【2nd May】Imaging Fluorescence Correlation Spectroscopy (FCS) investigates bilayers and live cell mem
日期:2013-05-02 阅读:1144

题目:Imaging Fluorescence Correlation Spectroscopy (FCS) investigates bilayers and live cell membranes

报告人:Professor Thorsten Wohland, Departments of Biological Sciences and Chemistry & Centre for Bioimaging Sciences, National University of Singapore

时间:513日(周一),上午10:00

地点: 化学楼演讲厅(化学化工学院A518室)

邀请人:任吉存 教授(化学化工学院)

 

Abstract: Fluorescence Correlation Spectroscopy (FCS) has been traditionally performed on single spots within a sample using confocal microscopy. With the advent of fast and sensitive array detectors and alternative illumination schemes this has changed and we now can measure millions of points simultaneously with millisecond time resolution, or thousands of points with a time resolution down to the microsecond range [1-2]. The measurements of an FCS image is sufficiently fast (< 20 s) to even allow the recording of FCS time series, observing the dynamics of cells and bilayers over an extended period of time.

In this seminar we will discuss the principles and theoretical and practical limits of imaging FCS approaches. Imaging FCS requires different illumination schemes to avoid the crosstalk between pinholes present in multi-confocal systems. They have to illuminate a whole plane in a sample but have to restrict the illumination in z-direction at the same time. For this purpose Total Internal Reflection (TIR) Microscopy can be used for illumination of samples close to the glass coverslip. When planes deeper in a cell or organisms are to be recorded a suitable illumination scheme is provided by single plane illumination microscopy (SPIM). We demonstrate the applicability of both systems for biological applications. We used imaging TIR-FCS (ITIR-FCS) to characterize the physico-chemical properties of supported lipid bilayers with 1-3 components and show that domains as well as underlying meshwork structures can be determined [2-4]. We apply the technology then to a range of different proteins on cell membranes to demonstrate cell membrane organization and show time lapse movies of membrane organization dynamics in the case of a membrane active peptide. SPIM-FCS provides similar data to ITIR-FCS but can perform measurements even deep within tissues [5-7]. We applied SPIM-FCS to a range of different proteins (fluorescent proteins, transcription factors and Rho-GTPases) in live cells and in zebrafish embryos to determine location dependent diffusive behavior of the proteins.

Imaging FCS provides now sufficient time resolution to observe molecular processes in cells and organisms similar to confocal FCS [8-9]. But its inherent multiplexing allows collecting much more data on a single sample simultaneously with reduced optical exposure and damage, allowing more measurements per sample.

References:

1. B. Kannan, et al., (2007), Anal Chem, 79, 4463-4470.

2. J. Sankaran, et al., (2009), Biophys J, 97, 2630-2639.

3. N. Bag et al., (2012), ChemPhysChem 13(11), 2784-94.

4. R. Kraut, et al., (2012) Methods in Cell Biology 108, 395-427.

5. J. Husiken et al, (2004), Science 305, 1007-1009.

6. T. Wohland et al., (2010), Opt Express, 18, 10627-10641.

7. J. Sankaran et al., (2010), Opt Express, 18, 25468-25481.

8. J. Sankaran et al., (2013), Anal Chem, in press (dx.doi.org/10.1021/ac303485t)

9. A.P. Singh et al., (2013), Opt Express, 21 (7) 8652-8668.

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