Limits...
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.

Show MeSH

Related in: MedlinePlus

Energy leveldiagrams used to model the MPT process. The firststep of MPB corresponds to a simultaneous 2PA, described by the crosssection σ2. After excitation of level m, the chromophore can relax nonradiatively (wavy blue arrow) to thelowest excited state 1 and then relax back to the ground state 0 orconvert photochemically to a different structure with the quantumyield φ1. Being excited to state m, the chromophore can be further promoted to a higher level, n, via excited-state transition with the one-photon crosssection σmn. In DsRed2 (a), thephotochemical reaction starting from level n is characterizedby quantum efficiency φn. In mFruits(b), the additional, fourth, step is the transition from the excitedstate n into a level c belongingto the continuum of quasi-free states with the corresponding effectivephotodetachment cross section σnc.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126731&req=5

fig7: Energy leveldiagrams used to model the MPT process. The firststep of MPB corresponds to a simultaneous 2PA, described by the crosssection σ2. After excitation of level m, the chromophore can relax nonradiatively (wavy blue arrow) to thelowest excited state 1 and then relax back to the ground state 0 orconvert photochemically to a different structure with the quantumyield φ1. Being excited to state m, the chromophore can be further promoted to a higher level, n, via excited-state transition with the one-photon crosssection σmn. In DsRed2 (a), thephotochemical reaction starting from level n is characterizedby quantum efficiency φn. In mFruits(b), the additional, fourth, step is the transition from the excitedstate n into a level c belongingto the continuum of quasi-free states with the corresponding effectivephotodetachment cross section σnc.

Mentions: The first step of theMPB process in DsRed2 can be presented bya three-level model shown in Figure 7a.


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)

Energy leveldiagrams used to model the MPT process. The firststep of MPB corresponds to a simultaneous 2PA, described by the crosssection σ2. After excitation of level m, the chromophore can relax nonradiatively (wavy blue arrow) to thelowest excited state 1 and then relax back to the ground state 0 orconvert photochemically to a different structure with the quantumyield φ1. Being excited to state m, the chromophore can be further promoted to a higher level, n, via excited-state transition with the one-photon crosssection σmn. In DsRed2 (a), thephotochemical reaction starting from level n is characterizedby quantum efficiency φn. In mFruits(b), the additional, fourth, step is the transition from the excitedstate n into a level c belongingto the continuum of quasi-free states with the corresponding effectivephotodetachment cross section σnc.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Energy leveldiagrams used to model the MPT process. The firststep of MPB corresponds to a simultaneous 2PA, described by the crosssection σ2. After excitation of level m, the chromophore can relax nonradiatively (wavy blue arrow) to thelowest excited state 1 and then relax back to the ground state 0 orconvert photochemically to a different structure with the quantumyield φ1. Being excited to state m, the chromophore can be further promoted to a higher level, n, via excited-state transition with the one-photon crosssection σmn. In DsRed2 (a), thephotochemical reaction starting from level n is characterizedby quantum efficiency φn. In mFruits(b), the additional, fourth, step is the transition from the excitedstate n into a level c belongingto the continuum of quasi-free states with the corresponding effectivephotodetachment cross section σnc.
Mentions: The first step of theMPB process in DsRed2 can be presented bya three-level model shown in Figure 7a.

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