High-resolution cytometry of FISH dots in interphase cell nuclei

Background: Flow cytometry (FCM) and laser scanning cytometry (LSCM) provid e indispensable tools for measuring large number of cells with low resoluti on. Confocal microscopy, on the other hand, is used for measuring small num ber of cells with high resolution. In this paper, we present a reasonable c ompromise between the two extremes. Methods: We have developed a completely automated, high-resolution system ( high-resolution cytometer, HRCM) capable of analyzing microscope slides wit h FISH-stained interphase nuclei in two dimensions as well as in three dime nsions using a fully motorized epi-fluorescence microscope and a cooled dig ital CCD camera fully controlled by a high-performance computer which perfo rms both acquisition and related on-line image analysis. The images of diff erent dyes are acquired sequentially using highly specific filters and supe rimposed in computer memory. For each nucleus and each hybridization dot, u ser-selected attributes (such as position, size, intensity, etc.) are compu ted off-line using another processor or computer connected with a network. Results: Using HRCM, it is possible to analyze multi-color preparations inc luding W-excited dyes as well as repeatedly hybridized preparations reacqui ring individual nuclei. The speed of the acquisition and analysis is about 50 nuclei per minute in two dimensions and 1 nucleus per minute in three di mensions, but depends on the density of nuclei on the slide; the precision of the lateral and axial measurements is approximately 100 nm. Conclusions: Thus, using overnight acquisition, quanti ties comparable to t hose of FCM or LSCM measurements can be analyzed with an accuracy comparabl e to confocal microscopy. HRCM is suitable for a number of clinical and sci entific tasks: routine diagnostics, follow-up of therapy, studies of chroma tin structure, and many other different aspects of cell research. (C) 1999 Wiley-Liss, Inc.