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 are possible until end of July.
Please contact us via email if you are interested in presenting a poster at the workshop.
- Abstracts can only be submitted along with the registration for 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 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: July 31, 2015
- Deadline for submission of abstracts: May 31, 2016
- Deadline for early bird registration: May 31, 2016
- Deadline for workshop registration: August 15, 2016
- Notification on acceptance of abstracts: early July 2016
- Program available: July 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 2016
Within the framework of the Biomedical Optics Symposium BiOS, PicoQuant is co-organizing the special conference "Single Molecule Spectroscopy and Superresolution Imaging IX" (BO403)." The call for papers is now open and abstracts are due August 3, 2015. 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 - single-molecule tools for molecular mechanisms”
- Yuval Ebenstein (Tel Aviv University, Israel)
“Beyond sequencing: Single-molecule genomics”
- Philipp Kukura (University of Oxford, UK)
“Single molecule imaging, localisation and tracking without fluorescence?”
- Melike Lakadamyali ( Institut de Ciències Fotòniques, Spain)
“Revealing new biology with super-resolution microscopy”
- Viola Vogel (ETH Zurich, Switzerland)
“Mechanobiology: the art of forming and breaking bonds”
- Katrin Willig (Max Planck Institute of Experimental Medicine, Germany)
“STED microscopy of the living mouse brain”
- Paul W. Wiseman (McGill University, USA)
“Pairing image correlation spectroscopy with super-resolution microscopy to map flows and transport in cells”
- 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)
The program consists of invited and contributed oral presentations, as well as poster presentations.
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.
Time schedule (as of August 12)
We have received an overwhelming large amount of abstracts for talks and posters. We thank all particpants for their contribution.
|12:00 - 13:00||Registration and collection of workshop material|
|13:00 - 13:15||Rainer Erdmann, Berlin, Germany|
|Session: Methods and techniques 1Chair: Rainer Erdmann|
|13:15 - 13:45|
Force and Function: How do Molecules do it?
Hermann E. Gaub
LMU, Angewandte Physik, Amalienstraße 54, 80799 München, Deutschland
Molecular interactions are the basis of life and forces play a crucial role in both the assembly and the structural integrity as well as the dynamics of all living systems. The regulation of bio-molecular complexes, the maintenance of cellular structures, and even cell signaling are controlled by forces. At the molecular level the relation between these forces and their biological functions have become accessible by the different single molecule force spectroscopy techniques, which evolved in recent years. A deeper understanding of the physics of this relation has emerged from the very fruitful combination of the high resolution and precision of such experiments with the insight in structural rearrangements from all-atom Molecular Dynamics Calculations. In this talk a general overview on this field will be given, followed by a report on recent discoveries: The activation mechanisms of two prominent intracellular force sensors, Myosin Light Chain Kinase and Titin Kinase were elucidated. The clamp-mechanism of catch bonds between Cohesin and Dockerins in the Cellulosome complexes could be resolved. Novel strategies for parallelization of force-measuring assays will be discussed and a new chip based strategy will be introduced starting out from genes providing direct access to the mechanics of the encoded proteins in a one step process. At last, the use of molecular force balances for the analysis of DNA-protein interactions will be presented.
|13:45 - 14:05|
Resolution enhancement for scanning microscopy by immersion at low temperature
Michael Metzger1, Alexander Konrad1, Alfred J. Meixner1, Marc Brecht2
1IPTC, Universität Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany
KEY WORDS: Confocal imaging, fluorescence spectroscopy, low-temperature microscopy, correlative light and electron microscopy (CLEM).
One convenient way to increase the resolution of e.g. fluorescence images is realized by confocal microscopy with an objective of high numerical aperture and immersion oil. The combination of immersion fluids with high-performance objectives is however exceedingly problematic under low temperature conditions [1,2].
A new construction of a scanning stage and a well-chosen immersion fluid enables us to immerse an objective together with the sample positioned inside a cryostat at cryogenic temperatures . Heating the sample chamber over the melting point of an appropriate chosen immersion fluid (e.g. 1-propanol) allows us to move the objective into the melted immersion droplet and thereby to increase the refractive index between the objective lens and the sample.
We recorded confocal fluorescence images of quantum dots at 160 K with a high NA objective. By determining the point spread function of imaged single quantum dots the effective numerical aperture was appointed to be larger than unity (1.08). The presented method provides new opportunities e.g. for studies on biological systems like vitrified cells at low temperature and is also of relevance for correlative light and electron cryo microscopy (cryoCLEM).
 M.A. Le Gros; G. McDermott; M. Uchida; C.G. Knoechel, and C.A. Larabell, Journal of Microscopy, 235, 1-8 (2009).
 R. Kaufmann, C. Hagen, and K. Grunewald, Current opinion in chemical biology, 20, 86–91 (2014).
 M. Hussels, A. Konrad, and M. Brecht, Review of Scientific Instruments, 83, 123706 (2012).
 C. van Rijnsoever; V. Oorschot, and J. Klumperman, Nature Methods, 5, 973-980 (2008).
|14:05 - 14:25|
Photo-reactivation of luminescent centers in single SiO2 nanoparticles
Alexey Chizhik1, Anna Chizhik1, Luigi Tarpani2, Loredana Latterini2, Ingo Gregor1, Jörg Enderlein1
1III. Institute of Physics, Georg August University, 37077 Göttingen, Germany
Photobleaching is one of the key limiting issues in nearly all fields of fluorescence imaging. So far, the major strategy in overcoming this limitation has been the enhancement of a fluorophore's photostability. We pesent a fundamentally new approach towards acquiring a higher number of photons from a single emitter.
 Godefroo, S., et al., Nat. Nanotechnol., 3, 174 (2008).
|14:25 - 14:45|
Accurate fluorescence quantum yield determination by fluorescence correlation spectroscopy
Daryan Kempe1, Antonie Schoene2, Jörg Fitter1,2, Matteo Gabba2
1I. Physikalisches Institut (IA), AG Biophysik, RWTH Aachen University, Aachen, Germany
We present a comparative method for the accurate determination of fluorescence quantum yields (QYs) by fluorescence correlation spectroscopy . By exploiting the high sensitivity of single-molecule spectroscopy, we obtain the QYs of samples in the microliter range and at (sub)nanomolar concentrations. Additionally, in combination with fluorescence lifetime measurements, our method allows the quantification of both static and collisional quenching constants. Thus, it opens up the possibility to photophysically characterize labeled biomolecules under application-relevant conditions and with low sample consumption, which is often important in single-molecule studies. Currently, we are evaluating the method with respect to solutions of higher refractive indices than water. This would extend the range of possible applications to the characterization of molecules exposed to chemical denaturants or crowding agents.
 D. Kempe, A. Schöne, J. Fitter, and M. Gabba, J. Phys. Chem. B, 119, 4668-4672, 2015
|14:45 - 15:20||COFFEE BREAK|
|Session: FRET/FCSChair: Hermann Gaub|
|15:20 - 15:50|
Ultrahigh resolution single molecule fluorescence-force spectroscopy and engineering of a superenzyme
University of Illinois Physics, 1110 W Green St. 133 Loomis, Urbana, IL 61801, USA
We developed an instrument that can probe the structure-function directly from a single enzyme during its function. It allows us to measure the conformation of the enzyme using fluorescence resonance energy transfer, and simultaneously detect the enzyme’s function, in this case, DNA unwinding at single basepair resolution, using ultrahigh resolution optical trap. This allowed us to show that bacterial helicases assume the closed form during unwinding and the open form during rezipping of DNA. By artificially constraining the protein to the closed form via crosslinking, we could engineer a superhelicase that can unwind thousands of basepairs processively even against a strong force. This superhelicase may be useful for nanopore-based single molecule sequencing and isothermal DNA amplification.
|15:50 - 16:10|
Using smFRET towards understanding structure, function and dynamics of molecular machines: The role of conformational changes for DNA supercoiling by gyrase
Airat Gubaev, Simon Hartmann, Daniela Weidlich & Dagmar Klostermeier
University of Muenster, Institute for Physical Chemistry, Corrensstrasse 30,
48149 Muenster, Germany
The topological state of DNA in the cell is important for replication, recombination and transcription, and is regulated by DNA topoisomerases. Gyrase is a bacterial DNA topoisomerase that catalyzes the ATP-dependent introduction of negative supercoils into DNA. The enzyme is not present in humans, and constitutes an attractive drug target for the treatment of infections. Using single molecule FRET experiments, we have shown that gyrase undergoes a cascade of DNA- and nucleotide-induced conformational changes at the beginning of its catalytic cycle. These conformational changes couple DNA binding to ATP hydrolysis, and are a prerequisite for ATP-dependent negative DNA supercoiling. Supercoiling is believed to occur by a strand passage mechanism that requires cleavage of both strands of the double-stranded DNA substrate. Surprisingly, a gyrase variant that does not cause double-strand breaks but only cleaves a single strand of the DNA still shows robust supercoiling activity, in contradiction to the current text-book model for gyrase activity. We present an alternative mechanism for DNA supercoiling involving trapping, segregation and relaxation of two positive DNA supercoils. This mechanism avoids dangerous double-strand breaks in the genome. Altogether, our results pave the way for finding novel mechanism-based gyrase inhibitors.
|16:10 - 16:30|
Quantitative experimental and theoretical investigation of diffusion of macromolecules through gel matrices by fluorescence methods
D. Sandrin1, D. Wagner2, C. Sitta3, R. Thoma4, S. Felekyan1, H. E. Hermes2, C. Janiak4, N.de Sousa Amadeu4, R. Kühnemuth1, H. Löwen3, S. U. Egelhaaf2, C. A. M. Seidel1
1Institut für Physikalische Chemie II, Molekulare Physikalische Chemie, Heinrich-Heine-Universität, Universitätsstr. 1, 40225 Düsseldorf, Germany
The dynamical hindrance of polymer molecules diffusing through a polyacrylamide gel is systematically explored using labelled dextrans of different molecular weights by applying three complementary methods. While Multiparameter Fluorescence Image Spectroscopy (MFIS) is applied to investigate the local diffusion of single molecules on a microscopic length scale inside the hydrogel, macroscopic transmission imaging (MTI) and nuclear magnetic resonance (PFG-NMR) are used to study the collective motion of particles through the gel matrix. We find quantitative agreement for the long-time diffusion coefficient of the dextran molecules. The measured diffusion coefficients decay markedly with increasing molecular weight and fall on a master curve. The trends are also described with Brownian dynamics simulations. On the microscopic scale by MFIS we observe different classes of diffusing species: a fast component (as also observed by the other two methods) and a distribution of temporarily trapped molecules. The interaction between gel and probe is studied under different salt conditions and clearly shows the dependency of the fraction of immobile molecules with the ionic strength of the solvent. Strong interaction between matrix and solute in particular for Alexa dye labeled particles was also indicated by MTI, showing higher equilibrium concentrations of dye inside the hydrogel as compared to the surrounding solution.
|16:30 - 16:50|
Pressure unfolding of a model folding protein followed by smFRET
Sven Schneider, Erik Hinze, Thorben Blömker, Janka Schwarzer, Christian Hübner
Institut für Physik, Universität zu Lübeck, Ratzeburger Allee 160, D-23562 Lübeck, Germany
Single molecule FRET has proven a powerful method for the study of the unfolded state of folding model proteins(1). Unfolding by temperature and chemical denaturants have been elucidated by smFRET, however, unfolding by high pressures has not been demonstrated to date.
