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Related Concept Videos

Flow Cytometry01:23

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The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
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Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction
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Fluorescent Proteins for Flow Cytometry.

Teresa S Hawley1, Robert G Hawley2, William G Telford3

  • 1Flow Cytometry Core Facility, Laboratory of Genome Integrity, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

Current Protocols in Cytometry
|April 4, 2017
PubMed
Summary

Spectral flow cytometry offers a promising solution for detecting multiple fluorescent proteins, overcoming challenges posed by spectral overlap in conventional methods. This technique enhances multicolor analysis in cell biology research.

Keywords:
conventional flow cytometryfluorescent proteinslasersspectral flow cytometry

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Area of Science:

  • Biotechnology
  • Cell Biology
  • Spectroscopy

Background:

  • Fluorescent proteins (FPs) are essential tools in cell and molecular biology, offering a wide spectrum of colors.
  • Advancements in solid-state lasers have improved excitation capabilities for FPs.
  • Simultaneous detection of multiple FPs is theoretically possible but practically challenging due to spectral overlap.

Purpose of the Study:

  • To review the development and applications of fluorescent proteins.
  • To discuss strategies for overcoming spectral overlap in multicolor detection.
  • To highlight the potential of spectral flow cytometry for enhanced FP analysis.

Main Methods:

  • Review of fluorescent protein development and characteristics.
  • Analysis of spectral overlap issues in conventional flow cytometry.
  • Introduction to spectral flow cytometry principles and advantages.

Main Results:

  • Broad excitation and emission spectra of FPs cause significant spectral overlap.
  • Conventional flow cytometry requires careful filter and wavelength selection.
  • Spectral flow cytometry provides a more facile detection of multiple FPs by moving beyond the "one color, one detector" limitation.

Conclusions:

  • Spectral flow cytometry is a promising emerging methodology for multicolor fluorescent protein detection.
  • This technique addresses the limitations of conventional flow cytometry in handling spectral overlap.
  • The chapter provides a comprehensive overview of FPs, detection strategies, and applications.