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Multiphoton photochemistry of red fluorescent proteins in solution and live cells.

Drobizhev M, Stoltzfus C, Topol I, Collins J, Wicks G, Mikhaylov A, Barnett L, Hughes TE, Rebane A - J Phys Chem B (2014)

Bottom Line: Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function.Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3-5 photon) absorption.The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and ‡Department of Cell Biology and Neuroscience, Montana State University , Bozeman, Montana 59717, United States.

ABSTRACT
Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3-5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue.

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Fluorescence spectral changes during multiphoton irradiationofRFPs. The average power density is P = 11.4 W/cm2. The top spectrum corresponds to irradiation time t = 0, and the bottom spectrum corresponds to the maximumtotal irradiation time of 3039 s for DsRed2, 1667 s for mCherry, 611s for mPlum, and 3583 s for mStrawberry. Intermediate spectra, fromtop to bottom, correspond to several intermediate time points. Dashedlines show spectral contributions from the initial (red) and intermediate(blue) forms at the late stage of bleaching.
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fig2: Fluorescence spectral changes during multiphoton irradiationofRFPs. The average power density is P = 11.4 W/cm2. The top spectrum corresponds to irradiation time t = 0, and the bottom spectrum corresponds to the maximumtotal irradiation time of 3039 s for DsRed2, 1667 s for mCherry, 611s for mPlum, and 3583 s for mStrawberry. Intermediate spectra, fromtop to bottom, correspond to several intermediate time points. Dashedlines show spectral contributions from the initial (red) and intermediate(blue) forms at the late stage of bleaching.

Mentions: In theseexperiments, we irradiate buffered solutions of RFPs with the Ti:Saamplifier laser system operated at 790 nm wavelength, 1 kHz repetitionrate, and 120 fs pulse duration, with a uniform distribution of thepower density across the sample (see the SupportingInformation for more details). We monitor the concentrationsof the initial and intermediate forms by recording time-dependentfluorescence spectra during MPB. Figure 2 showsthe series of selected fluorescence spectra of DsRed2 and mFruitsduring the irradiation process. Because the same laser is used forboth irradiation and interrogation, these 2PEF spectra reflect thetime-dependent evolution of the concentration of initial, as wellas photoconverted, fluorescent species, which can be excited by the790 nm pulses.


Multiphoton photochemistry of red fluorescent proteins in solution and live cells.

Drobizhev M, Stoltzfus C, Topol I, Collins J, Wicks G, Mikhaylov A, Barnett L, Hughes TE, Rebane A - J Phys Chem B (2014)

Fluorescence spectral changes during multiphoton irradiationofRFPs. The average power density is P = 11.4 W/cm2. The top spectrum corresponds to irradiation time t = 0, and the bottom spectrum corresponds to the maximumtotal irradiation time of 3039 s for DsRed2, 1667 s for mCherry, 611s for mPlum, and 3583 s for mStrawberry. Intermediate spectra, fromtop to bottom, correspond to several intermediate time points. Dashedlines show spectral contributions from the initial (red) and intermediate(blue) forms at the late stage of bleaching.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126731&req=5

fig2: Fluorescence spectral changes during multiphoton irradiationofRFPs. The average power density is P = 11.4 W/cm2. The top spectrum corresponds to irradiation time t = 0, and the bottom spectrum corresponds to the maximumtotal irradiation time of 3039 s for DsRed2, 1667 s for mCherry, 611s for mPlum, and 3583 s for mStrawberry. Intermediate spectra, fromtop to bottom, correspond to several intermediate time points. Dashedlines show spectral contributions from the initial (red) and intermediate(blue) forms at the late stage of bleaching.
Mentions: In theseexperiments, we irradiate buffered solutions of RFPs with the Ti:Saamplifier laser system operated at 790 nm wavelength, 1 kHz repetitionrate, and 120 fs pulse duration, with a uniform distribution of thepower density across the sample (see the SupportingInformation for more details). We monitor the concentrationsof the initial and intermediate forms by recording time-dependentfluorescence spectra during MPB. Figure 2 showsthe series of selected fluorescence spectra of DsRed2 and mFruitsduring the irradiation process. Because the same laser is used forboth irradiation and interrogation, these 2PEF spectra reflect thetime-dependent evolution of the concentration of initial, as wellas photoconverted, fluorescent species, which can be excited by the790 nm pulses.

Bottom Line: Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function.Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3-5 photon) absorption.The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and ‡Department of Cell Biology and Neuroscience, Montana State University , Bozeman, Montana 59717, United States.

ABSTRACT
Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3-5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue.

Show MeSH
Related in: MedlinePlus