Multipoint fluorescence correlation spectroscopy with total internal reflection fluorescence microscope.
We report simultaneous determination of diffusion coefficients at different points of a cell membrane using a multipoint fluorescence correlation spectroscopy (FCS) system. A system carrying seven detection areas in the evanescent field is achieved by using seven optical fibers on the image plane in the detection port of an objective-type total internal reflection FCS (TIR-FCS) system. Fluctuation of fluorescence intensity is monitored and evaluated using seven photomultiplier tubes (PMTs) and a newly constructed multichannel correlator. We demonstrate simultaneous-multipoint FCS, with a 3-mus time resolution, to investigate heterogeneous structures such as cell membranes and membrane-binding molecular dynamics near glass surfaces in live cells.
Ohsugi
Yu
Y
Hokkaido University, Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Sapporo 001-0021, Japan.
Kinjo
Masataka
M
D017076:Computer-Aided Design; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation*; D007090:Image Interpretation, Computer-Assisted / Q000295:instrumentation* / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted / Q000295:instrumentation*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods; D018511:Systems Integration