Science in your labs - Wuhan 2014
PicoQuant: Science in your labs
“Advanced fluorescence microscopy: from cells to single molecules”
June 18, 2014 in Wuhan, China
Joint event of Prof. Chun Tang from the Wuhan Institue of Physics and Mathematics of the Chinese Academy of Sciences, ETSC Technology, and PicoQuant GmbH
Aim and purpose
Single molecule detection techniques, especially Fluorescence Lifetime Imaging (FLIM), Förster Resonance Energy Transfer (FRET), and Fluorescence Correlation Spectroscopy (FCS) provide important insights in the field of life and material science, and are particularly well suited to monitor interactions between molecules both in cells and in vitro. One of the leading manufacturers of advanced imaging instruments, PicoQuant, is organizing a one-day-event in the lab of Prof. Chun Tang (Wuhan Institute of Physics and Mathematics, CAS). It includes talks and discussions about the principles and applications of FLIM, FRET, and FCS.
Therefore, outstanding scientists like Andong Xia (Institute of Chemistry, CAS, Beijing) and Xiaodong Su (BIOPIC, Beijing University) will introduce the concepts of time-resolved fluorescence microscopy and show examples of their use in scientific research.
In addition, the participants get the opportunity to attend an experimental hands-on session showing a Nikon A1 LSM upgraded towards FLIM and FCS.
Registration closes on June 15, 2014.
Information and correspondence
Rudower Chaussee 29
12489 Berlin, Germany
About 45 graduate students, postdocs, technicians, and professional researchers attended the joint event of Prof. Chun Tang from the Wuhan Institue of Physics and Mathematics of the Chinese Academy of Sciences, ETSC Technology, and PicoQuant GmbH. The one-day-event featured talks and discussions about the principles and applications of FLIM, FRET, and FCS. In addition, the participants got the opportunity to attend experimental hands-on sessions showing a Nikon A1 LSM upgraded towards FLIM and FCS.
- Manoel Veiga (PicoQuant, Berlin, Germany)
- Andong Xia (Chinese Academy of Science, The State Key Laboratory of Molecular Reaction Dynamics, Beijing, China)
- Xiaodong Su (BIOPIC, Beijing University, Beijing, China)
- Jiangyun Wang (Researcher in The Institute of Biophysics, Chinese Academy of Sciences, China)
Manoel Veiga (PicoQuant, Berlin, Germany)
"Advanced FLIM, FRET and Anisotropy Upgrade for Confocal Laser Scanning Microscopes"
Confocal laser scanning microscopes (CLSM) are an essential tool in biological and biomedical research. Their functionality can be further enhanced by adding sensitive time-resolved data acquisition capabilities, enabling Fluorescence Lifetime Imaging (FLIM), lifetime based Förster Resonance Energy Transfer (FRET) as well as aniostropy measurements. Complete and turn-key upgrade packages allow to apply these powerful techniques for all modern CLSMs. We will present the underlying method, actual instrumentation based on Time-Correlated Single Photon Counting (TCSPC) and discuss recent applications:
- The fluorescence lifetime of a dedicated fluorophore is strongly dependent on its photophysical properties and a wealth of environmental parameters such as pH, ion or oxygen concentration, molecular binding or the proximity of energy acceptors making it the technique of choice for functional imaging of many kinds. As the fluorescence lifetime does not depend on concentration, absorption by the sample, sample thickness, photo-bleaching and/or excitation intensity, it is more robust than intensity based methods. The fluorescence decay can act as a fingerprint for a dye in a certain condition. Bsed on this, we have developed a novel FLIM analysis method called Pattern Matching that allows for an excellent separation of fluorophores in FLIM images and their discrimination from autofluorescence in biological samples.
- Förster Resonance Energy Transfer (FRET) studies provide a very powerful tool for a broad range of biological applications since this technique enables to measure intra- and intermolecular distances down to several nanometres. In this way, molecular interactions can be determined in vitro as well as in living cells. In addition, so-called FRET sensors allow the monitoring of environmental conditions such as pH and ion concentration. FRET results in donor quenching and leads to changes in its fluorescence intensity and fluorescence lifetimes. Such distance measurements on a nanometer scale can be improved by applying FLIM-FRET. Here, changes in fluorescence lifetime of the donor are monitored which is in a broad range, concentration independent. This is advantageous since in biological systems like cells, the fluorophore concentration often cannot be accurately determined and compared amongst different cells. Other than intensity-based FRET measurements, FLIM can further reveal sub-populations; thus, allowing to determine the fraction of free donors compared to associated donor molecules within a complex. The result of such an analysis yields not only the lifetime distribution of FLIM-FRET images, but also the fraction and distribution of complete to incomplete FRET complexes.
