Detecting and Quantifying Colocalization of Cell Surface Molecules by Single Particle Fluorescence Imaging

Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles. The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian fu...

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Bibliographic Details
Published in:Biophysical journal 2003-12, Vol.85 (6), p.4110-4121
Main Authors: Morrison, Ian E.G., Karakikes, Ioannis, Barber, Rosamund E., Fernández, Nelson, Cherry, Richard J.
Format: Article
Language:English
Subjects:
Animals
Cell Biophysics
Cell Membrane - metabolism
Cells
CHO Cells
Chromosome Aberrations
Cricetinae
Escherichia coli - metabolism
Fluorescence
Fluorescent Dyes - pharmacology
Image Processing, Computer-Assisted
Lipopolysaccharide Receptors - biosynthesis
Lipopolysaccharides - chemistry
Microscopy, Confocal - methods
Microscopy, Fluorescence
Molecules
Normal Distribution
Polylysine - chemistry
Transfection
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Description
Summary:Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles. The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian function. We have exploited the positional information obtained from SPFI to develop a method for detecting colocalization of cell surface molecules. This involves labeling two different molecules with different colored fluorophores and determining their positions separately by dual wavelength imaging. The images are analyzed to quantify the overlap of the particle images and hence determine the extent of colocalization of the labeled molecules. Simulated images and experiments with a model system are used to investigate the extent to which colocalization occurs from chance proximity of randomly distributed molecules. A method of correcting for positional shifts that result from chromatic aberration is presented. The technique provides quantification of the extent of colocalization and can detect whether colocalized molecules occur singly or in clusters. We have obtained preliminary data for colocalization of molecules on intact cells. Cells often exhibit particulate autofluorescence that can interfere with the measurements; a method for overcoming this problem by triple wavelength imaging is described.
ISSN:0006-3495
1542-0086
DOI:10.1016/S0006-3495(03)74823-1