A Guide to Accurate Fluorescence Microscopy Colocalization Measurements

Biomolecular interactions are fundamental to the vast majority of cellular processes, and identification of the major interacting components is usually the first step toward an understanding of the mechanisms that govern various cell functions. Thus, statistical image analyses that can be performed...

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Bibliographic Details
Published in:Biophysical journal 2006-12, Vol.91 (12), p.4611-4622
Main Authors: Comeau, Jonathan W.D., Costantino, Santiago, Wiseman, Paul W.
Format: Article
Language:English
Subjects:
Accuracy
Algorithms
Animals
Antibodies, Monoclonal - metabolism
Biophysics
Cell Line
Cells
Computer Simulation
Fibroblasts - metabolism
Fluorescence
Fluorescent Dyes
Humans
Image Interpretation, Computer-Assisted
Mice
Microscopy
Microscopy, Fluorescence - methods
Receptor, Platelet-Derived Growth Factor beta - immunology
Spectrometry, Fluorescence
Spectroscopy, Imaging, Other Techniques
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Summary:Biomolecular interactions are fundamental to the vast majority of cellular processes, and identification of the major interacting components is usually the first step toward an understanding of the mechanisms that govern various cell functions. Thus, statistical image analyses that can be performed on fluorescence microscopy images of fixed or live cells have been routinely applied for biophysical and cell biological studies. These approaches measure the fraction of interacting particles by analyzing dual color fluorescence images for colocalized pixels. Colocalization algorithms have proven to be effective, although the dynamic range and accuracy of these measurements has never been well established. Spatial image cross-correlation spectroscopy (ICCS), which cross-correlates spatial intensity fluctuations recorded in images from two detection channels simultaneously, has also recently been shown to be an effective measure of colocalization as well. Through simulations, imaging of fluorescent antibodies adsorbed on glass and cell measurements, we show that ICCS performs much better than standard colocalization algorithms at moderate to high densities of particles, which are often encountered in cellular systems. Furthermore, it was found that the density ratio between the two labeled species of interest plays a major role in the accuracy of the colocalization analysis. By applying a direct and systematic comparison between the standard, fluorescence microscopy colocalization algorithm and spatial ICCS, we show regimes where each approach is applicable, and more importantly, where they fail to yield accurate results.
ISSN:0006-3495
1542-0086
DOI:10.1529/biophysj.106.089441