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. 2006;8:63-8.
doi: 10.1251/bpo119. Epub 2006 Jul 21.

Practical Three Color Live Cell Imaging by Widefield Microscopy

Free PMC article

Practical Three Color Live Cell Imaging by Widefield Microscopy

Jianrun Xia et al. Biol Proced Online. .
Free PMC article


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.


Fig. 1
Fig. 1. PSCFP2 Can be Distinctly Imaged with a DAPI/hoechst Filter Set.
Panels a-d, Transient transfection of 1ug each eGFP-ASF, PSCFP2-Mito localization signal, mRFP-huntingtin Exon1 Q138 fragment after 24 hours in NIH3T3 (non-plasmid replicating) cells. Less than 200 msec required for each channel of exposure. Scale bar is 10 um. Panels e-I, live cell four color imaging of PSCFP2-NXF1, mCerProfilin1, eYFP-Synapsin1, and mRFP-huntingtin exon1Q138. Distinct signals could be detected for each of the proteins. 4 channel color images and merge were assembled in Imaris 4.3 (Bitplane, Zurich, Switzerland). Scale bar is 10 μm.
Fig. 2
Fig. 2. Live cell Imaging over Time of RGB Fluorescent Proteins That Can form Bright Puncta.
A. Three color imaging using a triple cube filter set for DAPI/FITC/Texas Red (Semrock DA/FI/TX-B) and switching only excitation filters for three proteins that form independent bright puncta: huntingtinExon1Q138-mRFP, Mito-eGFP, and PSCFP2-NXf1. Bright puncta in each channels are highlighted with white arrows. B. Time course of mRFP-huntingtin Q138 exon1 fragment, prone to aggregation, eGFP-ASF2, nuclear speckle protein, and PSCFP2-NXF1, a mRNA export factor in STHdh cells. Three color images were captured every minute for 75 minutes. Developing polyglutamine inclusions of huntingtin can be seen in the red channel without bleed to green or blue channels and no artifactual accumulation of the other two fusion proteins into the large polyglutamine aggregates (top row). Scale bar is 10 μm. Also in supplemental video 1.
Fig. 3
Fig. 3. Under Wide Field Illumination, PSCFP2 is Photostable.
A. fluorescent proteins, without fused sequences, were continuously irradiated for 300 seconds with appropriate filter sets, with images captured every two seconds in live NIH 3T3 cells. Monochrome images were quantified using Image J (NIH), normalized to start fluorescent levels, and plotted over time. Deviations across three experiments for each fluorophore was <5%. B. PSCFP2 does not photoactivate with 175W unfiltered xenon light over 15 minutes constant irradiation. Light was provided without filtration through a liquid light guide and using a plan apochromat NA1.3 63X oil immersion objective (Nikon). While slight photobleaching was noted, (panels a versus c), no cyan fluorescence was detected above CCD camera noise (panels b,d). Scale bar is 10 μm.

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