Mr. Masayoshi Matsumoto Profile

Masayoshi Matsumoto

at Nara Institute of Science and Technology

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Proceedings Article | 13 July 2007 Paper

Spatially resolved fluorescence correlation spectroscopy based on electron multiplying CCD

Masayoshi Matsumoto, Tadao Sugiura, Kotaro Minato

Proceedings Volume 6630, 663017 (2007) https://doi.org/10.1117/12.728399

KEYWORDS: Fluorescence correlation spectroscopy, Molecules, Charge-coupled devices, Diffusion, Glasses, Luminescence, Spatial resolution, Particles, Sensors, Microscopes

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Fluorescence correlation spectroscopy (FCS) is widely used for investigation of concentration, diffusion coefficients and dynamics of single molecules. To introduce spatial resolution in FCS measurement, we develop a novel FCS system, which uses an electron-multiplying charge-coupled device (EM-CCD) to get FCS data at each pixel. We tested 3 samples, which have different concentrations of fluorescent beads, and successfully investigated the difference of correlation coefficients of FCS signal. In addition, we introduce a new illumination method for EM-CCD based FCS measurement, to limit depth of a observation volume. Although a evanescent field has a nature of limited penetration depth, the penetration depth which is 50 to 200nm in typical, is short in comparison with the resolution in the lateral direction. As a result FCS measurement becomes too sensitive in the depth direction, but worse in lateral direction. So we introduce a novel illumination method, in which a laser beam is incident with an angle slightly smaller than the critical angle to illuminate fluorescent molecule (critical-angle illumination). The depth of observation volume can be controlled with the angle of incidence. We expect this method to be applied to a measurement of local diffusion coefficient of molecules in living cells.

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