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Related Experiment Videos

Scanning fluorescent microscopy analysis is applicable for absolute and relative cell frequency determinations.

József Bocsi1, Viktor Sebestyén Varga, Béla Molnár

  • 11st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
|September 8, 2004
PubMed
Summary

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Scanning fluorescent microscopy (SFM) offers a reliable alternative for cell analysis, comparable to laser scanning cytometry (LSC) and flow cytometry (FCM). Slide-based cytometry (SBC) excels in detecting rare cells, with SFM being a promising adaptation for this purpose.

Area of Science:

  • Biomedical Engineering
  • Cell Biology
  • Analytical Chemistry

Background:

  • Flow cytometry (FCM) and laser scanning cytometry (LSC) are established methods for fluorescent cell analysis.
  • A novel scanning fluorescent microscopy (SFM) technique has been developed.
  • This study evaluates SFM against LSC and FCM in both experimental and clinical contexts.

Purpose of the Study:

  • To compare the performance of Scanning Fluorescent Microscopy (SFM) with Laser Scanning Cytometry (LSC) and Flow Cytometry (FCM).
  • To assess the suitability of SFM and LSC (slide-based cytometry techniques) for rare-cell detection.
  • To validate SFM as a reliable alternative for fluorescent cell analysis.

Main Methods:

  • Relative cell frequencies were determined using serially diluted HT29 colorectal cancer cells and Ficoll-separated blood mononuclear cells (FSBMCs) across SFM, LSC, and FCM.

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  • Absolute cell numbers for slide-based cytometry (SBC) were determined using smeared FSBMCs and micromanipulator-placed HT29 cells.
  • Clinical samples involved isolating circulating tumor cells from colorectal cancer patients' blood, with double-staining for CD45 and CAM5.2, and nuclear counterstaining with TOTO-3 and Hoechst 33258.

    • Main Results:

    • For relative cell frequency, SFM, LSC, and FCM showed correlations of r(2) = 0.79, 0.62, and 0.84, respectively (P < 0.01).
    • SFM and LSC demonstrated a high correlation (r(2) = 0.97; P < 0.01) for absolute cell frequency determination.
    • SFM proved comparable to LSC and FCM in experimental and clinical settings.

    Conclusions:

    • Scanning fluorescent microscopy (SFM) is a dependable method for fluorescent cell analysis, yielding results comparable to LSC and FCM.
    • Slide-based cytometry (SBC) techniques, including SFM, are more effective than FCM for detecting rare cells.
    • SFM, particularly with digital slides, represents a viable adaptation of conventional fluorescent microscopes for rare-cell detection.