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Biological Procedures Online

Open Access

Practical three color live cell imaging by widefield microscopy

  • Jianrun Xia1,
  • Song Hon H. Kim1,
  • Susan Macmillan1 and
  • Ray Truant1Email author
Biological Procedures Online8:81063

Received: 10 April 2006

Accepted: 13 June 2006


Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope.

Indexing terms

Green Fluorescent ProteinsBlue Fluorescent ProteinAequorea Victoria