Abstract
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.
Highlights
Multicolor imaging is one of the fundamental aspects of biological fluorescence microscopy, in cases where the spatial relationship of different molecules or structures is of major interest
The Z step size for reference and target images is preferably less than half of the optical resolution of the Z axis, which is calculated by for a diffractionlimited microscopy, where λ is the wavelength in nanometers and NA is the numerical aperture of the objective lens
The alignment parameter was applied to the target image (Figure 5C,D), which has exactly the same number of voxels as the reference image
Summary
Multicolor imaging is one of the fundamental aspects of biological fluorescence microscopy, in cases where the spatial relationship of different molecules or structures is of major interest. "Crosstalk reference images" have the highest correction accuracy and are relatively simple to accomplish[3 , 4] (Table 1) The drawback is their limitation in microscopy applications due to their incapability of measuring chromatic shifts in excitation paths. The accuracy depends on how much the imaging conditions are kept constant In this regard, the best performance is obtained when both target and reference samples are on the same slide, using, for example, 8-well chambered coverglasses (Table 1, right-most). Chromagnon can combine the microscope instrumental local distortion map and the global alignment parameters from the biological calibrations (Table 1) Using this method, it is expected that the average accuracy of biological calibration will be improved by an additional 1−2 nm. In the fourth section, we describe a method to complement the biological calibration reference images by using a microscope's instrumental local calibration
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