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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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Power dependence of the(a) first step rate k1 and (b) reversestep rate k2 andsecond step rate k3 for mPlum in solutionupon irradiation with a Ti:Sa amplifier.
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fig4: Power dependence of the(a) first step rate k1 and (b) reversestep rate k2 andsecond step rate k3 for mPlum in solutionupon irradiation with a Ti:Sa amplifier.

Mentions: To gain further insight into the mechanismsof MPB, we studied the dependence of individual MPT rates, involvedin eq 5, as a function of laser power. For theseexperiments, we selected mPlum because it bleaches quickly even atlow power. The fluorescence signal was collected in the region of630–680 nm, i.e., where the fluorescence of forms B and C is negligible (Figure 2). We first checked that the initial (unbleached) fluorescence dependedquadratically on power (Supporting Information Figure 11), implying that the simultaneous 2PA transition is notsaturated. Using the method of initial rates (see above), we obtained k1 as a function of power over a broad powerrange. Figure 4a shows the dependence of k1 on the average power P ina double-logarithmic scale. Describing the dependence with an empiricalpower law11we find α = 3.25, which means that afour-photon process is involved in the A → B transformation. The noninteger value of α suggeststhat either four photons, in total, are absorbed, but some of thetransitions are saturated, or that the four-photon process competeswith lower-order (two- or three-photon) processes.


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)

Power dependence of the(a) first step rate k1 and (b) reversestep rate k2 andsecond step rate k3 for mPlum in solutionupon irradiation with a Ti:Sa amplifier.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Power dependence of the(a) first step rate k1 and (b) reversestep rate k2 andsecond step rate k3 for mPlum in solutionupon irradiation with a Ti:Sa amplifier.
Mentions: To gain further insight into the mechanismsof MPB, we studied the dependence of individual MPT rates, involvedin eq 5, as a function of laser power. For theseexperiments, we selected mPlum because it bleaches quickly even atlow power. The fluorescence signal was collected in the region of630–680 nm, i.e., where the fluorescence of forms B and C is negligible (Figure 2). We first checked that the initial (unbleached) fluorescence dependedquadratically on power (Supporting Information Figure 11), implying that the simultaneous 2PA transition is notsaturated. Using the method of initial rates (see above), we obtained k1 as a function of power over a broad powerrange. Figure 4a shows the dependence of k1 on the average power P ina double-logarithmic scale. Describing the dependence with an empiricalpower law11we find α = 3.25, which means that afour-photon process is involved in the A → B transformation. The noninteger value of α suggeststhat either four photons, in total, are absorbed, but some of thetransitions are saturated, or that the four-photon process competeswith lower-order (two- or three-photon) processes.

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