Multiphoton photochemistry of red fluorescent proteins in solution and live cells.
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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.
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Affiliation: Department of Physics and ‡Department of Cell Biology and Neuroscience, Montana State University , Bozeman, Montana 59717, United States.
ABSTRACT
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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. Related in: MedlinePlus |
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Mentions: The power exponent α of the intensity dependence of photobleachingrates (Figure 5) in most cases is a nonintegernumber. Physically, this can be explained either by a competitionbetween parallel two-, three-, four-, and so on photon-induced processes37 or by saturation of a certain transition inthe multiphoton consecutive ladder process.38,39 In the former case, the microscopic rate k1 is described by a sum of the rates of processes with differentphoton orders3712After integration of the individual contributionsin eq 12 over space and explicitly expressingpower dependences of the corresponding process, we arrive at the polynomialfunction13where all of the coefficients an are positive. In the secondcase, ifany of the consecutive transitions involved in an n-photon absorption process is saturated, then, as can be shown (Supporting Information), the corresponding rates k1(n) and K(n) can be described by ananalytical function, which can be approximated in the limiting casesof the slight or moderately strong saturation by a polynomial functionof P, where some of the coefficients turn negative.Therefore, careful analysis of the experimental power dependence ofthe higher order (3–4 photons) MPT rate will allow one to distinguishbetween the two cases. In any of the above mechanisms, the photochemicalreaction starting from the S1 state that becomes populatedafter the initial 2PA step cannot be ruled out. We first calculatethe contribution K(2) in the whole powerrange of Figure 5. By neglecting saturationof the 2PA transition (F(0) depends quadraticallyon P), one can show (SupportingInformation) that14where σ2 is the two-photoncross-section at the excitation wavelength, φ1 isthe quantum yield of the reaction starting from the lowest excitedstate (S1), f is the pulse repetitionrate, h is Plank’s constant, ν is thephoton frequency, Δτ is the pulse duration, w0 is the laser beam width at the focal plane, and P is the average power. According to Kasha’s rule,φ1 does not depend on the method of excitation (one-or two-photon). To evaluate a2, we thereforecan use the values of φ1 previously determined inone-photon bleaching experiments,31 aswell as the known σ2 values35 and laser parameters. We then calculated the a2P2 contribution and subtractedit from the total rate K to obtain the power dependencesof the higher order (three- or four-photon) processes. Although K(2) turns out to be several times smaller than K in the whole power range, this correction still providesmore careful evaluations of K(3) and K(4) or of their combination. The results areshown in Figure 6, where the insets presentthe same plots on a double-logarithmic scale. For DsRed2, α= 2.84, from which we conclude that, in total, three photons are involvedin photobleaching and the third-photon absorption is slightly saturated.(The first, simultaneous 2PA transition is not saturated because theinitial fluorescence depends quadratically on power.) For mFruits,the analysis shows that a combination of the nonsaturated third andfourth power terms in eq 13 cannot explain theobserved power dependence (coefficient a4 turns negative), and we conclude that four photons are involvedand at least one of the subsequent excited-state transitions is moderatelystrongly saturated. |
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Affiliation: Department of Physics and ‡Department of Cell Biology and Neuroscience, Montana State University , Bozeman, Montana 59717, United States.