- Measurement of fluorescence anisotropy offers fascinating possibilities to study molecular orientation and mobility as well as processes that affect them. Except of special cases, anisotropy does not depend on the concentration of fluorophores, thus on the detected signal intensity. Owing to this similarity with the behavior of fluorescence lifetime, one can regard anisotropy as a yet another dimension of fluorescence information. Other potential applications include investigations of biomembrane fluidity and rigidity, study of receptor-ligand binding, alignment of molecules in various matrices and monitoring FRET between identical molecules (homo-FRET).
"Integration of Time-resolved Fluorescence Techniques FCS, FCCS and FLCS for Confocal Laser Scanning Microscopy"Fluorescence Correlation Spectroscopy (FCS) is nowadays a standard tool in biophysics and more and more used also in complex environments, like in cell biology and multi-label applications. It allows to measure diffusion rates and is even capable to detect and distinguish between single and multiple component diffusion. Furthermore, this technique enables the investigation of lateral and rotational diffusion of fluorophores as well as conformational dynamics providing e.g. information about hydrodynamic radii and singlet-triplet dynamics. In addition, FCS provides a direct and independent, calibration-free measure of molecular concentration within a sample. Based on the concentration measurement and cross-correlation between two fluorophores (dual-color FCCS), data are used to detect molecular association and dissociation and to determine the stoichiometry of molecular complexes. In this way it is possible to determine kinetic rate constants, i.e. on and off kinetics of complex formation, as well as enzyme dynamics and intramolecular dynamics in vitro and in living cells.
Common problems which complicate these experiments, like detector afterpulsing and spectral crosstalk, have recently be overcome by looking at the nanosecond arrival time of the detected photons after pulsed excitation. Thus, by using TCSPC, intensity based analysis schemes like FCS are significantly improved by sorting and weighting the detected photons according to their fluorescence lifetime. The combination of both techniques, Fluorescence Lifetime Correlation Spectroscopy (FLCS), allows to identify and suppress artifact signals as e.g. Raman scattering  and to study diffusion properties of different species with similar emission. It will be demonstrated how this method detects molecular binding in liquid environment at concentrations ranging from pM to µM. Furthermore, we will show current results for absolute concentrations measurements of diffusing proteins in live cells as well as dual color FCCS binding studies. Especially in dual color applications, when two pulsed lasers are not available, the decay pattern analysis allows quantitatively to separate the pulsed laser excited fluorescence from the cw excited one to overcome spectral bleedthrough problems.
Demo session: Demonstration of the LSM Upgrade Kit using an Nikon A1 LSMThe upgrade of a Confocal Laser Scanning Microscope towards a time-resolved system is based on picosecond pulsed lasers, fast and sensitive photon counting detectors and sophisticated time-correlated single photon counting (TCSPC) electronics for data acquisition. The underlying technique (Time-Tagged Time-Resolved (TTTR) data acquisition) allows to simultaneously record timing and fluorescence intensity information, both spectrally and spatially, on a single photon basis and on time scales from sub-nanoseconds to seconds.
Thus, new measurement modes like Fluorescence Lifetime Imaging (FLIM), lifetime based Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) become feasible. We will demonstrate the implementation of these techniques and the system performance using an upgraded Nikon A1 LSM.
Andong Xia (Chinese Academy of Science, The State Key Laboratory of Molecular Reaction Dynamics, Beijing, China)
"Conjugation Mediated Electronic Energy Transfer in Branched Chromophores Studied by Single Molecule Spectroscopy"In this report, we will introduce a single molecule analysis of superexchange mediated-electronic energy transfer in branched chromophores. The purpose of the present work is to explore the role played by through-space (TS) and through-bond (TB) couplings on exciton interactions by examining electronic absorption spectra and single molecule spectra of three different BODIPY-dimers, in which the pair of BODIPY chromophores are held at well-defined distances and orientations by three typical rigid bridges (para-, meta- and ortho-linkers) of variable length and orientations. The strong-coupling and stepwise photobleaching of the two branches, depending on different pi-bridges have been observed at SM level. The TS and TB interactions are identified according quantum chemical calculation and single molecule analysis.
Xiaodong Su (BIOPIC, Beijing University, Beijing, China)
"Probing DNA-protein interactions by single-molecule fluorescent technique and structural analyses"In proteins, conformational change impacting the function has been well studied in the past decades, which was named ‘Allostery’. But in DNA-protein interactions, the DNA affects a DNA binding protein only by ‘allosterism’ (different conformations) without sequence changed is not well understood. Predictably, it could exist in nucleosome, as nucleosomal DNA is much less flexible and accessible than free DNA as its bending, fixing and blocking by histone core. According to many high-resolution nucleosome structures, the unusual DNA conformational changes relative to naked DNA always happened induced by the bending of histone core..
Here we report the different average residence time (ART) of glucocorticoid receptor DNA binding domain (GRDBD) on nucleosomal glucocorticoid response element (GRE) using single molecule fluorescent assays. The results suggested that that GR binding on nucleosomal DNA is impacted not only by blocking of histone, but also conformational change of DNA, the binding stability is decreased as much as 3.7 fold even in the outwards position. The strongest binding occurred in nucleosomal DNA, which is increased 1.5 fold compared to naked DNA. We also used the structural modeling to propose how the conformational changes of the nucloesomal DNA affact the binding of GRDBD.
