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Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy

Lansford, Rusty and Bearman, Gregory and Fraser, Scott E. (2001) Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy. Journal of Biomedical Optics, 6 (3). pp. 311-318. ISSN 1083-3668. https://resolver.caltech.edu/CaltechAUTHORS:20160201-085751916

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Abstract

The imaging of living cells and tissues using laser-scanning microscopy is offering dramatic insights into the spatial and temporal controls of biological processes. The availability of genetically encoded labels such as green fluorescent protein (GFP) offers unique opportunities by which to trace cell movements, cell signaling or gene expression dynamically in developing embryos. Two-photon laser scanning microscopy (TPLSM) is ideally suited to imaging cells in vivo due to its deeper tissue penetration and reduced phototoxicity; however, in TPLSM the excitation and emission spectra of GFP and its color variants [e.g., CyanFP (CFP); yellowFP (YFP)] are insufficiently distinct to be uniquely imaged by conventional means. To surmount such difficulties, we have combined the technologies of TPLSM and imaging spectroscopy to unambiguously identify CFP, GFP, YFP, and redFP (RFP) as well as conventional dyes, and have tested the approach in cell lines. In our approach, a liquid crystal tunable filter was used to collect the emission spectrum of each pixel within the TPLSM image. Based on the fluorescent emission spectra, supervised classification and linear unmixing analysis algorithms were used to identify the nature and relative amounts of the fluorescent proteins expressed in the cells. In a most extreme case, we have used the approach to separate GFP and fluorescein, separated by only 7 nm, and appear somewhat indistinguishable by conventional techniques. This approach offers the needed ability to concurrently image multiple colored, spectrally overlapping marker proteins within living cells.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1117/1.1383780DOIArticle
http://biomedicaloptics.spiedigitallibrary.org/article.aspx?articleid=1101398PublisherArticle
ORCID:
AuthorORCID
Lansford, Rusty0000-0002-2159-3699
Fraser, Scott E.0000-0002-5377-0223
Additional Information:© 2001 SPIE. Paper JBO-FM-05 received Feb. 15, 2001; accepted for publication Mar. 20, 2001. A Silvio Conte Research Center grant (NIMH), a grant from the NCRR, The Beckman Institute, and a Muscular Dystrophy Association Research Development award to one of the authors (R.L.) supported this work.
Funders:
Funding AgencyGrant Number
National Institute of Mental Health (NIMH)UNSPECIFIED
National Center for Research Resources (NCRR)UNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Muscular Dystrophy AssociationUNSPECIFIED
NIHUNSPECIFIED
Subject Keywords:microscopy; tissues; tunable filters
Issue or Number:3
Record Number:CaltechAUTHORS:20160201-085751916
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160201-085751916
Official Citation:Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy Rusty Lansford; Gregory Bearman; Scott E. Fraser J. Biomed. Opt. 6 (3), 311-318 (July 01, 2001); doi: 10.1117/1.1383780
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:64112
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:03 Feb 2016 00:59
Last Modified:03 Oct 2019 09:34

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