The first part of the study describes EzColocalization, and the second part demonstrates its use for different sample types and for resolving common issues that prevent rapid and robust quantitative measurements of colocalization. In this study, an open source plugin for ImageJ called EzColocalization was developed so that researchers at all levels of proficiency can visualize the localization of signals and measure colocalization via an easy-to-use graphical user interface (GUI). Therefore there is a pressing need for a single application that provides all the tools for start to finish analysis of colocalization and can be easily customized. Many researchers do not have these skills or resources, and this is a likely reason that many studies evaluate colocalization by the simple, but often misleading, method of overlaying red and green color images 10, 11.
#ADDING SCALE BAR IN IMAGEJ SOFTWARE#
However, combining and customizing software requires proficiency in programming, experience with quantitative microscopy, comfort with mathematics and statistics, and other support. It is often possible to address the above challenges by combining multiple existing software programs and customizing them 8, 15. When this heterogeneity is present, software is need to provide measurements for each cell or defined subsets of cells in samples. A third factor is that there are often mixed localization patterns within cells and different localization patterns among cells in a sample 8, 11, 19. That is, software tools are needed to distinguish intracellular pixels from extracellular pixels, and to select signal intensity thresholds to limit analyses to a subset of intracellular pixels. The latter can occur because the probes or reporters are not sufficiently specific 16, are not adequately removed from cells or organisms 17, or have low signal relative to endogenous compounds ( i.e. “background”) 15, and where there are high levels of non-specific signal in cells 8. A second factor is that the software is often not suited to experiments that push the limits of detection, where the intensity of the intracellular signal is similar to the extracellular signal ( i.e. One factor is that customization of the software is often required for the equipment, reporters and samples 13, 14, and for automated analyses. Several factors limit the use of current software for visualizing the localization of reporters in biological samples and measuring colocalization 9, 10, 11, 12. colocalization, anticolocalization and noncolocalization respectively) in cells, tissues or organisms 8. In particular, it is often challenging to determine whether the different molecules of interest occur in the same locations, different locations or independent locations ( i.e. However, researchers often find it difficult to rigorously evaluate and interpret the images. These features make EzColocalization well-suited for experiments with low reporter signal, complex patterns of localization, and heterogeneous populations of cells and organisms.Īdvances in microscopy equipment and labeling techniques make it possible for researchers to image a variety of biological molecules in almost any cell, tissue, or organism 1, 2, 3, 4, 5, 6, 7.
Features of EzColocalization include: (i) tools to select individual cells and organisms from images (ii) filters to select specific types of cells and organisms based on physical parameters and signal intensity (iii) heat maps and scatterplots to visualize the localization patterns of reporters (iv) multiple metrics to measure colocalization for two or three reporters (v) metric matrices to systematically measure colocalization at multiple combinations of signal intensity thresholds and (vi) data tables that provide detailed information on each cell in a sample. EzColocalization is designed to be easy to use and customize for researchers with minimal experience in quantitative microscopy and computer programming. Here we describe an open source plugin for ImageJ called EzColocalization to visualize and measure colocalization in microscopy images. Insight into the function and regulation of biological molecules can often be obtained by determining which cell structures and other molecules they localize with ( i.e.
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