process will be published at a later date.
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
- a formal letter of recommendation from your department/institute
Deadline for a fee waiver application is May 31, 2016.
Please note that only one person per research group can be considered for a fee waiver.
Details about booking accommodations will be announced at a later date.
We have negotiated special rates for a limited number of rooms in two hotels/appartment block located close to the workshop location. The number of rooms and the booking time are limited and we therefore advise to reserve your room as soon as possible.
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.
- single room: 63 € (excl. breakfast)
- double room: 78 € (excl. breakfast)
- breakfast: 12 € per day and person
Booking code: 21. Workshop PicoQuant.
Please use the booking form to reserve a room.
The rooms are bookable at this rate until August 11, 2015. We can not guarantee any reservations to these prices or any reservation at all after this date.
- 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 10-15 € per person and day.
Wireless LAN is included in the room price.
Booking code: PicoQuant Workshop.
Please use the booking form to reserve a room.
The rooms are bookable at this rate until August 31, 2016 on a first come, first served basis. We cannot guarantee reservations at these prices or any reservations at all after this date.
- single room: 82 € (incl. breakfast)
- double room: 110 € (incl. breakfast)
Wireless LAN is included in the room price.
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 1, 2016 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 22nd Single Molecule Workshop!
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