High-resolution cytometry of FISH dots in interphase cell nuclei

Flow cytometry (FCM) and laser scanning cytometry (LSCM) provide indispensable tools for measuring large number of cells with low resolution. Confocal microscopy, on the other hand, is used for measuring small number of cells with high resolution. In this paper, we present a reasonable compromise between the two extremes.

We have developed a completely automated, high-resolution system (high-resolution cytometer, HRCM) capable of analyzing microscope slides with FISH-stained interphase nuclei in two dimensions as well as in three dimensions using a fully motorized epi-fluorescence microscope and a cooled digital CCD camera fully controlled by a high-performance computer which performs both acquisition and related on-line image analysis. The images of different dyes are acquired sequentially using highly specific filters and superimposed in computer memory. For each nucleus and each hybridization dot, user-selected attributes (such as position, size, intensity, etc.) are computed off-line using another processor or computer connected with a network.

Using HRCM, it is possible to analyze multi-color preparations including UV-excited dyes as well as repeatedly hybridized preparations reacquiring 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 dimensions, but depends on the density of nuclei on the slide; the precision of the lateral and axial measurements is approximately 100 nm.

Thus, using overnight acquisition, quantities comparable to those of FCM or LSCM measurements can be analyzed with an accuracy comparable to confocal microscopy. HRCM is suitable for a number of clinical and scientific tasks: routine diagnostics, follow-up of therapy, studies of chromatin structure, and many other different aspects of cell research.