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Related Experiment Videos

Practical three color live cell imaging by widefield microscopy.

Jianrun Xia1, Song Hon H Kim, Susan Macmillan

  • 1Department of Biochemistry and Biomedical Sciences, McMaster University, HSC 4H24A 1200 Main Street West, Hamilton, Ontario, L8N 3Z5, Canada.

Biological Procedures Online
|August 16, 2006
PubMed
Summary

PS-CFP2 is a photostable blue fluorescent protein ideal for live cell imaging. It enables distinct three-color imaging with standard microscopes, even with high protein expression.

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Microscopy

Background:

  • Fluorescent proteins are essential tools for live cell imaging.
  • Multi-color imaging using aequorea fluorescent protein (AFP) derivatives is powerful but can be limited by spectral overlap.
  • Sophisticated and expensive microscopes are often required for multi-color imaging.

Purpose of the Study:

  • To evaluate the aequorea coerulescens fluorescent protein derivative, PS-CFP2, as a blue fluorophore for live cell imaging.
  • To determine if PS-CFP2 can be used with standard filter sets and widefield microscopes.
  • To assess the photostability and potential for artifactual interactions of PS-CFP2 fusions.

Main Methods:

  • Live cell fluorescence microscopy.
  • Widefield illumination.

Related Experiment Videos

  • Fusion of PS-CFP2 to proteins forming cytoplasmic or nuclear puncta.
  • Imaging with standard filter sets.

    • Main Results:

    • PS-CFP2 exhibits excellent practical properties as a blue fluorophore with distinct spectral properties.
    • PS-CFP2 can be imaged using standard filter sets on a widefield microscope.
    • PS-CFP2 demonstrates high photostability under widefield illumination in live cells.
    • PS-CFP2 fusions do not artifactually interact with other AFP fusion proteins, even at high expression levels.

    Conclusions:

    • PS-CFP2 is a valuable blue fluorophore for distinct three-color imaging.
    • It is compatible with standard, inexpensive microscopy setups.
    • PS-CFP2 facilitates multi-color live cell imaging alongside eGFP and mRFP without spectral interference or artifacts.