12th Carl Zeiss sponsored workshop on Fluorescence Correlation Spectroscopy and related methods

Systems biology is the study of an organism, viewed as an integrated and interacting network of genes, proteins and biochemical reactions which give rise to life. Instead of analyzing individual components or aspects of the organism, systems biologists focus on all the components and the interactions among them, all as part of one system using computer-based or special algorithms-based analysis. Here we introduce a novel method for predicting genome-wide condition-specific subcellular locations of human proteins using only limited and condition-unspecified known locations. With systems biological analysis of glioma, the key target genes which involved in the control of the tumoregenesis process can be estimated. After systemic molecular biological experiments for target genes with immunohistochemical staining, FCCS, Olink system, western blotting, siRNA and growth/proliferation inhibition assay technique to determine protein binding sites on DNA and molecular imaging, the gene regulatory mechanism can be investigated and validated functionally based on key mechanisms. We discover that the relocated but still interacting PSPN and GFRa4 in endoplasmic reticulum do not interact with ret proto-oncogene (RET), whereas the three proteins are highly interact to each other in plasma membrane in normal brain tissues. The novel relocation of each gene discovered here can be a biomarker in glioma. FLUORESCENCE CORRELATION SPECTROSCOPY ON NANO-FAKIR SURFACES WITH TWO-PHOTON EXCITATION J. Delahaye*, S. Grésillon*, E. Fort*, N. Sojic** and S. Lévêque-Fort*** * Centre d’Imageries Plasmoniques Appliquées, Institut Langevin, ESPCI ParisTech, CNRS UMR 7587, 10 rue Vauquelin, 75 231 Paris Cedex 05, France **Institut des Sciences Moléculaires, UMR 5255 CNRS, Université Bordeaux 1 – ENSCPB, 16 Avenue Pey-Berland, 33607 Pessac Cedex, France ***Laboratoire de Photophysique Moléculaire, Université Paris sud, bat 210, 91405 Orsay, France [email protected] Single biomolecule behaviour can reveal crucial information about processes not accessible by ensemble measurements. It thus represents a real biotechnological challenge. Common optical microscopy approaches require picoto nano-molar concentrations in order to isolate an individual molecule in the observation volume. However, biologically relevant conditions often involve micromolar concentrations. These impose a drastic reduction of the conventional observation volume by at least three orders of magnitude. This confinement is also crucial for mapping subwavelength heterogeneities in cells which play an important role in many biological processes. We propose an original approach that couples Fluorescence Correlation Spectroscopy (FCS), powerful tool to retrieve essential information on single molecular behaviour, and nanostructured plasmonic substrates with strong field enhancements and confinements at their surface. These electromagnetic singularities at nanometer scale, called “hotspots”, are the result of the unique optical properties of surface plasmons. They provide an elegant way for studying single-molecule dynamics at high concentrations by reducing dramatically the excitation volume and enhancing the fluorophore signal by several orders of magnitude. Fig. 1 Scanning electron micrographs of a part of the nanotip array The nanostructured substrates are obtained by electrochemistry of an optical fibre bundle covered by a metallic thin film (see Figure 1) [1]. This technique is versatile and highly reproducible. It permits to create “nano-fakir surfaces” which cover a wide range of surface topographies to tune and tailor the hotspots density, localisation and size. We present two-photon FCS results on these nano-fakir substrates using fluorescent nanobeads. We show i) a dramatic reduction of the observation volume and ii) the ability to perform at high concentration measurements, which is promising for biological applications. [1] V. Guieu, F. Lagugné-Labarthet, L. Servant et al., “Ultrasharp Optical-Fiber Nanoprobe Array for Raman Local-Enhancement Imaging” Small, 4, 96-99 (2008). Full correlation FCS (fcFCS) to investigate the bulk polymerization of styrene Maren Dorfschmid, Andreas Zumbusch, Dominik Wöll Zukunftskolleg/Department of Chemistry, Universität Konstanz, 78457 Konstanz, Germany E-mail: [email protected] Radical polymerization represents a challenging topic for basic kinetic research. Proceedingfrom dilute to semi-dilute and finally concentrated polymer solutions, the mobility of monomers,polymer chains and other solutes becomes more and more restricted. This restriction starts todominate polymerization kinetics as soon as polymerization or termination rates become diffusion-controlled. In previous work, we have concentrated on studying the decrease of translationaldiffusion coefficients of single perylendiimide (PDI) molecules by fluorescence correlationspectroscopy (FCS) and wide-field fluorescence microscopy (WFM).[1]Here, we present our latest results on rotational and translational motion of different sizedPDIs during bulk polymerization of styrene. Rotational diffusion coefficients in dilute solutionswere determined by fluorescence anisotropy measurements. When rotational diffusion times clearlyexceed the fluorescence lifetime (ca. 5 ns for the PDI used), a situation which is reached at somepoint during increasing monomer-to-polymer conversion, this method cannot be applied anymore.However, in this case, rotational diffusion can be observed by fcFCS with correlations down tonanoseconds.[2,3] The crosscorrelation curves show antibunching, rotational and translationalmotion. Triplet contributions do not appear due to the low ISC rate of the PDI. With fcFCS we wereable to follow rotational motion of the probing dye over an extended range of monomer-to-polymerconversion. Thus, a direct comparison between rotational and translational motion, both observedby the same fcFCS experiment, is possible. This knowledge will be used to gain insight intochanges in mobility during radical polymerization and how they depend on the polymerizationconditions. References[1] D. Wöll, H. Uji-i, T. Schnitzler, J. Hotta, P. Dedecker, A. Herrmann, F. C. DeSchryver, K. Müllen, J. Hofkens, Angew. Chem. Int. Ed. 2008, 47, 783.[2] M. Wahl, H.-J. Rahn, I. Gregor, R. Erdmann, J. Enderlein, Rev. Sci. Instr. 2007, 78,033106.[3] S. Felekyan, R. Kühnemuth, V. Kudryavtsev, C. Sandhagen, W. Becker, C. A. M.Seidel, Rev. Sci. Instr. 2005, 76, 083104.