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Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.

Guan Y, Meurer M, Raghavan S, Rebane A, Lindquist JR, Santos S, Kats I, Davidson MW, Mazitschek R, Hughes TE, Drobizhev M, Knop M, Shah JV - Mol. Biol. Cell (2015)

Bottom Line: We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5.We also use MPE-FCCS to detect drug-protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair.The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115.

No MeSH data available.


Related in: MedlinePlus

Intracellular molecular brightness of 2P-excitable FPs. (A) Schematic of 2PE-FCS/FCCS setup. A Ti:sapphire femtosecond laser (tuned to 850 nm) is focused into the cellular cytoplasm through a high–numerical aperture objective. The fluorescence from the sample is collected back through the same objective and separated by a dichroic mirror to two detectors (photomultiplier tubes [PMTs]). Two-photon absorption reduces the excitation volume to <1 fl, <0.1% of the cell volume. (B) Fluorescence images of mTFP1 and hmKeima8.5 under identical excitation in individual cells. (C) FCS calculation. The autocorrelation algorithm is applied to fluctuating fluorescence signals, generating the FCS curve (in gray), G(τ). The fitting results (in red) yield the diffusion coefficient D (derived from the characteristic decay time) and the average molecular number N (derived from the inverse of the amplitude, G(0)). The average molecular brightness is obtained by average intensity <I(t)> divided by N. (D) Comparison of intracellular molecular brightness for cyan FPs and mKeima mutants as measured by FCS. mTFP1 and hmKeima8.5 were chosen for further characterization.
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Figure 2: Intracellular molecular brightness of 2P-excitable FPs. (A) Schematic of 2PE-FCS/FCCS setup. A Ti:sapphire femtosecond laser (tuned to 850 nm) is focused into the cellular cytoplasm through a high–numerical aperture objective. The fluorescence from the sample is collected back through the same objective and separated by a dichroic mirror to two detectors (photomultiplier tubes [PMTs]). Two-photon absorption reduces the excitation volume to <1 fl, <0.1% of the cell volume. (B) Fluorescence images of mTFP1 and hmKeima8.5 under identical excitation in individual cells. (C) FCS calculation. The autocorrelation algorithm is applied to fluctuating fluorescence signals, generating the FCS curve (in gray), G(τ). The fitting results (in red) yield the diffusion coefficient D (derived from the characteristic decay time) and the average molecular number N (derived from the inverse of the amplitude, G(0)). The average molecular brightness is obtained by average intensity <I(t)> divided by N. (D) Comparison of intracellular molecular brightness for cyan FPs and mKeima mutants as measured by FCS. mTFP1 and hmKeima8.5 were chosen for further characterization.

Mentions: To perform measurements for intracellular molecular brightness, we used a custom measurement setup (Figure 2A; Materials and Methods; Saunders et al., 2012; Gaglia et al., 2013; Kayatekin et al., 2014). Molecular brightness is a crucial parameter for FCS/FCCS. The particle brightness (molecular brightness) is calculated by division of the mean fluorescence intensity (<I(t)>, in counts per second [cps]) by the average number of particles/molecules (N) present in the observation volume (Figure 2D). The high molecular brightness of bright fluorophores provides higher signal-to-noise (S/N) ratios, especially in the intracellular environment in which autofluorescence adds to the background. In addition, brighter FPs require lower laser power and hence allow for reduced exposure of the cells to potentially harmful irradiation, which also reduces photobleaching.


Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.

Guan Y, Meurer M, Raghavan S, Rebane A, Lindquist JR, Santos S, Kats I, Davidson MW, Mazitschek R, Hughes TE, Drobizhev M, Knop M, Shah JV - Mol. Biol. Cell (2015)

Intracellular molecular brightness of 2P-excitable FPs. (A) Schematic of 2PE-FCS/FCCS setup. A Ti:sapphire femtosecond laser (tuned to 850 nm) is focused into the cellular cytoplasm through a high–numerical aperture objective. The fluorescence from the sample is collected back through the same objective and separated by a dichroic mirror to two detectors (photomultiplier tubes [PMTs]). Two-photon absorption reduces the excitation volume to <1 fl, <0.1% of the cell volume. (B) Fluorescence images of mTFP1 and hmKeima8.5 under identical excitation in individual cells. (C) FCS calculation. The autocorrelation algorithm is applied to fluctuating fluorescence signals, generating the FCS curve (in gray), G(τ). The fitting results (in red) yield the diffusion coefficient D (derived from the characteristic decay time) and the average molecular number N (derived from the inverse of the amplitude, G(0)). The average molecular brightness is obtained by average intensity <I(t)> divided by N. (D) Comparison of intracellular molecular brightness for cyan FPs and mKeima mutants as measured by FCS. mTFP1 and hmKeima8.5 were chosen for further characterization.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Intracellular molecular brightness of 2P-excitable FPs. (A) Schematic of 2PE-FCS/FCCS setup. A Ti:sapphire femtosecond laser (tuned to 850 nm) is focused into the cellular cytoplasm through a high–numerical aperture objective. The fluorescence from the sample is collected back through the same objective and separated by a dichroic mirror to two detectors (photomultiplier tubes [PMTs]). Two-photon absorption reduces the excitation volume to <1 fl, <0.1% of the cell volume. (B) Fluorescence images of mTFP1 and hmKeima8.5 under identical excitation in individual cells. (C) FCS calculation. The autocorrelation algorithm is applied to fluctuating fluorescence signals, generating the FCS curve (in gray), G(τ). The fitting results (in red) yield the diffusion coefficient D (derived from the characteristic decay time) and the average molecular number N (derived from the inverse of the amplitude, G(0)). The average molecular brightness is obtained by average intensity <I(t)> divided by N. (D) Comparison of intracellular molecular brightness for cyan FPs and mKeima mutants as measured by FCS. mTFP1 and hmKeima8.5 were chosen for further characterization.
Mentions: To perform measurements for intracellular molecular brightness, we used a custom measurement setup (Figure 2A; Materials and Methods; Saunders et al., 2012; Gaglia et al., 2013; Kayatekin et al., 2014). Molecular brightness is a crucial parameter for FCS/FCCS. The particle brightness (molecular brightness) is calculated by division of the mean fluorescence intensity (<I(t)>, in counts per second [cps]) by the average number of particles/molecules (N) present in the observation volume (Figure 2D). The high molecular brightness of bright fluorophores provides higher signal-to-noise (S/N) ratios, especially in the intracellular environment in which autofluorescence adds to the background. In addition, brighter FPs require lower laser power and hence allow for reduced exposure of the cells to potentially harmful irradiation, which also reduces photobleaching.

Bottom Line: We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5.We also use MPE-FCCS to detect drug-protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair.The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115.

No MeSH data available.


Related in: MedlinePlus