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The goal of this article is to provide cell biologists with some guidance on how to select the most appropriate method for evaluating colocalization in their research and an appreciation of the factors that need to be considered for meaningful interpretation of colocalization studies. While it is laudable that these tools are now widely available to biomedical researchers, they are frequently only vaguely described.
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(In fact, the reduced appetite for articles on the topic of colocalization is quantifiable a literature search shows that while the number of biomedical review articles including the term “fluorescence microscopy” has increased exponentially since 1980, the number including the term “colocalization” started saturating at the turn of the millennium.) This article is predicated upon the proliferation of software tools for analyzing colocalization that have been implemented in image analysis software packages available to cell biologists (see partial listing in appendix). Given the number of excellent reviews that have been published on the topic of colocalization analysis ( 7, 38, 50) and the absence of much that is fundamentally new for the past 20 years, a reader could justifiably wonder about the need for yet another review.
Rather, cell biologists frequently treat colocalization as a subjective feature, using something like Potter Stewart's criterion for defining obscenity “I know it when I see it.” This practice persists despite a relatively large literature in methods of quantitative colocalization analysis. However, the data collected in these studies are seldom rigorously evaluated. Such comparisons can be used to understand the function of a protein, as when the protein is found to colocalize with a marker of a particular organelle, or to understand intracellular transport, as when the protein is found to colocalize with a marker of a particular pathway. One of the most common applications of fluorescence microscopy is to compare the subcellular distributions of two fluorescently labeled molecules. Fueled by developments in molecular biology, electronics, and chemistry, fluorescence microscopy has flourished in the past 30 years.
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