
Proceedings Paper
Evaluation of optimal DNA staining for triggering by scanning fluorescence microscopy (SFM)Format | Member Price | Non-Member Price |
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Paper Abstract
In imaging and flow cytometry, DNA staining is a common trigger signal for cell identification. Selection of
the proper DNA dye is restricted by the hardware configuration of the instrument. The Zeiss Imaging Solutions
GmbH (München, Germany) introduced a new automated scanning fluorescence microscope - SFM (Axio
Imager.Z1) which combines fluorescence imaging with cytometric parameters measurement. The aim of the
study was to select optimal DNA dyes as trigger signal in leukocyte detection and subsequent cytometric
analysis of double-labeled leukocytes by SFM.
Seven DNA dyes (DAPI, Hoechst 33258, Hoechst 33342, POPO-3, PI,
7-AAD, and TOPRO-3) were tested and
found to be suitable for the implemented filtersets (fs) of the SFM (fs: 49, fs: 44, fs: 20). EDTA blood was
stained after erythrocyte lysis with DNA dye. Cells were transferred on microscopic slides and embedded in
fluorescent mounting medium. Quality of DNA fluorescence signal as well as spillover signals were analyzed
by SFM. CD45-APC and CD3-PE as well as CD4-FITC and CD8-APC were selected for immunophenotyping
and used in combination with Hoechst.
Within the tested DNA dyes DAPI showed relatively low spillover and the best CV value. Due to the low
spillover of UV DNA dyes a triple staining of Hoechst and APC and PE (or APC and FITC, respectively) could
be analyzed without difficulty. These results were confirmed by FCM measurements.
DNA fluorescence is applicable for identifying and triggering leukocytes in SFM analyses. Although some
DNA dyes exhibit strong spillover in other fluorescence channels, it was possible to immunophenotype
leukocytes. DAPI seems to be best suitable for use in the SFM system and will be used in protocol setups as
primary parameter.
Paper Details
Date Published: 12 February 2009
PDF: 12 pages
Proc. SPIE 7182, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VII, 71820L (12 February 2009); doi: 10.1117/12.808920
Published in SPIE Proceedings Vol. 7182:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VII
Daniel L. Farkas; Dan V. Nicolau; Robert C. Leif, Editor(s)
PDF: 12 pages
Proc. SPIE 7182, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VII, 71820L (12 February 2009); doi: 10.1117/12.808920
Show Author Affiliations
Anja Mittag, Univ. Leipzig (Germany)
Monika Marecka, Univ. Leipzig (Germany)
Arkadiusz Pierzchalski, Univ. Leipzig (Germany)
Monika Marecka, Univ. Leipzig (Germany)
Arkadiusz Pierzchalski, Univ. Leipzig (Germany)
Wolf Malkusch, Carl Zeiss Imaging Solutions GmbH (Germany)
József Bocsi, Univ. Leipzig (Germany)
Attila Tárnok, Univ. Leipzig (Germany)
József Bocsi, Univ. Leipzig (Germany)
Attila Tárnok, Univ. Leipzig (Germany)
Published in SPIE Proceedings Vol. 7182:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VII
Daniel L. Farkas; Dan V. Nicolau; Robert C. Leif, Editor(s)
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