Share Email Print

Proceedings Paper

Triggering of leukocytes by phase contrast in imaging cytometry with scanning fluorescence microscope (SFM)
Author(s): József Bocsi; Arkadiusz Pierzchalski; Monika Marecka; Wolf Malkusch; Attila Tárnok
Format Member Price Non-Member Price
PDF $17.00 $21.00

Paper Abstract

Slide-based cytometry (SBC) leads to breakthrough in cytometry of cells in tissues, culture and suspension. Carl Zeiss Imaging Solutions' new automated SFM combines imaging with cytometry. A critical step in image analysis is selection of appropriate triggering signal to detect all objects. Without correct target cell definition analysis is hampered. DNA-staining is among the most common triggering signals. However, the majority of DNA-dyes yield massive spillover into other fluorescence channels limiting their application. By microscopy objects of >5μm diameter can be easily detected by phase-contrast signal (PCS) without any staining. Aim was to establish PCS - triggering for cell identification. Axio Imager.Z1 motorized SFM was used (high-resolution digital camera, AxioCam MRm; AxioVision software: automatic multi-channel scanning, analysis). Leukocytes were stained with FITC (CD4, CD8) and APC (CD3) labelled antibodies in combinations using whole blood method. Samples were scanned in three channels (PCS/FITC/APC). Exposition-times for PCS were set as low as possible; the detection efficiency was verified by fluorescence. CD45-stained leukocytes were counted and compared to the number of PCS detected events. Leukocyte subtyping was compared with other cytometers. In focus the PCS of cells showed ring-form that was not optimal for cell definition. Out of focus PCS allows more effective qualitative and quantitative cell analyses. PCS was an accurate triggering signal for leukocytes enabling cell counting and discrimination of leukocytes from platelets. Leukocyte subpopulation frequencies were comparable to those obtained by other cytometers. In conclusion PCS is a suitable trigger-signal not interfering with fluorescence detection.

Paper Details

Date Published: 12 February 2009
PDF: 11 pages
Proc. SPIE 7182, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VII, 71821S (12 February 2009); doi: 10.1117/12.809191
Show Author Affiliations
József Bocsi, Univ. Leipzig (Germany)
Arkadiusz Pierzchalski, Univ. Leipzig (Germany)
Monika Marecka, Univ. Leipzig (Germany)
Wolf Malkusch, Carl Zeiss Imaging Solutions GmbH (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)

© SPIE. Terms of Use
Back to Top