Laser-scanning cytometry: A new instrumentation with many applications

The laser-scanning cytometer (LSC) is a microscope-based cytofluorometer wh ich has attributes of both dow and image cytometry. Laser-excited fluoresce nce emitted from fluorochromed individual cells on a microscope slide is me asured at multiple wavelengths rapidly with high sensitivity and accuracy. Though the instrument has been available commercially for only 3 years, it is already used in a variety of different applications in many laboratories . This review focuses on the following unique analytical capabilities of LS C which complement those of flow cytometry and fluorescence image analysis: (a) the cells are positioned on slides during measurement so they may be e xamined repeatedly over time, a feature useful for studies of enzyme kineti cs and other time-resolved processes; (b) sequential analysis of the same c ells can be carried out using different immuno- or cytochemical stains or g enetic probes, merging information on cell immunophenotype, cell functions, expression of particular proteins, DNA ploidy and cell cycle position, and /or cytogenetic profile for each measured cell; (c) any of the cells measur ed can be relocated to correlate with visual examination by fluorescence or brightfield microscopy or with any other parameter; (d) topographic distri bution of fluorescence measurements within the cell, in cytoplasm vs nucleu s, permits analysis of the translocation of regulatory molecules such as NF kappa B, p53, etc., and is essential for FISH analysis; (e) hyperchromicit y of nuclear DNA as measured by maximal pixel fluorescence intensity allows one to identify cell types differing in degree of chromatin condensation s uch as mitotic or apoptotic cells; (f) analysis of tissue section architect ure and of the constituents in transected cells within tissue sections by r atiometric assays normalized to DNA content extends applications of LSC in clinical pathology; (g) because cell loss during sample preparation and sta ining is minimal, samples with a paucity of cells can be analyzed; and (h) analyzed cells can be stored indefinitely, e.g., for archival preservation or additional analysis. Potential future applications of LSC are discussed, (C) 1999 Academic h ess.