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Immunofluorescence Microscopy01:12

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A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.
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A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules
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Published on: March 10, 2017

Fluorescence-based assays.

W Frank An1

  • 1Chemical Biology Platform, The Broad Institute of Harvard and MIT, Cambridge, MA, USA. fan@broad.harvard.edu

Methods in Molecular Biology (Clifton, N.J.)
|April 7, 2009
PubMed
Summary
This summary is machine-generated.

Fluorescence assays offer sensitive, versatile tools for high-throughput screening in life sciences. This chapter details two methods, total intensity and fluorescence resonance energy transfer, for cell viability and protein interactions, respectively.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Biotechnology

Background:

  • Fluorescence-based assays are essential for high-throughput screening (HTS) due to their sensitivity and versatility.
  • They enable monitoring of diverse biological processes including molecular interactions, enzyme activity, and cell health.
  • Applications span various life-science research areas, from molecular dynamics to cellular distribution studies.

Purpose of the Study:

  • To describe two key fluorescence-based techniques for cell-based HTS.
  • To illustrate the application of total intensity measurement for assessing cell viability.
  • To demonstrate the use of fluorescence resonance energy transfer (FRET) for studying protein folding and interactions.

Main Methods:

  • Total intensity measurement for cell viability assessment.
  • Fluorescence resonance energy transfer (FRET) for protein folding and interaction analysis.
  • Application in plate-reader and automated microscope-based HTS formats.

Main Results:

  • Total intensity provides a reliable readout for cell viability in HTS.
  • FRET effectively indicates protein folding status and interaction dynamics.
  • Both techniques are suitable for automated, high-throughput cell-based screening.

Conclusions:

  • Fluorescence assays are powerful, adaptable tools for life-science HTS.
  • Total intensity and FRET offer distinct yet complementary approaches for biological investigations.
  • These methods facilitate efficient, large-scale screening in both plate-reader and microscopy formats.