Microscopy on the Fly

CompuCyte's Laser Scanning Cytometer Brings Scanning Capabilities to Fluorescence Microscopy Anyone who has ever got a headache from staring into a microscope for an extended period to count fluorescently labeled cells will appreciate the utility of the Laser Scanning Cytometer LSC® rom CompuCyte. As a slide-based laser scanning instrument that offers the analytical and quantitative features of a flow cytometer, the LSC pioneers a new dimension in fluorescence microscopy. The LSC will automatically scan a microscope slide and identify, count, and record the positions of fluorescently labeled cells. This scanning produces data very similar to that of flow cytometry but with a significant added benefit. Since the cells are fixed to a solid support and their positions are known, any identified cell can be relocated and viewed again. This feature is especially useful for correlating more than one label or identifying a particular distinguishing characteristic of labeled cells. Laser Scanning Cytometry System Using technology similar to that found in most flow cytometers, LSC measures a cell's light scatter and fluorescence by shining a sharply defined laser beam at each cell. In flow cytometry, these aspects are recorded as cells flow past the laser. However, with LSC, the slide and laser beam move so that fluorescence is sampled at 0.5 µm intervals in the X and Y directions across the slide. Use of both argon and helium-neon lasers provides a wide range of excitation peaks, and four photomultiplier tubes collect emission data, each tube detecting fluorescence in a different range of wavelengths. In addition, fluorescent or bright-field cell images can be visualized through the eyepiece or collected using a CCD camera. Since the instrument performs the scanning automatically, data for thousands of cells can be collected. The LSC combines existing technology in a new way that offers analytic benefits not previously available. Being able to collect quantitative information similar to that obtained from flow cytometry, in addition to standard fluorescent microscopy imaging, provides a combination with capabilities that neither fluorescence microscopy nor flow cytometry offers alone. Gene Zwolinski, vice president of marketing for CompuCyte, pointed out that the software, which identifies and correlates specific features with particular cells that the researcher can go back and find, is really the unique feature of the instrument. Zwolinski also noted that the system provides much less subjective data since "it automatically scans cells so you don't have to choose cells like with image analysis." Dr. Michael Grace, a development fellow in the gene therapy effort at Schering-Plough, has been using the LSC instrument to analyze cells that express a particular nuclear transgene expressed during apoptosis. He said that he and his colleagues use the instrument to "scan 50,000 to 100,000 nuclei and generate a positional bitmap of the labeled cells in a tissue section." Before undertaking these studies, they compared LSC with standard flow cytometry and found that the data obtained from both was "highly correlative." They have used the instrument for double-labeling and are planning to try triple-labeling experiments in the near future. He added that he was "very impressed with the software; having worked with several very kludgy systems in the past, this is very good." In terms of improvements, he would like to see a UV laser added to the instrument. Dr. Zbigniew Darzynkiewicz, at the New York Medical College Cancer Research Institute, has used the LSC to look at the kinetics of amino peptidase and esterase activities in vivo. He mentioned that "the ability to measure a group of cells over and over again makes it possible to see changes in the cells over time," something that is not possible with a flow cytometer. He also uses the instrument for apoptosis studies. Apoptosis is actually described by morphological changes in cells, and cells can sometimes be misclassified by the standard apoptotic probe assays. He mentioned that LSC gives them the ability to "go back and relocate, to confirm a cell is apoptotic," which enables them to "identify false positives." Overall, he feels LSC provides the advantages of flow cytometry with morphological analysis, which offers unique capabilities. However, he would like to see a higher quality CCD camera incorporated in the instrument. In the future, Darzynkiewicz looks to a time when lifetime measurements of fluorochromes could be done, which would open up the possibility of studying molecular interactions. For additional information on this product, please circle readers service #71 or call CompuCyte at 800-840-1303 or visit the company web site: www.compucyte.com. Author Paul Diehl can be reached at paul_vfocus@msn.com.

Microscopy on the Fly

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