Phototoxicity in live fluorescence microscopy, and how to avoid it

Abstract

Phototoxicity frequently occurs during live fluorescence microscopy, and its consequences are often underestimated. Damage to cellular macromolecules upon excitation light illumination can impair sample physiology, and even lead to sample death. In this review, we explain how phototoxicity influences live samples, and we highlight that, besides the obvious effects of phototoxicity, there are often subtler consequences of illumination that are imperceptible when only the morphology of samples is examined. Such less apparent manifestations of phototoxicity are equally problematic, and can change the conclusions drawn from an experiment. Thus, limiting phototoxicity is a prerequisite for obtaining reproducible quantitative data on biological processes. We present strategies to reduce phototoxicity, e.g. limiting the illumination to the focal plane and suggest controls for phototoxicity effects. Overall, we argue that phototoxicity needs increased attention from researchers when designing experiments, and when evaluating research findings.

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“…In some cases, excessive imaging can cause cell retraction and even rupture. If problems arise using traditional laser scanning confocal methods, consider alternative imaging approaches such as spinning disc confocal, wide-field coupled with deconvolution, two-photon, total internal reflection or light sheet microscopy which may help reduce cellular stress and the mitochondrial fragmentation that may occur when imaging live cells [113]. Especially promising is the newest technology used to image mitochondria, lattice light-sheet microscopy, which allows faster image collection and reduces photodamage [114].…”

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