Multifluor-fluorescence in-situ hybridization (M-FISH) imaging techniques using multiple multiband filters with image registration

摘要

Techniques for acquiring and analyzing M-FISH images in a manner that allows for proper image registration; and techniques for combining a plurality of registered images to provide labeled images. Epi-illumination embodiments use multiple filter cubes for different subsets of the entire set of desired dyes. The registration problem is addressed by configuring the different multiband cubes so that pairs of cubes have a common dye for which they are configured. Accordingly, a first set of images generated with a first cube includes an image that corresponds to (contains the same features as) one of the images in a second set of images generated with a second cube. This allows the first set of images to be precisely and accurately aligned with the second set of images. A method for generating a labeled image typically includes a normalization of the pixel intensities within each image, followed by a transformation to ratio images.