Hyperchromatic laser scanning cytometry

Attila Tárnok; Anja Mittag

doi:10.1117/12.698597

6 February 2007 Hyperchromatic laser scanning cytometry

Attila Tárnok, Anja Mittag

Proceedings Volume 6430, Advanced Biomedical and Clinical Diagnostic Systems V; 643011 (2007) https://doi.org/10.1117/12.698597
Event: SPIE BiOS, 2007, San Jose, California, United States

Abstract

In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

Citation Download Citation

Attila Tárnok and Anja Mittag "Hyperchromatic laser scanning cytometry", Proc. SPIE 6430, Advanced Biomedical and Clinical Diagnostic Systems V, 643011 (6 February 2007); https://doi.org/10.1117/12.698597

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KEYWORDS

Luminescence

Biological research

Analytical research

Fluorescence resonance energy transfer

Laser scanners

Neodymium

Statistical analysis

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