Triggering of leukocytes by phase contrast in imaging cytometry with scanning fluorescence microscope (SFM)

József Bocsi; Arkadiusz Pierzchalski; Monika Marecka; Wolf Malkusch; Attila Tárnok

doi:10.1117/12.809191

12 February 2009 Triggering of leukocytes by phase contrast in imaging cytometry with scanning fluorescence microscope (SFM)

József Bocsi, Arkadiusz Pierzchalski, Monika Marecka, Wolf Malkusch, Attila Tárnok

Author Affiliations +

Proceedings Volume 7182, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VII; 71821S (2009) https://doi.org/10.1117/12.809191
Event: SPIE BiOS, 2009, San Jose, California, United States

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.

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József Bocsi, Arkadiusz Pierzchalski, Monika Marecka, Wolf Malkusch, and Attila Tárnok "Triggering of leukocytes by phase contrast in imaging cytometry with scanning fluorescence microscope (SFM)", Proc. SPIE 7182, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VII, 71821S (12 February 2009); https://doi.org/10.1117/12.809191

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KEYWORDS

Luminescence

Signal detection

Phase contrast

Atomic force microscopy

Blood

Microscopy

Microscopes

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