(2)Tecmen,Müller;AIP Review of scientific instruments;Vol 75 ;5143-5147(2004)
|16:50 - 17.10|
farFRET: Extending the range in single-molecule FRET experiments beyond 10 nanometer
Georg Krainer1,2, Andreas Hartmann1, Michael Schlierf1
1TU Dresden, B CUBE, Arnoldstr. 18, 01307 Dresden
Single-molecule Förster resonance energy transfer (smFRET) has become a powerful nanoscopic tool in studies of biomolecular structures and nanoscale objects; however, conventional smFRET measurements are generally blind to distances above 10 nm, thus, impeding the study of long-distance phenomena. Here, we report the development of farFRET, a technique that extends the range in single-molecule FRET (smFRET) measurements beyond the 10-nm line by enhanced energy transfer using multiple acceptors. We demonstrate that farFRET can be readily employed to quantify FRET efficiencies and conformational dynamics using double-stranded DNA molecules, RecA-filament formation on single-stranded DNA and Holliday junction dynamics. farFRET allows quantitative measurements of large biomolecular complexes and nanostructures, thus, bridging the remaining gap to superresolution microscopy.
|17.10 - 17.30|
Studying the function of BAP in the nucleotide cycle of BiP by spFRET using MFD-PIE
Daniela Wengler1, Mathias Rosam2, Jelle Hendrix3, Johannes Buchner2, Don C. Lamb1
1Physical Chemistry, Department of Chemistry, Ludwig-Maximilians-Universität München, Centre for Nanoscience, 81377 Munich, Germany
Heat shock proteins Hsp70 and Hsp90 act together as chaperones assisting nascent proteins to reach their final functional conformation. Understanding the underlying mechanisms of chaperone-assisted protein folding is thus important for development of treatment for various disorders, such as Alzheimer or Parkinson’s disease. The Hsp70 BiP is localized in the endoplasmic reticulum (ER), having two domains, the nucleotide binding domain (NBD) and the substrate binding domain (SBD) and a flexible lid. When nascent protein chains enter the ER, their charged regions are bound by BiP, protecting them from non-specific interactions with themselves. This process is regulated by the nucleotide exchange factor of BiP, named BAP, which accelerates the ATP/ADP exchange, thus controlling BiP binding and release from nascent proteins. In order to gain insights into conformational changes of BiP upon BAP binding, we employed solution single pair Förster Resonance Energy Transfer (spFRET) measurements using the Multi-parameter Fluorescence Detection (MFD) setup, combined with Pulsed Interleaved Excitation (PIE)1. Our results suggest that the lid interacts with BAP, pushing the SBD away from the NBD. ATP inhibits this interaction, preventing BAP from binding to BiP.
 Kudryavtsev, V.; Sikor, M.; Kalinin, S.; Mokranjac, D.; Seidel, C. A. & Lamb, D. C., Chemphyschem, 13, 1060-78 (2012).
|18:00 - …||WELCOME RECEPTION|
|Session: Methods and techniques 2Chair: Taekjip Ha|
|9:00 - 09:35|
Nanophotonic Promises for Single-Molecule Detection
Institute for Physical & Theoretical Chemistry – NanoBioScience, and LENA (Laboratory for Emerging Nanometrology), and BRICS (Braunschweig Integrated Center for Systems Biology), Braunschweig University of Technology, 38106 Braunschweig, Germany, Email: email@example.com
Single-molecule applications are always hungry for more signal, improved signal-to-noise ratios, as well as smaller and defined detection volumes1. The field of nanophotonics offers new ways of light control on the nanoscale2. With nanoantennas, for example, single-molecule signals can be enhanced and the concentration range of single-molecule experiments can be expanded3. We will present recent progress in single-molecule detection combining ideas from nanophotonics with tools from DNA nanotechnology.
(1) Holzmeister, P.; Acuna, G. P.; Grohmann, D.; Tinnefeld, P.: Chem Soc Rev 2014, 43, 1014-28.
(2) Acuna, G.; Grohmann, D.; Tinnefeld, P.: FEBS Lett 2014.
(3) Acuna, G. P.; Moller, F. M.; Holzmeister, P.et. al.: Science 2012, 338, 506-10.
|09:35 - 09:55|
Monitoring intracellular pH of living cells using nanodiamonds
Zhiqin Chu*, Torsten Rendler, Andrea Zappe, Jörg Wrachtrup
3. Physikalisches Institut, Universität Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany
Detection of intracellular pH variations in living cells can help understanding several critical physiological processes including inflammation, apoptosis, multidrug resistance and enzyme activities. In particular, a sensor with long-term stability being able to sense subtle pH changes at specific subcellular sites in living system is highly desired. Here we discuss the development of an ultrasensitive pH sensor. Sensing functions is realized by monitoring the spin relaxation time changes of electronic ground spin states associated with nitrogen-vacancy (NV) color centers in nanodiamonds. With the developed sensing scheme, we could measure the pH changes at subcellular resolution without being affected by surrounding environmental factors. By feeding cells with nanodiamonds via endocytosis, we could monitor the real-time pH changes of nanodiamonds trafficking in endocytic organelles such as endo-lysosomes. Furthermore, we demonstrated the intracellular pH mapping by simultaneously imaging many different nanodiamonds distributed throughout the whole cell. The current work supplies a novel approach for studying the intracellular pH variations in living systems, and underlying sensing mechanism can be expanded to detect other species such as ROS and ion concentrations.
|09:55 - 10:15|
UV fluorescence lifetime modification by Al and Mg plasmonic nanoapertures
Yunshan Wang1, Xiaojin Jiao1, Eric M. Peterson2, Joel M. Harris2, Steve Blair1
1University of Utah, Department of Electrical and Computer Engineering, 50 S. Central Campus Dr., Rm. 3280, Salt Lake City, UT 84112
The study of plasmonic enhancements in the UV part of the spectrum is an emerging area of research, with many applications in biomolecule detection/analysis, UV light emission and detection, and optical lithography. In order to study plasmonic enhancement, we have assembled a UV tunable time-resolved microscopy system in order to measure the fluorescence lifetime and number statistics of freely-diffusing biomolecules. We have specifically focused on plasmonic enhancement effects on the emission of biomolecules by nanoapertures and related structures as small as 40nm constructed in Al and Mg films. Mg shows the potential for higher localized surface plasmon resonance response than Al in the mid- to near-UV part of the spectrum, which is born out by fluorescence lifetime measurements which show that Mg can produce a higher Purcell factor for biomolecules that emit in this spectral range.
Xiaojin Jiao, Eric M. Peterson, Joel M. Harris, and Steve Blair, """UV fluorescence lifetime modification by aluminum nanoapertures,""" ACS Photonics 1, 1270-1277 (2014)
Xiaojin Jiao, Yunshan Wang, and Steve Blair, """UV fluorescenc enhancement by Al and Mg nanoapertures,""" Journal of Physics D: Applied Physics 48, 184007 (2015)
|10:15 - 10:35|
Straightforward upgrade of a time-resolved confocal scanning microscope to a diffraction-unlimited STED nanoscope benefiting from time gating
André Klauss, Carsten Hille
Physical Chemistry / ALS ComBi, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany
The technical complexity of super-resolution STED microscopy has steadily been reduced in the last years. New concepts including single-beam path and single laser source designs were introduced and new compact turnkey laser sources became available. As far-field microscopy is the most widely employed microscopy modality in life sciences, it seems to be an attractive option to upgrade already existing systems, in order to achieve diffraction-unlimited fluorescence imaging in a cost-effective manner. Recently, we have demonstrated the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout as previously proposed as easy-STED [1,2]. Performing excitation at 635 nm and applying a ps-pulsed fiber laser at 766 nm for stimulated emission, lateral resolution of ~50 nm could be achieved in images of 20 nm-sized fluorescent beads as reference system. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and AbberiorSTAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. In addition, the time-correlated single-photon counting (TCSPC) detection system of the microscope allowed for straightforward offline time gating to further improve the signal-to-background ratio of STED images.
 M. Reuss, J. Engelhardt, S.W. Hell, “Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation”, Opt. Exp. 18, 1049-1058 (2010).
 A. Klauss, M. König, C. Hille, “Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope“, PLoS ONE 10(6), e0130717 (2015).
|10:35 - 11:10||COFFEE BREAK|
|Session: Methods and techniques 3Chair: Atsushi Miyawaki|
|11:10 - 11:40|
Measuring binding affinities using force methods
Universitat de Barcelona, Small Biosystems Lab, Departament de Fisica Fonamental, Facultat de Fisica Diagonal 647, 08028 Barcelona
Intermolecular binding reactions drive a myriad of processes central to molecular biology such as gene regulation, recombination, ribosome assembly and immune response. We introduce a novel fluctuation theorem for ligand binding to measure binding energies of biomolecular reactions at the single-molecule level. We investigate single oligonucleotides, DNA restriction enzymes, and small ligands binding to DNA hairpins in single-molecule pulling experiments. Binding energies are directly measured as a function of ligand concentration providing a direct experimental verification of the law of mass action both for highly specific interactions and non-specific binding to multiple DNA sites. The possibility of monitoring single binding modes one at a time in single molecule experiments, in combination with the fluctuation theorem for ligand binding, makes now possible to determine the energetics of complex multimolecular assemblies with unprecedented reliability and accuracy.
|11:40 - 12:00|
Counting by Photon Statistics - Fluorescence Quantification based on Photon Antibunching
Kristin Grußmayer, Dirk-Peter Herten
Excellence Cluster CellNetworks and Institute for Physical Chemistry, Heidelberg University, Germany, Im Neuenheimer Feld 267, 69120 Heidelberg
Counting by Photon Statistics (CoPS) is a single-molecule fluorescence spectroscopy technique for quantification of fluorescent probes. CoPS exploits the photon antibunching effect, i.e. a dye as a quantum system may only emit one photon per excitation cycle. The simultaneous detection of multiple photons in a four - detector confocal microscope enables estimation of the number of fluorophores along with their molecular brightness [1, 2]. Since our method is directly based on the measured photon statistics, it does not require calibration. We investigated the minimal requirements of CoPS on fluorophore brightness and photostability and determined the minimum measurement time both in simulations and experiments. This provides a practical guideline for a choice of suitable dyes. We show that CoPS is feasible with many fluorophores in a wide range of applications. In the biological context, we investigated the label number distribution of several commonly used fluorescent markers, e.g., SNAP-tag and nanobodies . As well, we studied the behavior of conjugated polymers, materials important for organic semiconductor devices like polymer solar cells. Time-resolved CoPS measurements revealed heterogeneous dynamics in polymer chain photophysics.
