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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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Decaykinetics of the initial (red symbols) and intermediate (bluesymbols) forms. The solid continuous lines show the correspondingfitting functions (see the text for details). The dashed blue linerepresents the contribution of structure B and thedotted blue line represents the contribution of structure C to the fluorescence of intermediate form in the case ofthe mFruits (see the text for details).
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fig3: Decaykinetics of the initial (red symbols) and intermediate (bluesymbols) forms. The solid continuous lines show the correspondingfitting functions (see the text for details). The dashed blue linerepresents the contribution of structure B and thedotted blue line represents the contribution of structure C to the fluorescence of intermediate form in the case ofthe mFruits (see the text for details).

Mentions: Figure 3 shows the temporal changes of theamplitudes of the initial (red symbols) and intermediate (blue symbols)forms. The kinetics of the initial form can be well-described by abiexponential decay with zero offset for all four proteins. This suggestsat least a two-step consecutive reaction with a reversible first step.


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)

Decaykinetics of the initial (red symbols) and intermediate (bluesymbols) forms. The solid continuous lines show the correspondingfitting functions (see the text for details). The dashed blue linerepresents the contribution of structure B and thedotted blue line represents the contribution of structure C to the fluorescence of intermediate form in the case ofthe mFruits (see the text for details).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Decaykinetics of the initial (red symbols) and intermediate (bluesymbols) forms. The solid continuous lines show the correspondingfitting functions (see the text for details). The dashed blue linerepresents the contribution of structure B and thedotted blue line represents the contribution of structure C to the fluorescence of intermediate form in the case ofthe mFruits (see the text for details).
Mentions: Figure 3 shows the temporal changes of theamplitudes of the initial (red symbols) and intermediate (blue symbols)forms. The kinetics of the initial form can be well-described by abiexponential decay with zero offset for all four proteins. This suggestsat least a two-step consecutive reaction with a reversible first step.

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