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Quantitative multicolor fluorescence microscopy relies on the careful spatial matching of color channels acquired at different wavelengths. Due to chromatic aberration and the imperfect alignment of cameras, images acquired in each channel may be shifted, and magnified, as well as rotated relative to each other in any of the three dimensions. With the classical calibration method, chromatic shifts are measured by multicolor beads attached to the surface of a coverslip, and a number of software are available to measure the chromatic shifts from such calibration samples. However, chromatic aberration can vary with depth, change with observation conditions and be induced by the biological sample itself, thus hindering determination of the true amount of chromatic shift in the sample of interest and across the volume. Correcting chromatic shifts at higher accuracy is particularly relevant for super-resolution microscopy where only slight chromatic shifts may affect quantitative analyses and alter the interpretation of multicolor images. We have developed an open-source software Chromagnon and accompanying methods to measure and correct 3D chromatic shifts in biological samples. Here we provide a detailed application protocol that includes special requirements for sample preparation, data acquisition, and software processing to measure chromatic shifts in biological samples of interest.

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using <em>Chromagnon</em>