 H. Ta, J. Wolfrum, and D.-P. Herten, Laser Physics, 20, 119–124 (2010)
 H. Ta, A. Kiel, M. Wahl, and D.-P. Herten, Physical chemistry chemical physics : PCCP, 12, 10295–10300 (2010)
 K. S. Grußmayer, A. Kurz, D.-P. Herten, ChemPhysChem, 15, 734 –742 (2014)
|12:00 - 12:20|
Alexander Wolf, Berlin, Germany (Student Award)
Diffusion Analysis of NAnoscopic Ensembles (DANAE): A tracking-free assessment for spectrally resolved diffusive modes of densely and inhomogeneously distributed particles
|12:20 - 12:40|
Malte Wachsmuth, Heidelberg, Germany
Large-scale FCS to assess molecular diffusion and chromatin interaction in vivo
|12:40 - 12:50||GROUP PICTURE|
|12:50 - 14:00||LUNCH BREAK|
|Session: Biological applications 1Chair: Felix Ritort|
|14:00 - 14:30|
Cruising inside X
RIKEN Brain Science Institute , RIKEN Center for Advanced Photonics
The behavior of biochemical molecules moving around in cells makes me think of a school of whales wandering in the ocean, captured by the Argus system on the artificial satellite. When bringing a whale back into the sea --- with a transmitter on its dorsal fin, every staff member hopes that it will return safely to a school of its species. A transmitter is now minute in size, but it was not this way before. There used to be some concern that a whale fitted with a transmitter could be given the cold shoulder and thus ostracized by other whales for “wearing something annoying.” How is whale’s wandering related to the tide or a shoal of small fish? What kind of interaction is there among different species of whales? We human beings have attempted to fully understand this fellow creature in the sea both during and since the age of whale fishing.
In a live cell imaging experiment, a fluorescent probe replaces a transmitter. We label a fluorescent probe on a specific region of a biological molecule and bring it back into a cell. We can then visualize how the biological molecule behaves in response to external stimulation. Since fluorescence is a physical phenomenon, we can extract various kinds of information by making full use of its characteristics. For example, the excited energy of a fluorescent molecule donor transfers to an acceptor relative to the distance and orientation between the two fluorophores. This phenomenon can be used to identify interaction between biological molecules or structural change in biological molecules. Besides, we can apply all other characteristics of fluorescence, such as polarization, quenching, photobleaching, photoconversion, and photochromism, in experimentation.
Cruising inside cells in a supermicro corps, gliding down in a microtubule like a roller coaster, pushing our ways through a jungle of chromatin while hoisting a flag of nuclear localization signal --- we are reminded to retain a playful and adventurous perspective at all times. What matters is mobilizing all capabilities of science and giving full play to our imagination. We believe that such serendipitous findings can arise out of such a sportive mind, a frame of mind that prevails when enjoying whale-watching.
|14:30 - 14:50|
Mobility and spatial distribution of transfer RNA (tRNA) in live bacteria using single-molecule tracking
Anne Plochowietz1, Ian Farrell2,3, Zeev Smilansky2, Achillefs Kapanidis1
1Clarendon Laboratory, Department of Physics, Parks Road, OX1 3PU, Oxford, UK.
Transfer RNA (tRNA) is the stable RNA species linking messenger-RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite its importance in translation, little is known about its diffusion and spatial distribution in live cells.
Here, we internalized bulk tRNA labeled with Cy3, Cy5, and Cy5.5 into live E. coli using electroporation#,† and performed single-molecule fluorescence imaging. We controlled the number of internalized tRNA molecules per cell and tracked individual molecules for seconds. We observed two diffusive species: a mobile species (assigned to free tRNA) and a slow-diffusing species (assigned to tRNA bound to active ribosomal complexes). From our single-molecule tracking and Monte Carlo simulations, we obtained an accurate diffusion coefficient of (8.1±1.0) μm2/s for bulk tRNA-Cy5. We also studied the spatial distribution of mobile and bound tRNA in a unit cell. Interestingly, we observed a non-uniform distribution with 45% more bound tRNAs at the cell periphery and 11% more mobile tRNAs along mid-cell. This uneven distribution disappeared upon chloramphenicol treatment (which blocks translation) and matched distributions for non-specific RNA and fixed cell controls further supporting the presence of bound tRNAs within active ribosomes. This is the first comprehensive study of tRNA mobility and spatial organization in living bacteria.
#Crawford*, Torella*, Aigrain*, Plochowietz*, Gryte, Uphoff, Kapanidis, Biophys J, 105, 2439–2450 (2013). *equal contribution
†Plochowietz, Crawford, Kapanidis, Phys Chem Chem Phys, 16, 12688-12694 (2014).
|14:50 - 15:10|
Characterisation of the monomeric state of amyloid beta 42 with fluorescence-based techniques
Mario Schneider1, Stefan Walta2, Walter Richtering2, Dieter Willbold1,3
1Institut für Physikalische Biologie, Heinrich-Heine Universität Düsseldorf
Amyloid beta 42 (Aβ42) is believed to play a central role in the progression of Alzheimer's disease . It was found to be prone to aggregation in a concentration-dependent manner down to low micromolar concentrations in vitro . However, in vivo Aβ42 usually occurs in nanomolar concentrations . It is an important component of amyloid plaques found in the brains of Alzheimer's disease patients. We wanted to characterize monomeric Aβ42, under near-physiological conditions at nanomolar concentrations. Therefore we applied fluorescence based techniques like fluorescence correlation spectroscopy (FCS), photon counting histogram (PCH) analysis, as well as fluorescence lifetime based techniques (TCSPC) to Alexa Fluor 488-labeled Aβ42. FCS revealed a two-state unfolding pattern of the monomer in varying concentrations of the denaturant guanidinium hydrochloride indicating that secondary structural motifs (α-helix and/or β-sheet/β-turn) are present. The free energy of the unfolding process was low, but existing, which corresponds to a structure of the Aβ42 monomer, which is distinct from a completely disordered or unfolded state.
 Finder VH, Glockshuber R, Amyloid-beta aggregation, Neurodegenerative Diseases, 4(1), 13-27 (2007)
 Ronald B.DeMattos, Kelly R.Bales, Maia Parsadanian, Mark A.O’Dell, Eric M.Foss, Steven M.Paul and David M.Holtzman, Plaque-associated disruption of CSF and plasma amyloid-b (Ab) equilibrium in a mouse model of Alzheimer’s disease, Journal of Neurochemistry, 81, 229–236 (2002)
 Suman Nag, Bidyut Sarkar, Arkarup Bandyopadhyay, Bankanidhi Sahoo, Varun K. A. Sreenivasan, Mamata Kombrabail, Chandrakesan Muralidharan, and Sudipta Maiti, Nature of the Amyloid-β Monomer and the Monomer-Oligomer Equilibrium, Journal of Biological Chemistry, 286, 13827-13833 (2011)
|15:10 - 15:30|
Pulling it all Together: Single Molecule FRET Reports on Accurate Structure and Dynamics in Bacterial Transcription
David L.V. Bauer1, Diego Duchi1, Laurent Fernandez1, Geraint Evans1, Nicole Robb1, Ling Chin Hwang1, Kristofer Gryte1, Alexandra Tomescu1, Pawel Zawadski1, Zakia Morichaud2, Konstantin Brodolin2, Achillefs N. Kapanidis1
1Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, OX1 3PU, UK
Single Molecule FRET is valuable tool for studying the dynamics of biological processes , and advances in molecular modelling [2,3] can now provide precise information about the structure of biological complexes. Here, we present recent work that applies these smFRET techniques to study the early stages of bacterial transcription in which DNA is transcribed into RNA, resulting in a FRET change as a DNA template is transcribed . The mechanism of initial transcription is unclear, mainly due to experimental challenges arising from the presence of transient intermediates and molecular heterogeneity. Our work  has revealed that RNA polymerases pauses after producing a 6-mer RNA, and that the pause can serve as a regulatory checkpoint. We identify a structural region of the polymerase, sigma 3.2, which contains a loop blocking the RNA-exit channel, as a major pausing determinant, blocking RNA extension and stabilizing growing RNAs. We have used the information gained to develop a new working model for initial transcription. In addition to the insight we have gained into the dynamics and structure of transient intermediates, this project is an excellent case study in how single-molecule techniques can inform the design of classical biochemical experiments – and vice versa.
 Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).
 Muschielok, A. et al. A nano-positioning system for macromolecular structural analysis. Nat. Methods 5, 965–971 (2008).
 Kalinin, S. et al. A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Methods 9, 1218–1225 (2012).
 Kapanidis, A. N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147 (2006).
 Duchi, D. et al. RNA Polymerase Pausing During Initial Transcription. Submitted.
|15:30 - 15:50|
Multivalency and local competition of heterochromatin protein 1 governs dynamic protein turnover in stable heterochromatin domains
Sinan Kilic, Andreas Bachmann, Louise Bryan, Beat Fierz
Laboratory of Biophysical Chemistry of Macromolecules, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
Fidelity of chromatin silencing in eukaryotes is essential in preventing aberrant gene expression. Heterochromatin protein 1 (HP1) is a hallmark of silenced heterochromatin. The post-translational modification (PTM) of histone H3 trimethylated on lysine 9 (H3K9me3) recruits HP1 to chromatin. Recognition occurs through a chromo-domain with micromolar affinity. Further, a C-terminal chromo-shadow domain allows for homodimerization and provides an interaction site for auxiliary factors. Despite forming macroscopically stable foci, HP1 exhibits fast dynamics.
Here, we studied the mechanisms governing dynamic, yet efficient recruitment of HP1 to heterochromatin. We reconstituted H3K9me3 chromatin arrays, which were immobilized in a flow cell. Co-localization single-molecule total internal reflection fluorescence microscopy allowed us to obtain binding and dissociation kinetics of fluorescently labeled HP1 proteins with chromatin arrays of defined modification state. Using this approach we systematically explored how H3K9me3 density as well as the multimeric state and concentration of the effector affected binding kinetics to modified chromatin.
