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J Biomol Tech. 2010 September; 21(3 Suppl): S9–S10.
PMCID: PMC2918013

Live Cell Imaging and Measurements of Molecular Dynamics

M. Frigault,1 J. Lacoste,2 and J. Swift3
1McGill University, Molecular Oncology Group, Montreal, Quebec, Canada;
2McGill University, Cell Imaging and Analysis Network, Montreal, Quebec, Canada;
3McGill University, Department of Chemistry, Montreal, Quebec, Canada;
4McGill University, Imaging Facility, Montreal, Quebec, Canada

Abstract

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Live cell microscopy is becoming widespread across all fields of the life sciences, as well as, many areas of the physical sciences. In order to accurately obtain live cell microscopy data, the live specimens must be properly maintained on the imaging platform. In addition, the fluorescence light path must be optimized for efficient light transmission in order to reduce the intensity of excitation light impacting the living sample. With low incident light intensities the processes under study should not be altered due to phototoxic effects from the light allowing for the long term visualization of viable living samples. Aspects for maintaining a suitable environment for the living sample, minimizing incident light and maximizing detection efficiency will be presented for various fluorescence based live cell instruments. Raster Image Correlation Spectroscopy (RICS) is a technique that uses the intensity fluctuations within laser scanning confocal images, as well as the well characterized scanning dynamics of the laser beam, to extract the dynamics, concentrations and clustering of fluorescent molecules within the cell. In addition, two color cross-correlation RICS can be used to determine protein-protein interactions in living cells without the many technical difficulties encountered in FRET based measurements. RICS is an ideal live cell technique for measuring cellular dynamics because the potentially damaging high intensity laser bursts required for photobleaching recovery measurements are not required, rather low laser powers, suitable for imaging, can be used. The RICS theory will be presented along with examples of live cell applications.


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