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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.

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Diffusion coefficient (D) determined by FCS at optimal excitation intensities (see Table 1). (a) Ds for purified FPs in solution are all statistically the same, except for mApple, which has no flickering threshold and shows an apparent D 30% > than D for other FPs; (b) D for FPs determined in cytosol at optimal excitation intensities. The apparent D for mApple-BZip is significantly (*) higher than the other FP-fusion proteins. D for mRuby2 is significantly (#) lower than the other FPs; (c) D for FP-BZip fusion proteins determined in the cytosol. The apparent D for mApple-BZip is significantly (#) higher than the other FP-BZip fusion proteins; (d) D for FP-BZip fusion proteins in the nucleus (away from heterochromatin) is much slower than in the cytosol due to BZip interactions with DNA. Apparent D for mApple=BZip is significantly (*) higher. Significance determined by comparing each red FP against values found for mCerulean3 in solution and in live cells for multiple T tests using the Holm-Sidak method (* p < 0.001, # p < 0.0001).
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f3-ijms-14-20340: Diffusion coefficient (D) determined by FCS at optimal excitation intensities (see Table 1). (a) Ds for purified FPs in solution are all statistically the same, except for mApple, which has no flickering threshold and shows an apparent D 30% > than D for other FPs; (b) D for FPs determined in cytosol at optimal excitation intensities. The apparent D for mApple-BZip is significantly (*) higher than the other FP-fusion proteins. D for mRuby2 is significantly (#) lower than the other FPs; (c) D for FP-BZip fusion proteins determined in the cytosol. The apparent D for mApple-BZip is significantly (#) higher than the other FP-BZip fusion proteins; (d) D for FP-BZip fusion proteins in the nucleus (away from heterochromatin) is much slower than in the cytosol due to BZip interactions with DNA. Apparent D for mApple=BZip is significantly (*) higher. Significance determined by comparing each red FP against values found for mCerulean3 in solution and in live cells for multiple T tests using the Holm-Sidak method (* p < 0.001, # p < 0.0001).

Mentions: Table 1 and Figure 3 show values for D found in solution and in live cells for the five different red FPs. For comparison purposes, D was also determined for mCerulean3, a cyan FP that is quite photostable and is not excited by the 561 nm laser line [19]. Because its maximum emission is below 520 nm, and these red FPs do not emit below 520 nm when excited with a 561 nm laser, it is possible to select appropriate filters to use mCerulean3 sequentially with red FPs with no overlap of fluorescent signal, thus enabling dual color FCS studies [14]. Each of the purified red FPs, with the exception of mApple, had the same value for D as mCerulean3 (Table 1 and Figure 3a). In contrast, measurements of diffusion of the red FPs expressed in the cytoplasm of living cells demonstrated differences in the diffusion rate both slower and faster than that measured for mCerulean3 (Figure 3b) Data acquired in the nucleus gave similar diffusion values to data acquired in the cytoplasm (data not shown). mRuby2 diffused at a much slower rate in the cytoplasm of live cells (19.1 ± 3.6 μm2/s), whereas mApple gave a much larger D (37.8 ± 8.5 μm2/s). The larger D for mApple is likely due to the same flickering processes that caused the incorrect determination of D for mApple in solution. The much lower value of D for mRuby2 requires further analysis.


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)

Diffusion coefficient (D) determined by FCS at optimal excitation intensities (see Table 1). (a) Ds for purified FPs in solution are all statistically the same, except for mApple, which has no flickering threshold and shows an apparent D 30% > than D for other FPs; (b) D for FPs determined in cytosol at optimal excitation intensities. The apparent D for mApple-BZip is significantly (*) higher than the other FP-fusion proteins. D for mRuby2 is significantly (#) lower than the other FPs; (c) D for FP-BZip fusion proteins determined in the cytosol. The apparent D for mApple-BZip is significantly (#) higher than the other FP-BZip fusion proteins; (d) D for FP-BZip fusion proteins in the nucleus (away from heterochromatin) is much slower than in the cytosol due to BZip interactions with DNA. Apparent D for mApple=BZip is significantly (*) higher. Significance determined by comparing each red FP against values found for mCerulean3 in solution and in live cells for multiple T tests using the Holm-Sidak method (* p < 0.001, # p < 0.0001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ijms-14-20340: Diffusion coefficient (D) determined by FCS at optimal excitation intensities (see Table 1). (a) Ds for purified FPs in solution are all statistically the same, except for mApple, which has no flickering threshold and shows an apparent D 30% > than D for other FPs; (b) D for FPs determined in cytosol at optimal excitation intensities. The apparent D for mApple-BZip is significantly (*) higher than the other FP-fusion proteins. D for mRuby2 is significantly (#) lower than the other FPs; (c) D for FP-BZip fusion proteins determined in the cytosol. The apparent D for mApple-BZip is significantly (#) higher than the other FP-BZip fusion proteins; (d) D for FP-BZip fusion proteins in the nucleus (away from heterochromatin) is much slower than in the cytosol due to BZip interactions with DNA. Apparent D for mApple=BZip is significantly (*) higher. Significance determined by comparing each red FP against values found for mCerulean3 in solution and in live cells for multiple T tests using the Holm-Sidak method (* p < 0.001, # p < 0.0001).
Mentions: Table 1 and Figure 3 show values for D found in solution and in live cells for the five different red FPs. For comparison purposes, D was also determined for mCerulean3, a cyan FP that is quite photostable and is not excited by the 561 nm laser line [19]. Because its maximum emission is below 520 nm, and these red FPs do not emit below 520 nm when excited with a 561 nm laser, it is possible to select appropriate filters to use mCerulean3 sequentially with red FPs with no overlap of fluorescent signal, thus enabling dual color FCS studies [14]. Each of the purified red FPs, with the exception of mApple, had the same value for D as mCerulean3 (Table 1 and Figure 3a). In contrast, measurements of diffusion of the red FPs expressed in the cytoplasm of living cells demonstrated differences in the diffusion rate both slower and faster than that measured for mCerulean3 (Figure 3b) Data acquired in the nucleus gave similar diffusion values to data acquired in the cytoplasm (data not shown). mRuby2 diffused at a much slower rate in the cytoplasm of live cells (19.1 ± 3.6 μm2/s), whereas mApple gave a much larger D (37.8 ± 8.5 μm2/s). The larger D for mApple is likely due to the same flickering processes that caused the incorrect determination of D for mApple in solution. The much lower value of D for mRuby2 requires further analysis.

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