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

Siegel AP, Baird MA, Davidson MW, Day RN - Int J Mol Sci (2013)

Bottom Line: 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.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.All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., MS 333, Indianapolis, IN 46202, USA. rnday@iupui.edu.

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.

Show MeSH
The flickering threshold for four of the five red FPs tested. mApple was excluded because it flickers at all laser powers tested.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3821618&req=5

f2-ijms-14-20340: The flickering threshold for four of the five red FPs tested. mApple was excluded because it flickers at all laser powers tested.

Mentions: We next varied excitation intensities for the other red FPs to further investigate their flickering characteristics. For each of the four remaining red FPs, we were able to find a threshold below which change in excitation intensity did not change D. When the excitation intensity was increased above this threshold, which we define as the flickering threshold, an artificially high D was measured. Representative autocorrelation curves and a table showing values for D at different excitation intensities for the four red FPs at ranges of laser intensity are in the Supplementary Material. These data were used to determine the flickering thresholds for mCherry, TagRFP-T, FusionRed and mRuby2 (Figure 2 and Table 1). FusionRed was most susceptible to flickering (variations in D noted above 0.11 kW/cm2), followed by mRuby2 and mCherry, while TagRFP-T displayed very little flickering. Table 1 gives the D values for the purified proteins determined by averaging all the readings below the flickering thresholds, except for mApple, where the D value was determined at 0.075 kW/cm2. There was little evidence of photobleaching while acquiring these measurements. Photobleaching is a separate characteristic of the FPs and is not necessarily correlated to flickering [5]. This distinguishes these results from other studies performed at higher illuminations where flickering and photolysis (and triplet effects) occur simultaneously, and are separated using different analytical techniques [5,13,15].


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

Siegel AP, Baird MA, Davidson MW, Day RN - Int J Mol Sci (2013)

The flickering threshold for four of the five red FPs tested. mApple was excluded because it flickers at all laser powers tested.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3821618&req=5

f2-ijms-14-20340: The flickering threshold for four of the five red FPs tested. mApple was excluded because it flickers at all laser powers tested.
Mentions: We next varied excitation intensities for the other red FPs to further investigate their flickering characteristics. For each of the four remaining red FPs, we were able to find a threshold below which change in excitation intensity did not change D. When the excitation intensity was increased above this threshold, which we define as the flickering threshold, an artificially high D was measured. Representative autocorrelation curves and a table showing values for D at different excitation intensities for the four red FPs at ranges of laser intensity are in the Supplementary Material. These data were used to determine the flickering thresholds for mCherry, TagRFP-T, FusionRed and mRuby2 (Figure 2 and Table 1). FusionRed was most susceptible to flickering (variations in D noted above 0.11 kW/cm2), followed by mRuby2 and mCherry, while TagRFP-T displayed very little flickering. Table 1 gives the D values for the purified proteins determined by averaging all the readings below the flickering thresholds, except for mApple, where the D value was determined at 0.075 kW/cm2. There was little evidence of photobleaching while acquiring these measurements. Photobleaching is a separate characteristic of the FPs and is not necessarily correlated to flickering [5]. This distinguishes these results from other studies performed at higher illuminations where flickering and photolysis (and triplet effects) occur simultaneously, and are separated using different analytical techniques [5,13,15].

Bottom Line: 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.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.All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., MS 333, Indianapolis, IN 46202, USA. rnday@iupui.edu.

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.

Show MeSH