title: Multiphoton photochemistry of red fluorescent proteins in solution and live cells.
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.
authors.affiliation: Department of Physics and ‡Department of Cell Biology and Neuroscience, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Drobizhev
authors.forename: Caleb
authors.initials: C
authors.lastname: Stoltzfus
authors.forename: Igor
authors.initials: I
authors.lastname: Topol
authors.forename: Jack
authors.initials: J
authors.lastname: Collins
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Wicks
authors.forename: Alexander
authors.initials: A
authors.lastname: Mikhaylov
authors.forename: Lauren
authors.initials: L
authors.lastname: Barnett
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Hughes
authors.forename: Aleksander
authors.initials: A
authors.lastname: Rebane
mesh_terms: D000465:Algorithms; D002460:Cell Line; D004583:Electrons; D004926:Escherichia coli; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D055668:Photochemical Processes; D017785:Photons; D012996:Solutions; D014162:Transfection; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012996:Solutions; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein