Process for identifying FISH signals
The invention herein includes a method for combining immuno staining and FISH using covalently bound small molecule tags. Among embodiments included are methods of tagging immuno and FISH probes for treatment of biologic material. Embodiments comprise directing the fluorescently labeled immuno probe(s) to non-chromosomic portions of a biologic sample and directing labeled FISH probe(s) to chromosomic portions of the biologic sample for further identification of subcellular components. Embodiments also comprise the automatic connection of information regarding the sample to image analysis, to subcellular component identification and enumeration in order to affect biomedical decisions.
1. A process within an epi-fluorescence system for identifying and enumerating fluorescent in situ hybridization (“FISH”) signals produced with respect to nuclear components hybridized in situ with fluorescent markers comprising;
acquiring using an epi-fluorescence microscope a plurality of images at different focal planes in each fluorescence channel corresponding to the hybridized FISH markers;
selecting a best focused image from said plurality of images for each nucleus;
selecting for each nucleus the one focal plane above and the one focal plane below the focal plane of said best focused image in which the image is best focused;
combining said images from the one focal plane above and below said best focused image with said best focused image of each said nucleus to produce a combined image for said nucleus;
analyzing the combined image of each nucleus to separate background pixels from signal pixels, and to determine areas of produced signals corresponding to pre-set size and shape criteria corresponding to a non-artifactual target; and,
outputting results of said analyzing step on a display.