Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

Abstract

Super-Resolution Microscopy enables non-invasive, moleculespecific imaging of the internal structure and dynamics of cells with sub-diffraction limit spatial resolution. One of its major limitations is the requirement for high-intensity illumination, generating considerable cellular phototoxicity. This factor considerably limits the capacity for live-cell observations, particularly for extended periods of time. Here, we overview new developments in hardware, software and probe chemistry aiming to reduce phototoxicity. Additionally, we discuss how the choice of biological model and sample environment impacts the capacity for live-cell observations. Phototoxicity | Photodamage | Super-Resolution Microscopy | Fluorescence Correspondence: s.culley@ucl.ac.uk, r.henriques@ucl.ac.uk Tosheva et al. | 1-12Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted:

An antioxidant screen identifies ascorbic acid for prevention of light-induced mitotic prolongation in live cell imaging

“…Phototoxicity occurs through the light-induced formation of reactive oxygen species (ROS) (Laissue et al, 2017;Icha et al, 2017), which react with numerous cellular components and disrupt their structures and functions. Severe oxidization results in cellular abnormalities such as membrane blebbing and subsequent cell death (Zdolsek et al, 1990;Tosheva et al, 2020). Even without apparent changes in cellular morphology, high amounts of light illumination can cause substantial cell dysfunction such as abnormal intracellular calcium homeostasis (Roehlecke et al, 2009;Nishigaki et al, 2006).…”

“…The copyright holder for this preprint this version posted June 20, 2022. ; https://doi.org/10. 1101/2022 Several approaches have been proposed to decrease photodamage in fluorescence imaging of living cells, including the use of advanced microscopes with low phototoxicity such as light-sheet microscopes (Tosheva et al, 2020).…”

Super‐Resolution Fluorescence Microscopy Methods for Assessing Mouse Biology

“…When fluorophores are excited to reactive states, such as long‐lived triplet states, they form reactive oxygen species (ROS), a major cause of phototoxicity. Tosheva, Yuan, Pereira, Culley, & Henriques (2020) and Cordes, Maiser, Steinhauer, Schermelleh, & Tinnefeld (2011) have studied the effect of phototoxicity in super‐resolution microscopy and suggest several stratagems to reduce this, such as low phototoxicity fluorescent probes, using longer wavelength illumination with long exposures rather than short bursts, and including oxygen scavengers or bubbling nitrogen through the medium to reduce the formation of ROS.…”