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Spatially Resolved Total Internal Reflection Fluorescence Correlation Microscopy Using an Electron Multiplying Charge-Coupled Device Camera
journal contribution
posted on 2007-06-15, 00:00 authored by Balakrishnan Kannan, Lin Guo, Thankiah Sudhaharan, Sohail Ahmed, Ichiro Maruyama, Thorsten WohlandA spatially resolved total internal reflection fluorescence
correlation microscopy (TIR-FCM) system is constructed
with an electron multiplying charge-coupled device (EMCCD) camera. The system was used to determine diffusion
coefficients of lipid molecules in a planar lipid bilayer,
and lipids and epidermal growth factor receptor (EGFR)
proteins on cell membranes of Chinese Hamster Ovary
(CHO) cells. The evaluation of the “cross talk” between
neighboring pixels suggests that a higher degree of
multiplexing can be achieved than was previously proposed [Kannan, B. et al. Anal. Chem. 2006, 78, 3444−51] using the same camera with a focused laser excitation.
The best time resolution possible with this system is 4
ms for a region of interest comprising 20 lines in the CCD
and is good enough to determine membrane diffusion in
lipid bilayers and of membrane proteins in living cells.
In this work, using a TIR-FCM setup, 1600 autocorrelation functions were measured simultaneously with a time
resolution of 4.8 ms. This area corresponds to a 40 × 40
pixel region of interest with a dimension of 11.3 × 11.3
μm2 and is sufficiently large to allow the measurement of
the lower membrane of a whole cell simultaneously.