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Practical three color live cell imaging by widefield microscopy

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 simultane...

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Detalles Bibliográficos
Autores principales: Xia, Jianrun, Kim, Song Hon H., Macmillan, Susan, Truant, Ray
Formato: Texto
Lenguaje:English
Publicado: Biological Procedures Online 2006
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1523422/
https://www.ncbi.nlm.nih.gov/pubmed/16909160
http://dx.doi.org/10.1251/bpo119
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author Xia, Jianrun
Kim, Song Hon H.
Macmillan, Susan
Truant, Ray
author_facet Xia, Jianrun
Kim, Song Hon H.
Macmillan, Susan
Truant, Ray
author_sort Xia, Jianrun
collection PubMed
description 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.
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spelling pubmed-15234222006-08-11 Practical three color live cell imaging by widefield microscopy Xia, Jianrun Kim, Song Hon H. Macmillan, Susan Truant, Ray Biol Proced Online Research 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. Biological Procedures Online 2006-07-21 /pmc/articles/PMC1523422/ /pubmed/16909160 http://dx.doi.org/10.1251/bpo119 Text en Copyright © July 07, 2006, J Xia et al. This paper is Open Access and is published in Biological Procedures Online under license from the authors. Copying, printing, redistribution and storage permitted.
spellingShingle Research Article
Xia, Jianrun
Kim, Song Hon H.
Macmillan, Susan
Truant, Ray
Practical three color live cell imaging by widefield microscopy
title Practical three color live cell imaging by widefield microscopy
title_full Practical three color live cell imaging by widefield microscopy
title_fullStr Practical three color live cell imaging by widefield microscopy
title_full_unstemmed Practical three color live cell imaging by widefield microscopy
title_short Practical three color live cell imaging by widefield microscopy
title_sort practical three color live cell imaging by widefield microscopy
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1523422/
https://www.ncbi.nlm.nih.gov/pubmed/16909160
http://dx.doi.org/10.1251/bpo119
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AT kimsonghonh practicalthreecolorlivecellimagingbywidefieldmicroscopy
AT macmillansusan practicalthreecolorlivecellimagingbywidefieldmicroscopy
AT truantray practicalthreecolorlivecellimagingbywidefieldmicroscopy