Jiangyun Wang (Researcher in The Institute of Biophysics, Chinese Academy of Sciences, China)
"Genetic code expansion can significantly improve our ability to visualize the proteome "The ability to selectively modify proteins with fluorescent probes has greatly facilitated both in vitro and in vivo studies of protein structure and function. Here we report that genetic code expansion can significantly improve our ability to visualize the proteome.
We are the first to report the genetic incorporation of a fluorescent amino acid (CouAA) into proteins in E. coli in response to the amber stop codon (JACS 2006). Recently, we have successfully incorporated unnatural amino acids bearing cyclopropene or tetrazole functional groups (JACS 2010, ACIE 2012a, ACIE 2012b). Through photoclick reaction, we can site-specifically attach any biophysical probe to a specific site of a target protein in vivo. The photoclick reaction has the following advantages: its fast rate (50 M-1s-1) allows for efficient protein labeling in one minute; spatiotemporal control by laser. We are now applying this technology for super-resolution imaging, post-translational modification sensing, as well as imaging in living plants. We have significantly expanded the function of fluorescent proteins through the genetic incorporation of metal-chelating unnatural amino acids (ACIE 2012b, ACIE 2013b). In recent works, we have bestowed GFP with metal-sensing function, and emission spectra in the infrared range. We are now using these new GFP variant for deep-tissue imaging. Most recently, we have designed an acid turn-on fluorescent protein sensor based on genetic code expansion. We are currently using this method for protein and virus endocytosis. These unpublished works will be discussed in the conference.
|Wednesday, June 18
Venue: Wuhan Institue of Physics and Mathematics of the Chinese Academy of Sciences
The Building of Magnetic Resonance Research Center, room number 201
Welcome by PicoQuant, ETSC and Prof. Chun Tang, Wuhan Institue of Physics and Mathematics of the Chinese Academy of Sciences
Advanced FLIM, FRET and Anisotropy Upgrade for Confocal Laser Scanning Microscopes
|9:15 am||Coffe break|
Conjugation Mediated Electronic Energy Transfer in Branched Chromophores Studied by Single Molecule Spectroscopy
Probing DNA-protein interactions by single-molecule fluorescent technique and structural analyses
Integration of Time-resolved Fluorescence Techniques FCS, FCCS and FLCS for Confocal Laser Scanning Microscopy
Genetic code expansion can significantly improve our ability to visualize the proteome
|2:05 pm||Closing remarks|
|2:15 pm||Coffee break|
Demo sessions (by appointment)
The following international workshops or courses have been organized by PicoQuant GmbH along with a local research institute in the recent years.
July 30 - 31, 2019 | Missoula, MT, USA
April 5, 2019 | Vienna, Austria
March 15, 2019 | Frederiksberg, Denmark
January 17, 2019 | Vienna, Austria
December 11, 2018 | Moscow, Russia
November 28, 2018 | Brno, Czech Republic
November 27, 2018 | Prague, Czech Republic
November 26, 2018 | Vestec, Czech Republic
October 22, 2018 | Erlangen, Germany
October 17, 2018 | Zürich, Switzerland
August 11, 2017 | Tianjin, China
December 2, 2016 | St. Louis, MO, USA
October 12, 2016 | Moscow, Russia
May 30 - June 3, 2016 | Krakow, Poland
May 11, 2016 | Wuhan, China
July 6, 2015 | Daegu, South Korea
May 26 - 29, 2015 | Chorzow, Wroclaw, Poland
May 18 - 20, 2015 | Mexico City/Cuernavaca/San Luis Potosi, Mexico
April 17, 2015 | São Paulo, Brazil
March 17 - 18, 2015 | Lausanne, Switzerland
December 11, 2014 | St. Louis, MO, USA
November 18 - 19, 2014 | Vienna, Austria
June 18, 2014 | Wuhan, China
June 3, 2014 | Warsaw, Poland
December 12, 2013 | St. Louis, MO, USA
November 12 - 13, 2013 | Vienna, Austria
October 11, 2013 | Liverpool, United Kingdom
June 10, 2013 | San Diego, USA
September 28, 2012 | Houston, USA
July 16 - 20, 2012 | Singapore, Singapore
June 19 - 20, 2012 | Vienna, Austria
January 19, 2012 | Los Angeles, USA
June 20 - 24, 2011 | Singapore, Singapore
May 27, 2011 | Munich, Germany
May 9 - 19, 2011 | China
March 4, 2011 | Baltimore, USA
January 10, 2011 | Los Angeles, USA
April 26 - 28, 2010 | BNL, USA
January 22 - 23, 2009 | Sacramento, USA
January 17 - 18, 2008 | Sacramento, USA
August 30, 2007 | Fort Worth, USA
January 18 - 19, 2007 | Sacramento, USA
January 19 - 20, 2006 | Sacramento, USA
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