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

Abstract

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.

Wide-field three-photon excitation in biological samples

“…Literature results suggest that damage is primarily multiphoton in nature40–44; penetration depths of well over a millimeter were achieved without observable tissue damage3. Nevertheless, an experiment was performed to establish the primary damage mechanism.…”

Phototoxicity in live fluorescence microscopy, and how to avoid it

“…Not surprisingly, photobleaching rates in twophoton microscopy were shown to be higher than in singlephoton excitation at power levels typically used in biological imaging, likely caused by higher-order photon interactions [58]. In addition to linear damage, two-photon excitation results in non-linear damage mechanisms, which are major contributors to phototoxicity in cells [59,60]. The near-simultaneous absorption of two photons triggers ROS formation and can induce direct DNA damage and plasma formation [61,62].…”

Wavelength dependence of femtosecond laser-induced breakdown in water and implications for laser surgery

“…Several studies provided evidence that ultra-short pulsed laser-induced modification of biomolecules produces intermediates with different optical or electronic properties that accelerate further linear or nonlinear modification processes once their concentration is sufficiently high [124][125][126][127][128][129]. Thus, abstraction of excess electrons from biomolecules will either directly or indirectly enhance the photoionization channel of breakdown, which will lower the modification thresholds in biological media compared to the bubble formation threshold in pure water [2].…”