Mechanisms of high-order photobleaching and its relationship to intracellular ablation

Kalies, Stefan; Kuetemeyer, Kai; Heisterkamp, Alexander

doi:10.1364/boe.2.000805

Cited by 40 publications

(42 citation statements)

References 32 publications

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“…These results suggest that the excited-state absorption proceeds through the singlet manifold in the RFPs under study (similar to that of EGFP 16,17 ). Another important conclusion is that the rate of the process does not depend on the local environment (i.e., buffer vs E. coli cell).…”

Section: Resultsmentioning

confidence: 53%

“…Empirically, it has been demonstrated that the multiphoton photobleaching rate k of dyes and FPs (including RFPs) increases much more steeply with the increasing laser intensity than the expected quadratic law, i.e., as k ∼ P α with the exponent α = 2.5–5.3. 11−16 It has been also observed that the bleaching rate of DsRed can be significantly reduced by tuning the laser wavelength from 720–760 to 950–1100 nm. 10,13 The MPB rate can also depend on the biological environment, pulse duration, repetition rate, and so forth.…”

Section: Introductionmentioning

confidence: 99%

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Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells

Drobizhev

Stoltzfus²,

Topol

et al. 2014

J. Phys. Chem. B

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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.

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Section: Resultsmentioning

confidence: 53%

Section: Introductionmentioning

confidence: 99%

Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells

Drobizhev

Stoltzfus²,

Topol

et al. 2014

J. Phys. Chem. B

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“…The femtosecond source results in high peak intensities in the diffraction-limited focus and subsequent multi-photon absorption in the local environment. The mechanism of DSB-induction by MPL is not known; however, the generation of reactive oxygen species is thought to be a major contributor to DNA damage at the site of laser damage, one product of which will be the generation of chromosomal DSBs [40].…”

Section: Resultsmentioning

confidence: 99%

Impact of Histone H4 Lysine 20 Methylation on 53BP1 Responses to Chromosomal Double Strand Breaks

et al. 2012

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Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.

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“…Depending on the experimental parameters used in the cited studies, these various effects can arise from one, two or even three photon absorption by the tissue, and can be mediated to a various degree by the presence of a low density plasma (free electrons) [32]. They depend on the nature of the absorbing molecules involved, as their intracellular localisation will influence the type of perturbation.…”

Section: Review Of Photodamage Mechanismsmentioning

confidence: 99%

Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0–1.2 µm Wavelength Range

Débarre

Olivier²,

Supatto³

et al. 2014

PLoS ONE

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Light-induced toxicity is a fundamental bottleneck in microscopic imaging of live embryos. In this article, after a review of photodamage mechanisms in cells and tissues, we assess photo-perturbation under illumination conditions relevant for point-scanning multiphoton imaging of live Drosophila embryos. We use third-harmonic generation (THG) imaging of developmental processes in embryos excited by pulsed near-infrared light in the 1.0–1.2 µm range. We study the influence of imaging rate, wavelength, and pulse duration on the short-term and long-term perturbation of development and define criteria for safe imaging. We show that under illumination conditions typical for multiphoton imaging, photodamage in this system arises through 2- and/or 3-photon absorption processes and in a cumulative manner. Based on this analysis, we derive general guidelines for improving the signal-to-damage ratio in two-photon (2PEF/SHG) or THG imaging by adjusting the pulse duration and/or the imaging rate. Finally, we report label-free time-lapse 3D THG imaging of gastrulating Drosophila embryos with sampling appropriate for the visualisation of morphogenetic movements in wild-type and mutant embryos, and long-term multiharmonic (THG-SHG) imaging of development until hatching.

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Mechanisms of high-order photobleaching and its relationship to intracellular ablation

Cited by 40 publications

References 32 publications

Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells

Multiphoton Photochemistry of Red Fluorescent Proteins in Solution and Live Cells

Impact of Histone H4 Lysine 20 Methylation on 53BP1 Responses to Chromosomal Double Strand Breaks

Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0–1.2 µm Wavelength Range

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