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Background: The recently developed technique of high-resolution cytometry (HRCM) enables automated acquisition and analysis of fluorescent in situ hybridization (FISH)-stained cell nuclei using conventional wide-field fluorescence microscopy. The method has now been extended to confocal imaging and offers the opportunity to combine the advantages of confocal and wide-field modes., Methods: We have automated image acquisition and analysis from a standard inverted fluorescence microscope equipped with a confocal module with Nipkow disk and a cooled digital CCD camera. The system is fully controlled by a high-performance computer that performs both acquisition and related on-line image analysis. The system can be used either for an automatic two (2D) and three-dimensional (3D) analysis of FISH- stained interphase nuclei or for a semiautomatic 3D analysis of FISH-stained cells in tissues. The user can select which fluorochromes are acquired using wide-field mode and which using confocal mode. The wide-field and confocal images are overlaid automatically in computer memory. The developed software compensates automatically for both chromatic color shifts and spatial shifts caused by switching to a different imaging mode., Results: Using the combined confocal and wide-field HRCM technique, it is possible to take advantage of both imaging modes. Images of some dyes (such as small hybridization dots or counterstain images of individual interphase nuclei) do not require confocal quality and can be acquired quickly in wide-field mode. On the contrary, images of other dyes (such as chromosome territories or counterstain images of cells in tissues) do require improved quality and are acquired in confocal mode. The dual-mode approach is two to three times faster compared with the single-mode confocal approach and the spectrum of its applications is much broader compared with both single-mode confocal and single-mode wide-field systems., Conclusions: The combination of high speed specific to the wide-field mode and high quality specific to the confocal mode gives optimal system performance., (Copyright 2001 Wiley-Liss, Inc.)

Background: 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., Methods: 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., Results: 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., Conclusions: 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., (Copyright 1999 Wiley-Liss, Inc.)