Using Cell-ID-1.4 with R for microscope-based cytometry

ARIEL CHERNOMORETZ; ALAN BUSH; RICHARD YU; ANDREW GORDON; ALEJANDRO COLMAN-LERNER

Current protocols in molecular biology

Wiley-Interscience

Año: 2008

1934-3639

This unit describes a method to quantify, from sets of microscope images, various cellular parameters from individual cells, and includes procedures to track cells over time. For example, the user can measure cell volume, total and subcellular localization (nuclear, plasma membrane) of fluorescence for multiple fluorescence channels. This method uses the image processing capabilities of Cell-ID (Gordon et al., 2007) and data analysis by the statistical programming framework R, both open source software packages. The first step for successful cytometry entails acquiring at least one SET of images for each field of cells. Each SET is composed of one purposefully defocused transmission image (sometimes referred to as brightfield, or BF) that will be used to locate each cell, and one fluorescence image for each of the color channels to be analyzed. Images may be conventional wide-field epifluorescence or confocal microscopy images. Cell-ID processes the images and outputs a tab-delimited file with information extracted from each cell, for each time point and each fluorescence channel. Finally, the user analyzes the data using R (R-Development-Team, 2008), which we have supplemented with a package tailored to analyze Cell-ID output.