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Related Experiment Videos

A microscope-based flow cytophotometer.

H B Steen

    The Histochemical Journal
    |February 1, 1983
    PubMed
    Summary
    This summary is machine-generated.

    A novel flow chamber transforms a standard fluorescence microscope into a high-resolution flow cytophotometer. This system enables simultaneous measurement of cellular fluorescence and light scatter for detailed cell analysis.

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    Area of Science:

    • Biophysics
    • Cell Biology
    • Analytical Chemistry

    Background:

    • Standard fluorescence microscopy lacks the throughput for high-volume cell analysis.
    • Existing flow cytometry methods may have limitations in resolution or sensitivity for certain cellular components.

    Purpose of the Study:

    • To develop a versatile flow cytophotometer by modifying a standard fluorescence microscope.
    • To enable simultaneous measurement of multiple cellular parameters, including fluorescence and light scatter, with high resolution and sensitivity.

    Main Methods:

    • A new flow chamber design was implemented, integrating with a fluorescence microscope and oil immersion objective.
    • Cells were analyzed in a laminar flow stream, with simultaneous detection of fluorescence and dark-field scattered light.

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  • Epi-illumination and parfocal optics simplified instrument adjustment and high-magnification viewing.

    • Main Results:

    • The system achieved high resolution and sensitivity, capable of measuring bacterial DNA content (approx. 5 x 10(-15) g/cell) with a 6% coefficient of variance (CV).
    • Mammalian cell DNA histograms were generated with CVs below 1%.
    • Light scatter detection, integrated from 0-90 degrees, provided a signal proportional to cell dry weight, eliminating diffraction effects.

    Conclusions:

    • The developed flow cytophotometer offers a sensitive, high-resolution, and user-friendly platform for cellular analysis.
    • Simultaneous multi-parameter measurements, including fluorescence and light scatter, provide comprehensive cellular data.
    • The system's performance can be further enhanced by using an argon laser excitation source, increasing sensitivity and measurement rates.