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.

Faculty of Informatics Masaryk University Brno Czech Republic

Citace poskytuje Crossref.org

Effect of gadolinium-based nanoparticles on nuclear DNA damage and repair in glioblastoma tumor cells

Inhibition of ATR kinase with the selective inhibitor VE-821 results in radiosensitization of cells of promyelocytic leukaemia (HL-60)

Irradiated stem cells and ageing of the haematopoietic system

Prediction of localization and interactions of apoptotic proteins

Single-cell c-myc gene expression in relationship to nuclear domains

Differentiation-specific association of HP1alpha and HP1beta with chromocentres is correlated with clustering of TIF1beta at these sites

Topography of genetic loci in the nuclei of cells of colorectal carcinoma and adjacent tissue of colonic epithelium

Cytogenetics and cytology of retinoblastomas

The 3D structure of human chromosomes in cell nuclei

Spatial arrangement of genes, centromeres and chromosomes in human blood cell nuclei and its changes during the cell cycle, differentiation and after irradiation