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Int. J. Mol. Sci. 2013, 14(10), 20340-20358; doi:10.3390/ijms141020340

Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy

1
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., MS 333, Indianapolis, IN 46202, USA
2
National High Magnetic Field Laboratory and Department of Biological Science, 1800 E. Paul Dirac Dr., Florida State University, Tallahassee, FL 32310, USA
*
Author to whom correspondence should be addressed.
Received: 15 July 2013 / Revised: 13 September 2013 / Accepted: 23 September 2013 / Published: 14 October 2013
(This article belongs to the Special Issue Frontiers of Micro-Spectroscopy in Biological Applications)
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Abstract

The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy. View Full-Text
Keywords: fluorescent protein; fluorescent correlation spectroscopy (FCS); diffusion; intrinsic brightness; flickering fluorescent protein; fluorescent correlation spectroscopy (FCS); diffusion; intrinsic brightness; flickering
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MDPI and ACS Style

Siegel, A.P.; Baird, M.A.; Davidson, M.W.; Day, R.N. Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy. Int. J. Mol. Sci. 2013, 14, 20340-20358.

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