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Related Concept Videos

Immunofluorescence Microscopy01:12

Immunofluorescence Microscopy

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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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Cyclic Immunofluorescence (CycIF), A Highly Multiplexed Method for Single-cell Imaging.

Jia-Ren Lin1, Mohammad Fallahi-Sichani1, Jia-Yun Chen1

  • 1HMS LINCS Center, Laboratory of Systems Pharmacology, Department of Systems Biology, Harvard Medical School, Boston, Massachusetts.

Current Protocols in Chemical Biology
|December 8, 2016
PubMed
Summary
This summary is machine-generated.

Cyclic Immunofluorescence (CycIF) enables highly multiplexed imaging on standard microscopes. This method preserves cell morphology and enhances signal-to-noise ratios for detailed cellular analysis.

Keywords:
CycIFhigh-content imagingimmunofluorescencemultiplexingsystems biology

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Area of Science:

  • Cell biology
  • Microscopy techniques
  • Immunofluorescence imaging

Background:

  • Multiplexed immunofluorescence is crucial for deep biological insights.
  • Existing methods often require specialized equipment or harsh conditions.
  • There is a need for accessible, high-content imaging techniques.

Purpose of the Study:

  • To introduce Cyclic Immunofluorescence (CycIF), a public-domain method for multiplexed immunofluorescence.
  • To describe variants of CycIF suitable for different biological applications.
  • To demonstrate the preservation of cell morphology and signal enhancement.

Main Methods:

  • Sequential imaging of 4- to 6-color fluorescence followed by fluorophore inactivation.
  • Direct conjugation of antibodies to Alexa Fluor dyes for fixed-cell imaging.
  • Chemical inactivation of fluorophores using a mild base, hydrogen peroxide, and light.
  • Adaptations for indirect immunofluorescence and genetically encoded fluorescent proteins.

Main Results:

  • CycIF allows imaging of up to 30 channels using conventional epifluorescence microscopes.
  • Cell morphology is maintained across multiple imaging rounds.
  • Signal-to-noise ratios improve with repeated CycIF cycles.
  • Gentle protocol suitable for cultured cell monolayers, unlike antibody-stripping methods.

Conclusions:

  • CycIF provides a versatile and accessible platform for high-content multiplexed immunofluorescence.
  • The method is adaptable for various immunofluorescence and live-cell imaging applications.
  • CycIF enhances imaging capabilities without compromising cellular integrity.