Our results show that HP1 is efficiently recruited to heterochromatin through re-association at proximal H3K9me3 sites. Facilitated dissociation maintains the dynamic turnover of the protein. Multivalent PTM binding results in accelerated and prolonged association of HP1 and allows for a combined 10-fold increase of affinity towards densely modified chromatin.
|15:50 - 16:10||COFFEE BREAK|
|16:10 - 18:40||POSTER SESSION|
|20:00 - 23:00||DINNER|
|Session: Super-resolution 1Chair: Katharina Gaus|
|9:00 - 09:35|
Seeing is believing
EPFL STI IMT LOB, BM 5.143 (Bâtiment BM), Station 17, CH-1015 Lausanne
This talk invites for looking into cell and subcellular organelles with a resolution below 100 nm (see image aside).
|09:35 - 09:55|
Investigating the active role of mechanical force during T-cell activation by super-resolved traction force microscopy
Huw Colin-York, Marco Fritzsche, Christian Eggeling
The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS
It is becoming increasingly clear that mechanical force plays an active role in the adaptive immune response, specifically in the formation of the so called immunological synapse that arises between the T-cell and antigen presenting cell. Despite its importance, there is a lack of high resolution techniques capable of measuring the relevant forces during these cellular interactions. To this end, an improvement on the well-established technique of traction force microscopy, using super-resolution Stimulated Emission Depletion (STED) microscopy, is introduced as a method of measuring the relevant forces. The success of this novel technique is demonstrated by combining computer-simulations and biological model systems, namely focal adhesions. Ultimately, the development of a tunable 1G4 T-cell system is introduced as a method of probing force generation in a physiologically relevant system.
|09:55 - 10:15|
Jasper H. M. van der Velde, Groningen, Netherlands (Student Award)
Super-Resolution with Self-Healing Organic Fluorophores
|10:15 - 10:35|
Advanced Pulse Pattern Generation and Fine Tuning for STED Microscopy
Rhys Dowler, Marcelle König, Paja Reisch, Alexander Glatz, Sebastian Tannert, Thomas Schönau, Romano Härtel, Tino Röhlicke, Marcus Sackrow, 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 a well established method offering optical superresolution below 50 nm. Optimal optical resolution can be achieved through running both excitation and depletion lasers in picosecond pulsed mode as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection or a more detailed fluorescence decay analysis. Furthermore, these two methods allow for accurate separation of different fluorescent species.
We present here a new generation of our VisIR 765 ”STED” depletion laser, featuring a pulse length and beam shape optimized for STED microscopy. The temporal overlap between excitation and STED laser pulses can be adapted specifically to different fluorescence lifetimes thanks to our fully computer controlled multichannel delay generator SOM-D. The SOM-D allows for the easy introduction of electronic delays between laser channels with time resolutions below 50 ps.
Interleaved pulse patterns can also be realized with the SOM-D, allowing to cycle between STED and non-STED illumination in a measurement. Such interleaved excitation patterns are advantageous in the identification and elimination of unwanted STED induced processes on the nanosecond timescale. Examples from blinking and photobleaching in single molecule imaging as well as in fluorescence correlation spectroscopy (STED-FCS) will be given.
This extended STED functionality along with improved data throughput are the latest extensions to the confocal microscope platform MicroTime 200 and will soon be available as upgrade for existing systems.
|10:35 - 11:10||COFFEE BREAK|
|Session: Biological applications 2Chair: Ingmar Schoen|
|11:10 - 11:40|
Molecular insights into the regulation of T cell signalling
EMBL Australia Node in Single Molecule Science, and
The hallmark of adaptive immunity is antigen recognitions. To distinguish antigens from non-antigenic molecules (for example peptides derived from cancer cells versus peptides derived from the host), T lymphocytes are equipped with highly specific receptors, the so-called T cell antigen receptor (TCR). The mission of cytotoxic T lymphocytes (CTLs) is to recognise antigens from damaged cells such as cancer cells, or antigens from foreign microorganisms such as viruses, bacteria and fungi and then kill the cancer or infected cell. Thus, cytotoxic T lymphocytes need to translate a molecular binding event, antigen binding to the T cell receptor, into a deadly immune response.
We have established single molecule localization microscopy to determine how TCR engagement reorganizes signalling proteins on the molecular scale. 2-colour single molecule imaging allows us to distinguish signalling from non-signalling receptors in an activated T cell. Thus, we can create TCR triggering maps that show that i) antigens reorganise the TCR into clusters and only clustered TCR signal; ii) only a proportion of TCRs signal even with high antigen doses; iii) TCRs within a cluster are either all signalling or are all non-signalling; and iv) the density of TCR clusters determines the signalling efficient. Thus, single molecule localization microscopy data has revealed details about the TCR signalling mechanism that were not visible in ensemble measurements.
|11:40 - 12:00|
Motors, gears and controls of FoF1-ATP synthase monitored by single-molecule Förster resonance energy transfer
Michael Börsch, Thomas Heitkamp, Maria Dienerowitz, Ilka Starke, Nawid Zarrabi, Bertram Su
Single-Molecule Microscopy Group, Jena University Hospital, Nonnenplan 4, 07743 Jena
Catalytic activities of enzymes are associated with elastic conformational changes of the protein backbone. The Escherichia coli FoF1-ATP synthase consists of a membrane-bound Fo motor where proton translocation through Fo drives a 10-stepped rotary motion. An internal central stalk transduces the energy of this rotation to the F1 motor, where ATP is synthesized in an 120° rotary stepping cycle[2, 3]. To prevent wasteful hydrolysis of ATP, FoF1-ATP synthase utilizes different autoinhibitory mechanisms including mechanical blocking of subunit rotation. These conformational changes can be monitored in real time by single-molecule Förster resonance energy transfer (smFRET). The rotary mechanics of proton-driven FoF1-ATP synthase will be discussed and a smFRET approach to observe both rotations in a single FoF1-ATP synthase at work will be presented. The internal mechanical controls to prevent wasteful ATP hydrolysis by the enzyme can be monitored by smFRET as well.
 M. G. Düser, N. Zarrabi, D. J. Cipriano, S. Ernst, G. D. Glick, S. D. Dunn, M. Börsch, 36° step size of proton-driven c-ring rotation in FoF1-ATP synthase, EMBO J 28. 2689-2696 (2009).
|12:00 - 12:20|
A macromolecular crowding study of RNA folding and activity – polymer pore size matters!
Richard Börner1, Erica Fiorini1, Bishnu Paudel2, David Rueda2, Roland K.O. Sigel1
1Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Catalytic RNAs, like the group IIB intron ribozyme of S. cerevesiae, require a high magnesium(II) concentration to show folding and function in vitro . In contrast, in vivo conditions are characterized by a highly crowded cellular environment and much lower ion concentration. Molecular crowding agents are a widespread tool to mimic cellular crowding . However, particular physical/chemical properties explaining the crowders influence are mostly not understood. In this study, we gain new insights on how polymer properties like viscosity, pore size etc. influence the activity and folding of a large RNA.
We combined bulk activity assays and single-molecule Förster Resonance Energy Transfer experiments, screening the PEG volume fraction (%) and molecular weight (MW). Our results revealed that upon the influence of crowding agents, a compaction of the underlying structure depends on the PEG % and the presence of different PEG MW and % unveiled an optimal pore size in terms of catalytic activity. In summary, an increasing density of the crowding environment shifts the RNA towards the most compact state, but the ribozyme is only active if the crowders network matches its size . We interpret the most compact state as necessary, but not sufficient, to keep the ribozyme active.
Financial support from the European Research Council (MIRNA N° 259092, to RKOS), the Swiss National Fund (SNF), and the Forschungskredit Grant of the University of Zürich (FK-14-096 to RB) are gratefully acknowledged.
 Swisher J.F., Su L.J., Brenowitz M., Anderson V.E., Pyle A.M., J. Mol. Bio., 315, 297-310 (2002).
|12:20 - 12:40|
3D Real-Time Orbital tracking in zebrafish embryos: High spatiotemporal analysis of mitchondrial dynamics in neurons
Fabian Wehnekamp1, Gabriela Plucinska2, Rachel Thong2, Thomas Misgeld2, Don C. Lamb1
1Fablab, Butenandstraße 11, 81377 München
The main function of mitochondria is to provide cells with adenosintriphosphate (ATP) in regions with high-energy demand. A complex machinery of motor proteins and signaling molecules are responsible for the distribution and recycling of mitochondria in cells. A malfunction in the dynamics of these complexes is one possible reason for neurodegenerative diseases.
To follow the trajectory of individual mitochondria in rohon-beard sensory neurons, we use a home-built three-dimensional real-time orbital-tracking microscope with a spatial resolution of a few and an acquisition speed of up to 500 Hz. Environmental information is recorded simultaneously with a built-in widefield microscope.
By using photoactivation, we are able to track single mitochondria over distances of more than 100 µm. Due to our high spatial and temporal resolution, we can identify several different dynamic populations involved in mitochondrial transport. The environmental information gives insight into the interactions between stationary and moving mitochondria. Combining the results from the fast and precise tracking microscope with the widefield data we obtain an in vivo overview over the dynamic processes in rohon-beard sensory neurons which can be used to study the effects of neurodegenerative diseases.
|12:40 - 13:00|
Darkfield hyperspectral imaging of Au-NPs in A549 cells
Thomas Ruckelshausen, Lars Leibrock, Annette Kraegeloh
Nano Cell Interactions group, INM - Leibniz Institute for New Materials, Campus D2 2, 66123 Saarbrücken, Germany
Engineered nanomaterials (ENMs) are promising tools for various commercial applications including biomedical applications. In order to design safe ENMs, it is necessary to identify nanoparticle properties influencing the interactions between nanoparticles and cells. Gold nanoparticles (Au-NPs) are a versatile tool for bioimaging: In electron microscopy their high atomic number leads to great contrast and their plasmonic resonance can be used for biosensing. Other applications for Au-NPs are photodynamic therapy and drug delivery. Despite their wide range of applications possible size dependent toxicity and their cellular distribution are still under investigation.
A first step was the characterization of different sized AU-NPs (50nm, 75nm and 100nm) with darkfield microscopy combined with hyperspectral imaging. This technique has the advantage to enable imaging of unlabeled particles, which is important in the context of cytotoxicity testing. Increasing particle size leads to a shift of the mean maximum of the scatter spectrum towards longer wavelengths.
In a first in-situ study we exposed the AU-NPs to human lung epithelial cells (A549). The nanoparticles could be detected intracellularly.
In a correlative study with electron microscopy positions of Au-NP signals were compared and scatter spectra of single nanoparticles were analyzed in comparison to those of small nanoparticle agglomerates.
|13:00 - 13:10||STUDENT AWARD PRESENTATION|
|13:10 - 14:20||LUNCH BREAK|
|Session: Super-resolution 2Chair: Theo Lasser|
|14:20 - 14:50|
What superresolution microscopy can teach us about biomolecular structures in fixed cell cultures
Laboratory of Applied Mechanobiology, Department of Health Sciences and Technology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland
A major challenge in cell biology is to obtain quantitative information about the number, conformation, and relative organization of proteins in larger complexes. Molecular specificity and sub-molecular resolution are key requirements for the successful elucidation of these structures, preferentially in their native environment. Here we show how site-specific labeling strategies together with localization-based single-molecule fluorescence microscopy techniques achieved this formidable task in the case of fibronectin fibrils in the extracellular matrix of fixed cells . Taking advantage of the complementary strengths of bioconjugate chemistry  and immunolabeling, as well as of stepwise photobleaching  and direct STORM, the extension of single fibronectin molecules in fibrils and their periodic arrangement along fibrils were retrieved. The consistent results yielded novel insights into intermolecular interactions and the hierarchical fibril organization. Whereas these findings have direct implications for the mechanical and functional properties of fibronectin fibrils, the presented methodologies offer diverse options to investigate also other proteins, protein fibrils, or structural complexes.
