Strengths and weaknesses of recently engineered red fluorescent proteins evaluated in live cells using fluorescence correlation spectroscopy

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...

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Bibliographic Details
Published in:International journal of molecular sciences 2013-10, Vol.14 (10), p.20340-20358
Main Authors: Siegel, Amanda P, Baird, Michelle A, Davidson, Michael W, Day, Richard N
Format: Article
Language:English
Subjects:
Animals
Cell Line
Data collection
diffusion
flickering
Fluorescence
fluorescent correlation spectroscopy (FCS)
fluorescent protein
intrinsic brightness
Lasers
Luminescent Proteins - metabolism
Mice
Proteins
Red Fluorescent Protein
Spectrometry, Fluorescence - methods
Spectrum analysis
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Summary: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.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms141020340