Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells.

Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells. Research Abstract Details 

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Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells. Abstract Text:

Lindsey N Hillesheim,Yan Chen,Joachim D ,

We investigate the potential of dual-color photon counting histogram (PCH) analysis to resolve fluorescent protein mixtures directly inside cells. Because of their small spectral overlap, we have chosen to look at the fluorescent proteins EGFP and mRFP1. We experimentally demonstrate that dual-color PCH quantitatively resolves a mixture of EGFP and mRFP1 in cells from a single measurement. To mimic the effect of protein association, we constructed a fusion protein of EGFP and mRFP1 (denoted EGFP-mRFP1). Fluorescence resonant energy transfer within the fusion protein alters the dual-channel brightness of the fluorophores. We describe a model for fluorescence resonant energy transfer effects on the brightness and incorporate it into dual-color PCH analysis. The model is verified using fluorescence lifetime measurements. Dual-color PCH analysis demonstrated that not all of the expressed EGFP-mRFP1 fusion proteins contained a fluorescent mRFP1 molecule. Fluorescence lifetime and emission spectra measurements confirmed this surprising result. Additional experiments show that the missing fluorescent fraction of mRFP1 is consistent with a dark state population of mRFP1. We successfully resolved this mixture of fusion proteins with a single dual-color PCH measurement. These results highlight the potential of dual-color PCH to directly detect and quantify protein mixtures in living cells.

Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells. Publishing Authors By Initials

LN Hillesheim,Y Chen,JD ,

For similar investigative techniques: chemistry, analytical: photometry: luminescent measurements: fluorometry: spectrometry, fluorescence research abstracts see: investigative techniques: chemistry, analytical: photometry: luminescent measurements: fluorometry: spectrometry, fluorescence research

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Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells. Journal Published:

PUBLICATION TYPE: Research Support, U.S. Gov't,

Journal: Biophysical journal

VOLUME: 91

Page Numbers: 4273-84

Journal Abbreviation: Biophys. J.

ISSN: 0006-3495

DAY: 15

MONTH: 09

YEAR: 2006

Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 370626

Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells. Keywords Mesh Terms:

KEYWORDS: Spectrometry, Fluorescence

MESH TERMS: methods

Chemical & Substance for Abstract: Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells. Information

Substance Name: Green Fluorescent Proteins

Registry Number: 147336-22-9

Grant and Affiliation Information for Dual-color photon counting histogram analysis of mRFP1 and EGFP in living cells.

AFFILIATION: School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA.

Country: United States

AGENCY: United States NIGMS

GRANT: GM64589

ACRONYM: GM

MEDLINETA: Biophys J

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