 S. M. Früh*, I. Schoen*, J. Ries, and V. Vogel, Nature Communications, 6, 7275 (2015).
 S. M. Früh, P. R. Spycher, M. Mitsi, M. A. Burkhardt, V. Vogel, and I. Schoen. ChemBioChem, 15, 1481 (2014).
 I. Schoen, Biophysical Journal, 107, 2122 (2014).
|14:50 - 15:10|
Quantitative Single-Molecule Localization Microscopy
Sebastian van de Linde1, Romain F. Laine2, Christian Franke1, Anna Löschberger1, Nadine Ehmann3, Robert Kittel3, Clemens F. Kaminski2, Markus Sauer1
1 Department of Biotechnology & Biophysics, Am Hubland / Biozentrum, University of Würzburg, Germany
Single-molecule localization microscopy does not only allow for studying cellular structures at high spatial resolution but also provides quantitative information. To demonstrate quantitative molecular labeling and localization of cellular proteins the technology can be combined with scanning electron microscopy . Further, computational methods such as particle averaging, which is originally applied in EM, can be used to generate accumulated super-resolved images from single-molecule coordinates . Principles and applications to the nuclear pore complex, envelope proteins in Herpes simplex virus type-1 (HSV-1), and the presynaptic protein Bruchpilot in Drosophila  will be described.
 Löschberger A. et al., J. Cell Sci., 127:4351 (2014)
 Laine R.F. et al., Nat. Commun., 6:5980 (2015)
 Ehmann E. et al., Nat. Commun., 5:4650 (2014)
|15:10 - 15:30|
Filling the usability gap: Bioinformatics solutions for Image-Scanning Microscopy, Stochastic Optical Fluctuation Imaging, and Surface Single Molecule Experiments
Dirk Hähnel, Narain Karedla, Anna Chizhik, Alexey Chizhik, Simon Christoph Stein, Anja Huss, Sebastian Isbaner, Qui Van, Ingo Gregor, Jörg Enderlein
III. Institute of Physics – Biophysics, Georg-August-University Göttingen, Friedrich-Hund-Platz 1, D-37077 Göttingen, Germany
Recent years have seen a tremendous increase of new and novel methods in the field of superresolution fluorescence microscopy. Furthermore even better methods for increasing axial resolution of fluorescence imaging have been introduced by our group very recently. Our group has developed powerful methods: Confocal Spinning Disc Image-Scanning Microscopy (CSDISM)(1)(2), Superresolution Optical Fluctuation Imaging (SOFI)(3)(4)(5) , and Metal Induced Energy Transfer (MIET) (2)(8). However, new microscopy techniques that provide not only enhanced image quality and resolution, but they are also simple enough for finding broad application. To bridge the ultimate usability gap for end-users, we present simple soft- and hardware solutions for CSDISM and SOFI which enable potential users to implement them in an easy and straightforward way into their existing microscopy systems. In the case of CSDISM, we have integrated the method into the environment of the widely used and popular MicroManager Open Source Imaging platform. This allows any researcher who already has a commercial Confocal Spinning Disk microscope to easily implement the image-scanning option and thus to double the spatial resolution. For SOFI, we have developed a dedicated hardware based on a Freely Programmable Gate Array (FPGA) which converts, in real time, image movies taken by high-speed CCD systems into SOFI cumulant images. Thus, all algorithmic complexities and numerical workload of SOFI calculations are taken care of. Furthermore we will present our recently developed software tool for smart automated single molecule on surface experiments termed (SIMA). This is an effective tool to save time and enables the researcher to conduct complex measurements. SIMA increases the comparability of single molecule measurements, and reduces bleaching to the absolute possible minimum.
 Müller and Enderlein, “Image Scanning Microscopy”;
 Schulz, Pieper, and Clever, “Resolution Doubling in Fluorescence Microscopy with Confocal Spinning-Disk Image Scanning Microscopy”;
 Dertinger et al., “Achieving Increased Resolution and More Pixels with Superresolution Optical Fluctuation Imaging (SOFI)”;
 Dertinger et al., “SOFI-Based 3D Superresolution Sectioning with a Widefield Microscope”;
 Dertinger et al., “Advances in Superresolution Optical Fluctuation Imaging (SOFI).”; Dertinger et al., “Fluctuation Imaging ( SOFI )”
 Geissbuehler et.al., “Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging”;
 Chizhik et.al. “Metal-induced energy transfer for live cell nanoscopy”;
 Karedla et.al. “Single-Molecule Metal-Induced Energy Transfer (smMIET): Resolving Nanometer Distances at the Single-Molecule Level”;
|15:30 - 15:50|
Single Molecule Blinking and Localization Based Super Resolution Imaging using Carbon Dots
Syamantak Khan, Navneet Chandra Verma, Abhishek Gupta, Chayan Kanti Nandi
School of Basic Sciences, Indian Institute of Technology Mandi, Mandi, Himachal Pradesh, India, 175001
The application of super resolution microscopy is limited by the availability of fewer numbers of reversible photoswitchable fluorophores. Here, we demonstrated the reversible photoswitching and long lived blinking state of the red emission of the multicolor carbon dots. A mechanism of electron transfer is proposed. The cationic dark state formed by the exposure of red light is revived back to the bright state with the very short exposure of blue light. Additionally, the natural on-off state of carbon dot fluorescence was tuned using an electron acceptor. The localization based Stochastic Optical Reconstruction Microscopy (STORM) of E. coli bacterial cell has been imaged, which shows the resolution down to approximately 40 nm. Our observation can make the carbon dots as an excellent candidate for the super-resolution imaging of nanoscale biomolecules within the cell.
Syamantak Khan, Navneet Chandra Verma, Abhishek Gupta and Chayan Kanti Nandi Nat. Sci. Rep. (2015, In press)
|15:50 - 16:00||CONCLUDING REMARKS|
|16:00 -||END OF WORKSHOP|
Super-resolution fluoresence imaging of nanoscale archeticture of HSC homing and migration molecules.
Karmen AbuZineh, Bader Alwan, Fajr Aleisa, Kosuke Sakashita, Samir Hamdan, Jasmeen Merzban, Satoshi Habuchi
Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia
Selectins (E-, P- and L-) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this study, using the hematopoietic stem cell (HSC) as a working model, we applied super-resolution fluorescence imaging techniques to visualize PSGL-1, a well-defined E-selectin ligand on HSC-model cell line, KG1a. Here we present information about the nanoscale architecture and spatial distribution of the PSLG-1 on the surface of the KG1a cells. The data revealed a unique clustering behavior of PSGL-1 with a cluster size of approximately 100 nm. We aim to visualize and determine how such cluster formation is influenced by the interaction of E-selectin on endothelial cells with PSGL-1 during the cell migration process. We anticipate that this will lead to a more comprehensive molecular level understanding of the nanoscale architecture of the ligands (PSGL-1) on the surface of HSC and how this architecture may change in response to binding their receptors (E-selectin). Our results will likely help suggest a more detailed mechanism through which selectins mediate binding to their ligands in the control of cell migration.
Singlet oxygen detection with spatial resolution by NIR phosphorescence with Microtime 200
A highly sensitive FLIM based assay for the detection of reactive oxygen species
Detection of coordinated motions in proteins by three-color FRET
Anders Barth, Lena Voith von Voithenberg, Don C. Lamb
Butenandtstr. 5-13, 81377 München
Single-pair FRET (spFRET) has become a standard tool to monitor conformational changes of biomolecules in vitro. In solution-based burst analysis, the burst of photons emanating from molecules diffusing through the confocal volume gives access to the underlying distribution of the FRET efficiency and other parameters of interest. A promising approach to address the coordination of motion within complex biological systems is multicolor FRET, allowing multiple distances to be monitored simultaneously. While several three- or four-color studies have already been performed, quantitative analysis of multicolor single molecule data is still limited to the extraction of average values due to the complexity of the analysis, losing additional information encoded in the distribution of FRET efficiencies. For spFRET, a rigorous statistical description of photon emission (Photon Distribution Analysis, PDA ) allows to distinguish shot-noise broadening of the FRET efficiency histogram from physically relevant heterogeneity of the sample, e.g. due to the existence of conformational substates.
We show the extension of PDA to three-color FRET systems. Based on simulated and experimental data, we demonstrate that the underlying probability distributions of the three interdye distances can be extracted to high accuracy, and that information about the coordination of intramolecular movements can be extracted.
 Antonik, M., Felekyan, S., Gaiduk, A., & Seidel, C. A. M., J. Phys. Chem. B, 110, 6970–6978 (2006).
Metal-induced energy transfer for novel applications in nano-spectroscopy
Alexey Chizhik1, Daja Ruhlandt1, Narain Karedla1, Anna Chizhik1, Ingo Gregor1, Jan Rother2, Andreas Janshoff2, Jörg Enderlein1
1III. Institute of Physics, Georg August University, 37077 Göttingen, Germany
Placing a chromophore in close proximity to the metal surface modifies its fluorescence lifetime, which is known as the Purcell effect. The phenomenon is based on the energy transfer from the excited emitter into plasmons of the metal. We present new applications of the fluorophore-metal interaction in nanoscopy.
Our new nanocavity-based method of measuring the fluorescence quantum yield requires only few microliters of low-concentrated solution , and is applicable even to single emitters . It allows for measuring the quantum yield of fluorophores placed inside complex systems, such as a mixture of several types of emitters with strongly overlapping absorption spectra  or even with complete overlap of both emission and absorption spectra, which is impossible to do by any other existing technique.
Also, we introduce a new technique for axial localization of fluorophores with nanometer accuracy using a metal-induced energy transfer . We demonstrate the power of this method by profiling the basal lipid membrane of living cells. The simplicity and high accuracy of the presented techniques allow for using them in a big variety of fluorescence studies.
 Chizhik, et al. Phys. Rev. Lett., 108, 163002 (2012).
 Chizhik, et al. Nano Lett., 11, 1700 (2011).
 Chizhik, et al. Nano Lett., 13, 1348 (2013).
 Chizhik, et al. Nature Photon., 8, 124 (2014).
STED Add-on for a Standard Time-resolved Confocal Microscope
Rhys Dowler, Marcelle König, Benedikt Krämer, Sebastian Tannert, Matthias Patting, Felix Koberling, Rainer Erdmann
PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany, email@example.com
Superresolution microscopy is about to evolve into a standard tool for biological research. Overcoming the diffraction limit for fluorescence imaging has been shown to be crucial for addressing various relevant biological questions. Here, we show how superresolution, namely Stimulated Emission Depletion (STED), can be easily added to a standard confocal time-resolved fluorescence microscope, the MicroTime 200.
