SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Dual-Color Photon Counting Histogram Analysis of mRFP1 and EGFP in Living Cells
Lindsey Hillesheim 1*, Yan Chen 2 and Joachim Mueller 2
1 University of St. Thomas
2 University of Minnesota
* To whom correspondence should be addressed. E-mail: lhilles{at}physics.umn.edu.
Submitted on March 24, 2006
Revised on May 3, 2006
Accepted on 29 August 2006
 |
Abstract |
|---|
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 (FRET) within the fusion protein alters the dual-channel brightness of the fluorophores. We describe a model for FRET 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.
Key Words:
FRET, fluorescence correlation spectroscopy, fluorescence lifetime, fluorescence microscopy, single molecule spectroscopy, two-photon excitation
