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Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

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Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial

C M Brown¹, R B Dalal, B Hebert

¹McGill University Life Sciences Complex Imaging Facility, Department of Biochemistry, Montreal, Quebec, Canada. claire.brown@mcgill.ca

Journal of Microscopy

|January 5, 2008

Summary

This summary is machine-generated.

Raster image correlation spectroscopy (RICS) provides guidelines for measuring molecular dynamics and concentrations in living cells using confocal microscopy. This novel technique enables accurate quantification of protein diffusion and concentration, even with complex cellular structures.

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Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Raster image correlation spectroscopy (RICS) is an emerging technique for analyzing molecular dynamics and concentrations.
Commercial confocal microscopy systems are widely available for biological imaging.
Analyzing complex cellular environments presents challenges for RICS.

Purpose of the Study:

To develop and present guidelines for performing RICS analysis on commercial laser scanning confocal microscopes.
To establish instrument and image acquisition settings for accurate RICS measurements.
To provide methods for analyzing RICS data in the presence of cellular complexity.

Main Methods:

Development of guidelines for instrument settings, image acquisition, and detector characterization.
Application of RICS to measure diffusion coefficients and concentrations of fluorescent dyes and proteins.
Implementation of algorithms to handle bright immobile structures and slow cellular movement.

Main Results:

Demonstrated ability to detect protein concentrations as low as approximately 2 nM using standard curves.
Accurate determination of paxillin-enhanced-green fluorescent protein (EGFP) diffusion in cellular cytosol and near adhesions.
Successful extraction of fast protein dynamics despite the presence of immobile cellular structures.

Conclusions:

RICS is a viable technique for quantifying molecular dynamics and concentrations in living cells using commercial confocal microscopes.
The presented guidelines facilitate RICS analysis, offering a starting point for various confocal systems.
Methods are provided to overcome common challenges in RICS analysis, enhancing its applicability.