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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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(a) TPLSM imaging of HEK cells cotransfected with EGFP(green)and mCherry (red). The frames from top to bottom correspond to 0,25, 50, and 125 s of raster scanning, with the rate of 2 frames/s.(b) Temporal decay of the fluorescence signal of EGFP (green) andmCherry (red). The biexponential fit to the latter is shown by a continuousblack line.
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fig1: (a) TPLSM imaging of HEK cells cotransfected with EGFP(green)and mCherry (red). The frames from top to bottom correspond to 0,25, 50, and 125 s of raster scanning, with the rate of 2 frames/s.(b) Temporal decay of the fluorescence signal of EGFP (green) andmCherry (red). The biexponential fit to the latter is shown by a continuousblack line.

Mentions: Red fluorescentproteins (RFPs), such as DsRed and its monomericderivatives, are attractive for deep imaging because of their red-shiftedfluorescence, but they bleach quickly compared to EGFP under typicalTPLSM conditions, see Figure 1 and refs (4 and 10). Empirically, it has been demonstratedthat the multiphoton photobleaching rate k of dyesand FPs (including RFPs) increases much more steeply with the increasinglaser intensity than the expected quadratic law, i.e., as k ∼ Pα with theexponent α = 2.5–5.3.11−16 It has been also observed that the bleaching rate of DsRed can besignificantly reduced by tuning the laser wavelength from 720–760to 950–1100 nm.10,13 The MPB rate can also dependon the biological environment, pulse duration, repetition rate, andso forth.17−19 Although MPB has been observed by several groups,the underlying photophysics, particularly in RFPs, is not understood.A faster-than-quadratic power dependence of the photobleaching ratesuggests an involvement of more than two photons in the process, butfundamental questions remain unanswered: Is the process instantaneous(three-, four-, or even more photon absorption) or stepwise, i.e.,a sequential absorption of photon(s) after an initial simultaneoustwo-photon transition? If the process is stepwise, then does it involvea climbing upon higher singlet or triplet states? If it is a singlet–singletexcitation, then is there any energetic relaxation involved betweenthe two-photon absorption (2PA) event and the additional photon(s)absorption? Why is the power exponent of the intensity dependenceoften a noninteger number? Is this because of a competition betweendifferent-order processes (e.g., two- and three-photon absorption)or because of saturation of one of the transitions?


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)

(a) TPLSM imaging of HEK cells cotransfected with EGFP(green)and mCherry (red). The frames from top to bottom correspond to 0,25, 50, and 125 s of raster scanning, with the rate of 2 frames/s.(b) Temporal decay of the fluorescence signal of EGFP (green) andmCherry (red). The biexponential fit to the latter is shown by a continuousblack line.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: (a) TPLSM imaging of HEK cells cotransfected with EGFP(green)and mCherry (red). The frames from top to bottom correspond to 0,25, 50, and 125 s of raster scanning, with the rate of 2 frames/s.(b) Temporal decay of the fluorescence signal of EGFP (green) andmCherry (red). The biexponential fit to the latter is shown by a continuousblack line.
Mentions: Red fluorescentproteins (RFPs), such as DsRed and its monomericderivatives, are attractive for deep imaging because of their red-shiftedfluorescence, but they bleach quickly compared to EGFP under typicalTPLSM conditions, see Figure 1 and refs (4 and 10). Empirically, it has been demonstratedthat the multiphoton photobleaching rate k of dyesand FPs (including RFPs) increases much more steeply with the increasinglaser intensity than the expected quadratic law, i.e., as k ∼ Pα with theexponent α = 2.5–5.3.11−16 It has been also observed that the bleaching rate of DsRed can besignificantly reduced by tuning the laser wavelength from 720–760to 950–1100 nm.10,13 The MPB rate can also dependon the biological environment, pulse duration, repetition rate, andso forth.17−19 Although MPB has been observed by several groups,the underlying photophysics, particularly in RFPs, is not understood.A faster-than-quadratic power dependence of the photobleaching ratesuggests an involvement of more than two photons in the process, butfundamental questions remain unanswered: Is the process instantaneous(three-, four-, or even more photon absorption) or stepwise, i.e.,a sequential absorption of photon(s) after an initial simultaneoustwo-photon transition? If the process is stepwise, then does it involvea climbing upon higher singlet or triplet states? If it is a singlet–singletexcitation, then is there any energetic relaxation involved betweenthe two-photon absorption (2PA) event and the additional photon(s)absorption? Why is the power exponent of the intensity dependenceoften a noninteger number? Is this because of a competition betweendifferent-order processes (e.g., two- and three-photon absorption)or because of saturation of one of the transitions?

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