Signal to noise analysis of multiple color fluorescence imaging microscopy

Background: Various approaches that were recently developed demonstrate the ability to simultaneously detect all human (or other species) chromosomes by using combinatorial labeling and fluorescence in situ hybridization (FIS H). With the growing interest in this field, it is important to develop too ls for optimizing and estimating the accuracy of different experimental met hods. Methods: We have analyzed the principles of multiple color fluorescence ima ging microscopy. First, formalism based on the physical principles of fluor escence microscopy and noise analysis is introduced. Next, a signal to nois e (S/N) analysis is performed and summarized in a simple accuracy criterion . The analysis assumes shot noise to be the dominant source of noise. Results: The accuracy criterion was used to calculate the S/N of multicolor FISH (IM-FISH), spectral karyotyping, ratio imaging, and a method based on using a set of broad band filters. Spectral karyotyping is tested on vario us types of samples and shows accurate classifications. We ha iie also test ed classification accuracy as a function of total measurement time. Conclusions: The accuracy criterion th:lt we have developed can be used for optimizing and analyzing different multiple color fluorescence microscopy methods. The assumption that shot noise is dominant in these measurements i s supported by our measurements. (C) 1999 Wiley-Liss, Inc.