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(American Journal of Pathology. 2003;162:373-379.)
© 2003 American Society for Investigative Pathology

Technical Advance

Multicolor Deconvolution Microscopy of Thick Biological Specimens

Christine Maierhofer*{dagger}, Rainer Gangnus*{dagger}, Joachim Diebold{ddagger} and Michael R. Speicher*{dagger}

From the Institut für Humangenetik,* Technische Universität München, München; the Institute of Pathology,{ddagger} Universität München, München; and the Institut für Humangenetik,{dagger} Gesellschaft für Strahlenforschung Forschungszentrum für Umwelt und Gesundheit, Neuherberg, Germany

One limitation in understanding disease at the cellular level has been the inability to efficiently analyze DNA on a cell-to-cell basis within the natural tissue context. However, DNA analyses at a single-cell resolution should be instrumental for the understanding of cancer cell biology, cancer evolution, for chromosomal mosaic analysis and rare cell events, and should provide otherwise inaccessible information on essential biological processes. Here we present a fluorescence in situ hybridization-based multicolor deconvolution technique for three-dimensional microscopy. We use up to seven different color channels for probe detection, which allows the simultaneous high-resolution localization of multiple point-like sources within a biological specimen with a thickness of up to 30 µm. In addition, a DNA counterstain is used for volume labeling of the nuclei offering the opportunity for a simultaneous segmentation of nuclei. Furthermore, as the instrumentation consists of a standard fluorescence microscope it represents a low-cost method as compared to confocal microscopy.




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