An EASYDOnut phaseplate converts the STED laser beam into the required donut-shaped focal spot while leaving the excitation beam unaffected . An alignment of the STED donut in respect to the excitation spot is not necessary since both, STED and excitation beam are delivered by the same optical single mode fiber. A resolution below 50 nm FWHM is achieved.
Externally triggered pulsed lasers and confocal detection with time-correlated single photon counting (TCSPC) allow this system to take advantage of various excitation schemes like bunched excitation, pulsed interleaved excitation (PIE) and detection modalities such as gated STED. This inherent flexibility makes it easy to modify experiments to address highly specific biological questions. Combinations of STED with other time-resolved techniques such as FLIM (Fluorescence Lifetime Imaging Microscopy) or FLCS (Fluorescence Lifetime Correlation Spectroscopy) as well as investigations down to the single molecule level are feasible.
Multi-label STED imaging is possible using one STED depletion wavelength. The fluorophores are excited by nearby excitation wavelengths in PIE mode. Using this technique highly accurate co-localization measurements are possible. Small differences in absorption and emission spectra as well as in the fluorescence lifetimes can be utilized to create fluorescence patterns which act like a fingerprint. The labels are distinguished by applying fluorescence Pattern Matching analysis which takes into account the full spectral and temporal information. The principle will be shown on double labeled biological cells.
 M. Reuss et. al. """ Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation""", Opt. Exp. 18, 1049-1058 (2010)
Studying the Hepatitis C Virus Replication Machinery by Single-Molecule Fluorescence Methods
Yasser Gidi1, Anupriya Kulkarni2, Anaïs Robert1, Matthias Götte3, Gonzalo Cosa1
1Department of Chemistry, McGill University
The Hepatitis C virus (HCV) is recognised as a major human pathogen. According to the World Health Organization, 150 million people worldwide are currently affected, with over 240,000 of them living in Canada. Recent progress in the development of drugs that directly target key enzymes, such as the polymerase and protease of HCV may offer promising therapies for the disease. However, fast turnover in virus production and error-prone viral replication machinery often leads to the emergence of mutants that are resistant to all known drug therapies. In order to develop new, more efficient antiviral drugs, we need to better understand the mechanism of the viral replication process including the workings of the key viral enzymes involved such as NS5B polymerase.
Single-molecule fluorescence techniques are ideally positioned to approach these problems as they allow for real-time monitoring of dynamic complexes of viral enzymes and nucleic acids. Here we describe state-of-the-art single molecule fluorescence studies on the binding of HCV NS5B to its RNA substrate and on the ensuing dynamics of the enzyme-oligonucleotide complex. A single-molecule Protein Induced Fluorescence Enhancement PIFE assay was developed and successfully applied to study the binding and sliding dynamics of NS5B polymerase on the RNA/DNA template. The approach does not require enzyme labelling or dual labelling of the substrate, which is an advantage in terms of monitoring enzyme mutants in high throughput assays. In the long term, we propose that our single-molecule platform will be used to gain new insights into the workings of this viral enzyme as well as to study it in the presence of inhibitors and potential antiviral drugs.
Structural changes in conjugated polymers close to substrate surfaces probed by single molecule spectroscopy
Francineide L. Araújo, Gustavo T. Valente, Roberto M. Faria, Francisco E. G. Guimaraes
Universidade de São Paulo, Instituto de Física de São Carlos, SP, Brazil
Optical and structural properties of ultrathin films and isolated molecules of poly (9,9 dioctylfluorene) (PFO) are investigated in this work. Ultrathin films (<10 nm) and isolated molecules were deposited by spin-coating on top of quartz substrates. We employed confocal fluorescence microscopy equipped with spectral and time-lapse fluorescence imaging to characterize samples. We observed a gradual planarization (β phase) of the fluorine repeat units with the decrease of PFO film thickness in the range between 5 nm and 0,7 nm at room temperature, even though the β phase is not expected when chloroform is used as solvent. Low temperature (5 K) luminescence of these ultra thin films reveals a well resolved zero-phonon emission band between the energies of the β phase and the amorphous phase that was associated to perturbations of the planar structure by primary chain conformations. However, the emission of these intermediated states are not observed for film thicknesses higher than 5 nm, which suggest that they behave as light-harvesting states responsible for guiding the excited state from higher energy non planar to lower energy planar phase states via energy transfer process. Single PFO molecules have frozen β phase structure at room temperature on the quartz substrate.
Super-Resolution and Single-Color Multiplexing Microscopy by chemically switchable Fluorescent Probes
Andreas Haderspeck, Dominik Brox, Dirk-Peter Herten
Institute for Physical Chemistry, Heidelberg University, Germany
Single-molecule based super-resolution microscopy is meanwhile well established for investigating cellular structures below the Abbe limit. Current methods make use of light-driven on/off-switching to enable imaging and subsequent localization of individual fluorophores with nanometer accuracy. Previously, we reported that switching between bright and dark states can also be achieved by chemical reactions, e.g. reversible coordination of metal ions to a ligand . The stochastic nature of the association/dissociation process can be used in localization microscopy to decouple switching from excitation, making additional laser irradiation obsolete and highly reducing phototoxicity . More recently, we could show that the same principles can be employed to establish a novel mode of multiplexing in fluorescence microscopy . Therefore, we currently work on modular systems allowing facile combination of different dye and receptor moieties to establish fluorescent probes that can be switched by chemical reactions. Here, we present new fluorescent probes which we recently synthesized and characterized in respect of their photophysical properties in bulk solution as well as on a single molecule level. We furthermore demonstrate their potential as fluorescent probe in localization microscopy imaging and their application in chemical multiplexing.
 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).
Millisecond Dynamics of Membrane-Protein Folding from Single-Molecule FRET Spectroscopy
Andreas Hartmann1, Georg Krainer1,2, Michael Schlierf1
1B CUBE-Center for Molecular Bioengineering, Technische Universität Dresden, Arnoldstr. 18, 01307 Dresden, Germany
We present single-molecule Förster resonance energy transfer (smFRET) confocal spectroscopy as a powerful tool in kinetic studies of membrane-protein folding. To this end, we performed urea-induced folding and unfolding experiments on the α‑helical membrane protein Mistic in the presence of the zwitterionic detergent n‑dodecylphosphocholine (DPC) to probe Mistic’s conformational switching while folding into and out of detergent micelles. Two-state millisecond interconversion dynamics between folded and unfolded states were observed during diffusion of the protein through the confocal detection volume. We demonstrate how to identify and quantify such dynamic processes using a set of qualitative, semi-quantitative, and quantitative analysis tools. Extracted rate constants were compared and tested against simulations. The results demonstrate that smFRET is an excellent method for probing the chemical physics of membrane-protein structure and dynamics in the complex and anisotropic environment of a hydrophilic/hydrophobic interface, providing insights into protein subpopulations and their unsynchronized interconversion dynamics.
Direct observation of the first steps in gelsolin-mediated actin filament nucleation
Alvaro Crevenna, Maria Hoyer, Don Lamb
Butenandtstr.11, 81377 München, Germany
Actin filament elongation has been extensively studied in bulk assays. The direct observation of the nucleation process on the single molecule level, however, has not been possible due to the relatively large concentrations needed for actin filament formation. Zero-mode waveguides provide a very small observation volume, which allows the measurement of concentrations up to the micromolar range. By using zero-mode waveguides, we directly observe the binding of individual fluorescently labeled actin monomers during filament formation.
Here, we present measurements of actin filament nucleation mediated by gelsolin, a barbed-end binding protein. Our data reveal the existence of kinetic intermediates during the binding of the first two monomers to gelsolin and a concentration-dependent transition to grow beyond 5 monomers. Blocking the conformational transition associated with filament formation slows down nucleation and stops elongation. Actin filament nucleation requires flattening of the actin monomer, which facilitates association and allows further elongation.
Dissecting the Stimulation of FEN1 Activity by PCNA on Flap Substrates at the Single Molecule Level
Luay Joudeh, Fahad Rashid, Samir Hamdan
Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia
Human proliferating cell nuclear antigen (PCNA) encircles double-stranded DNA and acts as a scaffold to recruit and coordinate the activities of a large number of DNA processing proteins involved in replication, repair, recombination and chromatin remodeling. In this study, we are focusing on characterizing the mechanism by which PCNA recruits and stimulates the structure specific flap endonuclease 1 (FEN1) to process the aberrant double flap (DF) structures that are produced during maturation of Okazaki fragment on the lagging strand and in long patch base excision repair. FEN1 distorts the DF structures into a bent conformer to place the scissile phosphate into the active site for cleavage. The product is a nick substrate that can be sealed by the DNA ligase I whose recruitment is also mediated by its interaction with PCNA. Here, I will present single-molecule Förster resonance energy transfer measurements that simultaneously monitored bending and cleavage of various DF substrates by FEN1 alone or in the presence of PCNA. This enabled us to dissect the effect of PCNA on the structure of the bent DNA conformer, rate of DNA bending and unbending and single turnover catalytic rates of FEN1.
High efficiency FRET sensor for monitoring caspase-3 activity in live cells
Maria Khrenova1,2, Alexander Goryashchenko2, Alexander Savitsky1,2
1Lomonosov Moscow State University,
Förster resonance energy transfer (FRET) is a powerful tool to investigate biochemical and biophysical processes in vitro and in vivo. FRET biosensors change their fluorescence lifetime when interacting with the target molecules. Sensors on caspase typically have specifically recognized motif in the linker connecting donor and acceptor that is cleaved by the enzyme thus leading to the loss of FRET. The latter results in the changes of the fluorescence lifetime distribution that can be detected precisely applying single molecule detection approach or measuring properties of ensemble. Lifetime distribution depends on conformational diversity of fuse protein: wide distribution lower the accuracy of sensors, therefore it is of great importance to formulate the ways of improvement of this property. As an example we take FRET pair composed of the fluorescent protein TagRFP as energy donor and the chromoprotein KFP as energy acceptor. Starting with molecular dynamics simulations we designed linker that tends to bend forming two antiparallel β-sheets bound together with the caspase-3 recognition DEVD motif being on the turning point of the linker. Thus constructed FRET sensor is characterized by two exponential fluorescence decay with the comparable intensities and lifetime’s ratio more than two.
