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

Dependence of the initial fluorescence signal F(0) (blue symbols) and initial photobleaching rate K (red symbols) on average laser power plotted on a double-logarithmicscale. Both F(0) and K were fittedwith the empirical power law function Pα. The best fit values of α are depicted for each protein. Bleachingwas monitored in live E. coli colonies.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Dependence of the initial fluorescence signal F(0) (blue symbols) and initial photobleaching rate K (red symbols) on average laser power plotted on a double-logarithmicscale. Both F(0) and K were fittedwith the empirical power law function Pα. The best fit values of α are depicted for each protein. Bleachingwas monitored in live E. coli colonies.

Mentions: In this set of experiments,we measured the fluorescence bleaching kinetics F(t) in live E. coli bacteria using the two-photon microscope setup with a high repetitionrate (76 MHz) (see the Supporting Information). Because of the strong nonuniformity of the laser intensity acrossthe focal volume and the non-mono-exponential character of the intrinsicbleaching kinetics (see above), the observed decay curves could notbe described with a sum of a reasonable number of exponents (Supporting Information Figure 13). Therefore,here, we use the method of initial rates to measure an effective rate K of the first, A → B, step as a function of laser power. The thus obtained value of K is a result of averaging of the local initial rates k1 over space across the focal volume. Figure 5 shows the dependence of both K and the initial fluorescence signal, F(0), on powerfor DsRed2 and mFruits. Although F(0) increases nearlyquadratically, the photobleaching rate shows a much steeper dependenceon power. The power exponent of eq 11 variesfrom α = 2.79 to 3.41. The value obtained for mPlum (α= 3.20) is close to what was measured at 1 kHz excitation (cf. Figure 4a), suggesting that the order of the processes (numberof photons involved) does not depend on the repetition rate or onthe local protein environment (water vs cell). Because α isalways larger than 2, we can conclude that the three- or four-photonexcitation of higher excited states is responsible for multiphotontransformations.


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)

Dependence of the initial fluorescence signal F(0) (blue symbols) and initial photobleaching rate K (red symbols) on average laser power plotted on a double-logarithmicscale. Both F(0) and K were fittedwith the empirical power law function Pα. The best fit values of α are depicted for each protein. Bleachingwas monitored in live E. coli colonies.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Dependence of the initial fluorescence signal F(0) (blue symbols) and initial photobleaching rate K (red symbols) on average laser power plotted on a double-logarithmicscale. Both F(0) and K were fittedwith the empirical power law function Pα. The best fit values of α are depicted for each protein. Bleachingwas monitored in live E. coli colonies.
Mentions: In this set of experiments,we measured the fluorescence bleaching kinetics F(t) in live E. coli bacteria using the two-photon microscope setup with a high repetitionrate (76 MHz) (see the Supporting Information). Because of the strong nonuniformity of the laser intensity acrossthe focal volume and the non-mono-exponential character of the intrinsicbleaching kinetics (see above), the observed decay curves could notbe described with a sum of a reasonable number of exponents (Supporting Information Figure 13). Therefore,here, we use the method of initial rates to measure an effective rate K of the first, A → B, step as a function of laser power. The thus obtained value of K is a result of averaging of the local initial rates k1 over space across the focal volume. Figure 5 shows the dependence of both K and the initial fluorescence signal, F(0), on powerfor DsRed2 and mFruits. Although F(0) increases nearlyquadratically, the photobleaching rate shows a much steeper dependenceon power. The power exponent of eq 11 variesfrom α = 2.79 to 3.41. The value obtained for mPlum (α= 3.20) is close to what was measured at 1 kHz excitation (cf. Figure 4a), suggesting that the order of the processes (numberof photons involved) does not depend on the repetition rate or onthe local protein environment (water vs cell). Because α isalways larger than 2, we can conclude that the three- or four-photonexcitation of higher excited states is responsible for multiphotontransformations.

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