Abstract
Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.
Highlights
Multiphoton microscopy comprises a series of techniques that enable the investigation of native cells and tissue without the need for any labeling [1]
Endogenous fluorophores within the cytoplasm can be addressed by the acquisition of Two-photon excited fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS) imaging delivers morphochemical information
We performed additional repeated scanning on the same area like required for temporally resolved imaging or the acquisition of z-stacks. This produced an explicit increase in endogenous fluorescence detected by TPEF (Fig. 1B, C)
Summary
Multiphoton microscopy comprises a series of techniques that enable the investigation of native cells and tissue without the need for any labeling [1]. In order to efficiently excite endogenous nonlinear processes, a very high optical peak power is necessary [12]. For this reason, multiphoton techniques use ultrashort pulsed laser sources with near infrared emission [4]. In consequence of the high laser irradiance, the photodamage of cells and tissue is a still open issue. These techniques draw increasing attention for in vivo use, where their intrinsic characteristics – omitting labels or dyes, and confocality – are better exploited
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