Single-molecule FRET reveals conformational heterogeneity in the transcription initiation complex
Sarah Schulz1, Katharina Kraatz1, Finn Werner2, Philip Tinnefeld1, Dina Grohmann3
1Physikalische und Theoretische Chemie, NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, 38106 Braunschweig, Germany
In all living organisms transcription is the first step in gene expression. Here, multisubunit RNA polymerases (RNAP) utilize genomic DNA as a template for RNA synthesis. Transcription can be understood as a cyclic process that can be divided into three steps: Initiation, elongation and termination . The transition from one step to the next demands the disruption and constitution of inter- and intramolecular interactions. Therefore, transcription is an inherently dynamic process that cannot be sufficiently described by X-ray crystallography or NMR. In this context, single-molecule experiments represent a powerful approach to understand the dynamic nature of the transcriptional machinery. Here, we exploited the fully recombinant and fluorescently labelled archaeal transcription system  to investigate the initiation phase of transcription. During initiation the RNAP, promoter DNA and transcription factors TBP, TFB and TFE form the pre-initiation complex (PIC). In order for transcription to commence, the promoter DNA is melted and the transcription bubble is formed. TFE is a transcription initiation factor that stabilizes the PIC, stimulates the catalytic activity of the RNAP and interacts with the RNAP clamp domain . The clamp represents the main flexible element in RNAPs from all three domains of life and it has been speculated that TFE modulates the position of the clamp. In order to monitor the conformational status of the RNAP clamp in the PIC in the presence and absence of TFE, we site-specifically incorporated a donor and acceptor fluorophore into the RNAP via unnatural amino acids followed by biorthogonal labeling. Employing single-molecule Förster resonance energy transfer experiments using a two-color prism-TIRF-microscope with alternating laser excitation, we found that the RNAP clamp adopts two distinct conformations in the PIC. We furthermore show that the distribution between a more open and closed status of the clamp is dependent on the melting status of the promoter DNA. Interestingly, TFE shifts the equilibrium towards the open clamp state most likely by stabilizing the non-template strand upstream end of the transcription bubble .
 Werner F. and Grohmann D. Nature Reviews Microbiology, 9:85-98 (2011).
 Schulz S., Kramm K., Werner F., Grohmann D. Methods (epub ahead of print), (2015).
 Grohmann D., Nagy J., Chakraborty A., Klose D., Fielden D., Ebright R.H., Michaelis J., Werner F. Molecular Cell, 43:263-74 (2011).
 Nagy J., Grohmann D., Cheung A.C.M., Schulz S., Smollett K., Werner F. & Michaelis J. Nature Communications, 6:6161 (2015).
Towards optical gating with single molecules
Johannes Maier1, Tina Weller2, Mukundan Thelakkat2, Martti Pärs1, Jürgen Köhler1
1Experimental Physics IV and Bayreuth Institute of Macromolecular Research (BIMF), 95440 Bayreuth, Germany
Photochromic molecules can be interconverted between two metastable states by light [1,2]. We synthesized a triad consisting of a photochromic unit (Dithienylperfluorocyclopentene, DCP) and two highly fluorescent chromophores (Perylene Bisimide, PBI) and monitored the PBI emission intensity as a function of the state of the switchable unit.
In order to exploit these effects on individual triads for optical gating, we worked at cryogenic temperatures, which ensures the requested photostability of the triads.
Therefore we have developed solid immersion lens (SIL) optics that enhanced the light collection efficiency by a factor of 1.8 with respect to a situation without SIL. 
In a first proof-of-principle experiment this allows us to detect the changes of the fluorescence intensity of individual triads with a contrast ratio of more than 80%.
 Pärs, M., Hofmann, C.C., Willinger, K., Bauer, P., Thelakkat, M., Köhler, J., Angew. Chem. Int. Ed., 50, 11405-11408 (2011).
 Pärs, M., Gräf, K., Bauer, P., Thelakkat, M., Köhler, J., Appl. Phys. Lett., 103, 221115 (2013).
 Jasny, J., Sepiol, J., Irngartinger, T., Traber, M., Renn, A., Wild, U.P., Rev. Sci. Instrum., 67, 1425-1430 (1996).
Register of Viral Capsid Proteins Oligomerisation by Single Molecule Fluorescence
M.A. Martínez-Partida, E. Reynaga-Hernández, A.M. Longoria-Hernández, N. LeijaMartínez, E. Gómez-García, J. Ruiz-García
Laboratory of Biological Physics, Physics Institute, Universidad Autónoma de San Luis Potosí, Av. Dr. Manuel Nava No. 64, San Luis Potosí, S.L.P. 78000 México firstname.lastname@example.org
In this work, we studied real-time viral capsid formation around its genome by single molecule fluorescence. First, we have focused on determining the size of the protein oligomers that eventually form the capsid, this was achieved by registering fluorescence from free diffusion cromophore-labeled capsid protein. Subsequently, we intend to record real-time arrival of the labeled proteins to the immobilized viral-RNA. An icosahedral plant virus was used as a model. Capsid proteins was cloned and mutated. Single amino acid mutation allows specific fluorescent labeling of capsid protein with a commercial dye. One of the viral-RNA was immobilized inside of a custom designed sample chamber. Single photon emission from the labeled protein arriving to an immobilized RNA was acquired with a modified epifluorescence microscope.
Probing the same inter-dye-distance of 6 nm by using the super- resolution DNA-PAINT and FRET technique on DNA Origami
Julia Molle, Mario Raab, Ija Jusuk, Sarah Schulz, Philip Tinnefeld & Dina Grohmann
Technische Universität Braunschweig, Institut für Physikalische und Theoretische Chemie, NanoBioSciences, Hans-Sommer-Straße 10, 38106 Braunschweig, Germany
Distances between fluorescent dyes specifically positioned on DNA Origami nanostructures are commonly examined using super-resolution fluorescence microscopy techniques [1,2]. In this work we focus on the transient binding of short oligonucleotides that are labeled with fluorescent dyes (DNA-PAINT, point accumulation for imaging in nanoscale topography) to resolve sub-10-nm distances where the so-called Förster resonance energy transfer (FRET) takes place . This effect relies on the distance dependent energy transfer from a donor fluorophore to an acceptor through non-radiative dipole-dipole coupling [3, 4]. In this approach we measure the same distance between two dyes with DNA- PAINT as well as FRET to evaluate the precision of both measurement techniques. Therefore we use rectangular DNA Origamis with binding sites at distances of 6 nm. In consequence of different complementary docking strands for DNA- PAINT (same dyes on both sides) as well as for FRET measurements (different dyes) we improved the measurement procedure by including washing steps to exchange the labeling solutions without further movement of the sample.
 M. Raab, J.J. Schmied, I. Jusuk, C. Forthmann, P. Tinnefeld, Fluorescence microscopy with 6 nm resolution on DNA origami, Chemphyschem a European journal of chemical physics and physical chemistry 15 (2014) 2431–2435.
In planta FRET-FLIM analyses uncover mechanisms how light-activated phytochrome mediates the reorganization of the COP1/SPA complex to promote photomorphogenesis
Sven zur Oven-Krockhaus2,3, Chiara Menon1, David Sheerin1,2, Beatrix Enderle1, Ling Zhu4, Philipp Johnen2, Frank Schleifenbaum2,3, York-Dieter Stierhof2, Enamul Huq4, Andreas Hiltbrunner1,5
1Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
In plants, the developmental program in light (photomorphogenesis) is regulated by several photoreceptors. One family of photoreceptors - the phytochromes - detect spectral changes via their phytochromobilin chromophore especially in the red/far-red region. By default, photomorphogenic growth is suppressed by the action of a protein complex comprised of CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and SUPPRESSOR OF phyA-105 (SPA). For dark-grown plant seedlings, this leads to a growth pattern in which they mainly elongate to emerge from the soil. Upon exposure to the sun’s radiation, phytochromes are now able to compensate the action of the COP1/SPA complex. The ensuing accumulation of transcription factors trigger photomorphogenic growth like leaf development and chlorophyll production. However, the mechanism by which phytochromes inactivate COP1/SPA activity is unknown. Our investigation involved FRET-FLIM analyses to show co-localization and interaction of those proteins in sub-compartiments of plant nuclei – so called nuclear bodies. Furthermore, we devised a FRET-FLIM scheme in which we used phytochromes as a direct modulator for the integrity of the COP1/SPA complex in planta. Supported by results of molecular biological assays, this provides a mechanism for the reorganization of COP1 and SPA by phytochrome mediated light perception.
Benchmarking Single Molecule Fluorescence Instruments: Detection in Solution
Jan Pavlita, Verena Hirschfeld, Christian Hübner
Universität zu Lübeck, Institut für Physik, Ratzeburger Allee 160, 23538 Lübeck
Single molecule fluorescence microscopy is a frequently used technique for the study of transport mechanisms, signal processing or internal dynamics, giving access to physiological, physical and biochemical parameters inaccessible via ensemble methods. Although single molecule fluorescence detection is widely used, there is a lack of a robust, reliable, and easy to implement method for analyzing and benchmarking these systems. Despite the wide availability of commercial systems capable of single molecule imaging, most laboratories work with home-build systems. Thereby determining the imaging properties of the respective microscope is a crucial factor if one wants to obtain high quality data and comparability to other systems.
We have developed a method based on photophysical properties of widely used dyes being capable of determining the excitation rate with its corresponding laser power, which, in conjunction with the detection rate enables for full assessment of the excitation/detection characteristics of the confocal microscope. This method is particularly suited for commercial microscopes, where the excitation power is often given in arbitrary units or just a percentage of the maximum.
To this end, the excitation power is varied and the triplet photo physics measured. Since the rate of intersystem crossing is a constant for a dye under defined conditions, the dependence of the triplet fraction on the laser power enables the unambiguous determination of the saturation power, which can then be compared with the saturation intensity providing information on the excitation spot size. The detected photon rate at saturation power, in turn, allows for determining the detection efficiency of the system. We use our method to compare three home-built microscopes in our group under different experimental conditions.
Single molecule imaging of genome activation in live Zebrafish embryos
Broadband fluorescent enhancement by means of silver nanoparticles assembled onto a DNA origami.
Maria Sanz, Carolin Vietz, Dongfang Wang, Guillermo Acuña, Philip Tinnefeld
Institute for Physical & Theoretical Chemistry, and BRICS (Braunschweig Integrated Center for Systems Biology), and LENA (Laboratory for Emerging Nanometrology), Braunschweig University of Technology, Hans-Sommer-Str. 15, 38106 Braunschweig, Germany
In this contribution we show how DNA origami structures can be used as breadboards for studying plasmonic interactions, since they allow the organization of metallic nanoparticles and single organic fluorophores with nanometer precision in three dimensions. So far, most studies have focused on the incorporation of gold nanoparticles due to the relatively simple functionalization with DNA through thiol modifications. Here, the hybridization of silver nanoparticles into DNA origami will be demonstrated in order to build optical nano-antennas. The plasmonic properties of silver will lead to a broadband enhancement of the fluorescence intensity at the nanoantenna hotspot. Such broadband fluorescence enhancement is of utmost relevance for applications in the fields of biosensing and for single molecule measurements at biologically relevant concentrations.
Correcting 3D Superresolution with DNA Origami References
Daniel Schmitt-Monreal, Mario Raab, Jürgen J. Schmied, Philip Tinnefeld
Institute for Physical & Theoretical Chemistry and Braunschweig Integrated Center for Systems Biology (BRICS), TU Braunschweig, Hans-Sommer-Strasse 10, 38106 Braunschweig, Germany.
