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Biophys. J. BioFAST: First Published July 22, 2005. doi:10.1529/biophysj.105.061853
© 2005 by the Biophysical Society.

A more recent version of this article appeared on October 1, 2005.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Quantitative Multiphoton Spectral Imaging and its use for Measuring Resonance Energy Transfer

Christopher Thaler 1, Srinagesh V Koushik 1, Paul S Blank 2 and Steven S Vogel 1*

1 NIAAA/NIH
2 NICHD/NIH

* To whom correspondence should be addressed. E-mail: stevevog{at}mail.nih.gov.

Submitted on February 23, 2005
Revised on April 11, 2005
Accepted on 7 July 2005


  Abstract
Protein labeling with GFP-derivatives has become an invaluable tool in cell-biology. Protein quantification, however, is difficult when cells express constructs with overlapping fluorescent emissions. Under these conditions, signal separation using emission filters is inherently inefficient. Spectral imaging solves this problem by recording emission spectra directly. Unfortunately, linear unmixing, the algorithm used for quantifying individual fluorophores from emission spectra fails when resonance energy transfer (RET) is present. We therefore sought to develop an unmixing algorithm that incorporates RET. An equation for spectral emission incorporating RET was derived and an assay based on this formalism, "spectral RET" (sRET), was developed. Standards with defined RET efficiencies, and with known Cerulean to Venus ratios were constructed and used to test sRET. We demonstrate that sRET analysis is a comprehensive, photon-efficient method for imaging RET efficiencies, and accurately determines donor and acceptor concentrations in living cells.

Key Words: Cerulean, FLIM, FRET, Venus, linear-unmixing, two-photon




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