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Related Experiment Videos

A flow cytometer designed for fluorescence calibration

H M Shapiro1, N G Perlmutter, P G Stein

  • 1Howard M. Shapiro, P.C., West Newton, Massachusetts, USA. hms@shapirolab.com

Cytometry
|October 17, 1998
PubMed
Summary
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This study presents a novel flow cytometer designed to minimize errors in fluorescence measurements. By using high-precision peak detectors and software-based compensation, the instrument achieves improved linearity for developing fluorescence standards.

Area of Science:

  • Biotechnology
  • Analytical Chemistry
  • Instrumentation

Background:

  • Accurate fluorescence measurements are crucial for developing standards and calibration materials.
  • Logarithmic amplifiers (log amps) and fluorescence compensation circuitry can introduce significant errors in flow cytometry.
  • Existing flow cytometer electronics may not provide the necessary linearity for precise measurements.

Purpose of the Study:

  • To develop a flow cytometer with enhanced linearity for accurate fluorescence measurements.
  • To minimize the impact of logarithmic amplifiers and compensation circuitry on measurement accuracy.
  • To create a reliable instrument for the development of fluorescence standards and calibration materials.

Main Methods:

  • Constructed a flow cytometer utilizing high-precision peak detectors (2 mV to 10 V range).

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  • Developed custom data acquisition software for transferring peak values to a 16-bit system.
  • Implemented software-based fluorescence compensation and a look-up table for logarithmic data transformation.

    • Main Results:

    • The developed instrument demonstrates substantially better linearity compared to conventional flow cytometers with log amps.
    • High sensitivity is achievable through photomultiplier tube gain adjustment.
    • Potential for full 4-decade dynamic range operation with improved data acquisition systems or digital processing.

    Conclusions:

    • The improved linearity of this flow cytometer is critical for accurate fluorescence standards development.
    • The instrument offers a more reliable platform for precise fluorescence measurements.
    • Further advancements in data acquisition and digital processing can enhance the instrument's dynamic range.