Various approaches have been presented to extend localization based superresolution microscopy to the third dimension. To obtain quantitative values the z-scale has to be corrected. This is because of a magnification induced by the diffraction index mismatch between the cover slip and the solution in which the sample is embedded. We use a DNA-origami tetrahedron with 100 nm edge length in combination with DNA-PAINT1 to experimentally determine the necessary correction factor. The sensitivity to various parameters including buffer composition demonstrates the necessity of experimental correction factor determination.2
(1) Raab, M.; Schmied, J. J.; Jusuk, I.et. al.: Chemphyschem, 15, 2431-5, (2014).
(2) Schmied, J. J.; Forthmann, C.; Pibiri, E.et. al.: Nano Lett, 13, 781-5, (2013).
Characterizing Dye Incorporation In DNA Origami
Tim Schröder, Max Boy Scheible, Jürgen Schmied, Susanne Beater, Birka Lalkens, Philip Tinnefeld
Physical and Theoretical Chemistry, TU Braunschweig, Hans-Sommer-Strasse 10, 38106 Braunschweig, Germany
DNA origami is a powerful tool to place a defined number of molecules e.g. organic dyes, at a defined distance . We investigated the incorporation efficiency of ATTO647N in a modified version of Rothemund’s rectangular DNA origami by measuring the fluorescence intensity as a function of the number of incorporated dyes at different positions and using different incorporation protocols. Due to the designed distance of 12 to 6 nm dye-dye-interaction is avoided and the intensity scales linearly with the number of dyes incorporated . This allowed us to attach up to 80 dyes on a DNA origami in an ultra-small volume. The bead-like structures are advantageous point-like light sources to characterize superresolution microscopes e.g. for STED microscopy.
 J. J. Schmied, A. Gietl, P. Holzmeister, C. Forthmann, C. Steinhauer, T. Dammeyer, P. Tinnefeld, Nat. Methods 9, 1133–1134 (2012).
The Power of Two: Covalent Coupling of Photostabilizers for Fluorescence Applications
Jasper H. M. van der velde1, Jens Oelerich2, Jingyi Huang3, Jochem H. Smit1, Matthias Hiermaier1, Evelyn Ploetz1, Andreas Herrmann3, Gerard Roelfes2, Thorben Cordes1
1Molecular Microscopy Research Group & Single-molecule Biophysics, Zernike Institute for Advanced Materials, Rijksuniversiteit Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Fluorescence is a versatile tool for spectroscopic investigations and imaging of dynamic processes and structures across various scientific disciplines. The photophysical performance of the employed fluorophores is a major limiting factor. Organic photostabilizers are frequently used to support fluorophores and suppress bleaching and blinking. We present a general concept to covalently link different molecules to form a combined photostabilizer with new properties. The direct linkage of different molecules has the potential to obtain combined or synergetic effects in fluorophore stabilization. The idea was explored by synthesizing a molecule with a reducing and oxidizing moiety (iROXS). Using single-molecule fluorescence microscopy, inter- and intramolecular photostabilization of iROXS resulted in reduced blinking and increased photostability of the cyanine fluorophore Cy5. The results indicate that covalently coupled photostabilizers can replace mixtures of photostabilizers in a buffer and might represent a simpler approach compared to using mixtures. When using iROXS in intramolecular photostabilization of Cy5, we find an unprecedented photostability increase of >100-fold which is competitive with solution based healing. The suggested synthetic concept and the proof-of-concept experiments represent the starting point for the quest to identify optimal combinations of linked photostabilizers for various fluorescence applications.
Carbocyanines revisited – experiment meets simulation
Fabio D. Steffen, Roland K.O. Sigel, Richard Börner
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
The popularity of carbocyanine dyes in single molecule spectroscopy of nucleic acids is unbroken . Studying the dynamics of large RNA constructs and the kinetics of the exon/intron binding site interaction in the group II intron of S. cerevisiae [2,3] have motivated a thorough photophysical characterization of the FRET pair Cy3/Cy5 in context of nucleic acids and RNA in particular. We show that Mg2+ as a mediator of RNA-dye interactions enhances the cyanine fluorescence lifetime. The increasing window for depolarization, monitored by time-resolved anisotropy, further reveals a dynamic equilibrium between free tumbling and stacking onto the RNA backbone, with the stacked conformation preventing photoisomerization . Tracking the mobility of fluorophores covalently bound to RNA on an atomistic level by means of molecular dynamics  allows to disentangle different types of dye-dye and dye-RNA interactions. Our hybrid approach combining time-correlated single photon counting and computer simulations will benefit the interpretation of absolute distance measurement by smFRET.
 M. Levitus and S. Ranjit, Q. Rev. Biophys, 44, 123-151 (2011).
 D. Kowerko, S.L.B König, M. Skilandat, D. Kruschel, M.C.A.A Hadzic, L. Cardo and R.K.O. Sigel, PNAS, 112, 3403-3408 (2015).
 M. Khier, D. Kowerko, F.D. Steffen, R. Börner and R.K.O. Sigel, in preparation.
 F.D. Steffen, R.K.O. Sigel, R.Börner, in preparation.
 R. Best, H. Hofmann, D. Nettels, B. Schuler, Biophys. J., 11, 2721-2731 (2015).
Single molecule fluorescence studies of nucleosome dynamics
Application of single molecule fluorescence microscopy to characterize the penetration of a large amphiphilic molecule in the stratum corneum of human skin
Unfolding of amyloid-beta 42 monomer investigated by two-focus fluorescence correlation spectroscopy
Stefan Walta1, Mario Schneider2, Dieter Willbold2,3, Walter Richtering1
1RWTH Aachen University, Institute of Physical Chemistry, Landoltweg 2, 52074 Aachen, Germany.
The amyloid-beta (Aβ42) peptide is associated with amyloid plaques found in the brains of Alzheimer patients . The aggregation of monomeric Aβ42 into toxic oligomers is considered to play a key role in Alzheimer’s disease . However, the structures of the monomer have remained rather unknown so far. Therefore it is important to investigate the early stages in Aβ42 aggregation. Because Aβ42 precipitates at micromolar concentration , sensitive techniques are necessary to characterize the monomeric state and monomer-oligomer transition.
Fluorescence correlation spectroscopy (FCS) is such a sensitive tool for measuring even at subnanomolar concentrations and thus for dealing with fast aggregating amyloid peptides . Furthermore two-focus fluorescence correlation spectroscopy (2fFCS) enables the absolute and precise determination of diffusion coefficients , which is an important feature to make small size changes during the process of unfolding visible. Recent experiments in our group have already shown the utility of 2fFCS in biomaterials research [6,7].
In this contribution we demonstrate that 2fFCS is capable to follow Aβ-42 unfolding. Guanidine hydrochloride was used as denaturant in the absence and presence of trifluoroethanol to get a hint which conformer is more prone to aggregation. The folding stability of Aβ-42 was calculated by applying a 2-state equilibrium unfolding model.
 C. L. Masters, G, Simms, N. A. Weinman, G. Multhaup, B. L. McDonald and K. Bayreuther, Proc. Natl. Acad. Sci. U.S.A., 82, 4245-4249 (1985).
 R. Reisende, E. Ferreiro, C. Pereira and C. Resende de Oliveira, Neuroscience 155, 725-737 (2008).
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 C. L. Ni, H. P. Shi, H. M. Yu, Y. C. Chang and Y. R. Chen, FASEB J., 25, 1390-1401 (2011).
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 S. Lehmann, S. Seiffert and W. Richtering, J. Am. Chem. Soc., 134, 15963-15969 (2012).
 H. Park, S. Walta, R. R. Rosencrantz, L. Elling, W. Richtering and A. Böker (submitted).
Towards 3d single particle orbit tracking in diblock copolymer membranes
Daniel Zalami1, Christian Pietsch2, Felix Schacher2, Jürgen Köhler1, Uwe Gerken1
1Experimental Physics IV and Bayreuth Institute of Macromolecular Research (BIMF), University of Bayreuth, Germany
The method of single particle orbit tracking (SPOT) permits to record trajectories of fluorescent particles for long observation times . In our setup we achieve time traces with 105 data points, a recording rate of 250 Hz and an accuracy for the spatial position of the particle better than 10 nm . This versatile technique can be applied in the field of life sciences as well as in the material research. We want to investigate the diffusion behavior of tracer particles within membranes with controllable pore size. Therefore we will expand our 2d SPOT setup to acquire fully three dimensional trajectories within a polystyrene-block-poly(N,N-dimethylaminoethyl methacrylate) (PS-b-PDMAEMA) membrane .
 Katayama, Y., Burkacky, O., Meyer, M., Bräuchle, C., Gratton, E., Lamb, D. C., ChemPhysChem, 10, 2458–64 (2009).
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Objective diagnosis of cancer by sub-cellular Multiparameter Fluorescence Image Spectroscopy
Peter Zentis1, Ville Rantanen2, Stefanie Weidtkamp-Peters1, Suren Felekyan1, Ralf Kühnemuth1, Evangelos Sisamakis3, Lei Xu3, Anna Perols4, Jutta Arden-Jacob5, Alexander Zilles5, Martina Oberländer6, Britta Fritzsche6, Per-Åke Nygren4, Amelie Eriksson Karlström4, Sampsa Hautaniemi2, Karl Heinz Drexhage5, Gert Auer7, Jens Habermann6, Jerker Widengren3, Claus Seidel1
1Institute of Molecular Physical Chemistry, Heinrich Heine University, Düsseldorf, Germany
We present an approach for multiplex imaging of immuno-fluorescent stained cells to enable objective automated diagnosis of breast cancer. In four representative cell lines we analyzed seven tumor biomarkers in parallel by a combination of indirect immuno-labeling with primary and secondary antibodies and direct labeling with dye conjugated Affibody molecules. To allow for separation of fluorescence photons from different markers, we selected dyes that can be discriminated by exploiting their spectral properties as well as their fluorescence decay time using a pattern-matching maximum likelihood fit. The fluorescence signal was detected by a confocal laser scanning microscope equipped with a custom extension for multi-parameter fluorescence image spectroscopy (MFIS) . To handle the full extent of information contained in the identified spatial intensity features and their correlations we used advanced bioinformatic image processing tools, i.e. the MFIS-files were processed by the image analysis workflow environment Anima  to train a classifier for cancer diagnosis.
The method discriminates non-malignant breast- and different breast cancer cell lines with high accuracy and sensitivity and can potentially be transferred to different samples and markers without necessitating major adaptions of setup and analysis workflow.
 Weidtkamp-Peters, S., Felekyan, S., Bleckmann, A., Simon, R., Becker, W., Kühnemuth, R., Seidel, C. A. M., Photochem. Photobiol. Sci., 8,470-480 (2009)
 Rantanen, V., Valori, M., Hautaniemi, S., Front Bioeng Biotechnol, 2, 25 (2014)
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- 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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