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

Abstract

Phototoxicity is an important issue in fluorescence live imaging of light-sensitive cellular processes such as mitosis, especially in high spatiotemporal resolution microscopy that often requires high-intensity illumination. Among several approaches to reduce phototoxicity, the addition of antioxidants to the imaging media has been used as a simple and effective method. However, it remains unknown what are the optimal antioxidants that could prevent phototoxicity- induced defects during mitosis in fluorescence live cell microscopy. In this study, we analyzed the impact of phototoxicity on the mitotic progression in fluorescence live imaging of human diploid cells and performed a screen to identify the most efficient antioxidative agents that reduce it. Quantitative analysis shows that high amounts of light illumination cause various mitotic defects such as prolonged mitosis and delays of chromosome alignment and centrosome separation. Among several antioxidants known to reduce cellular phototoxicity, our screen reveals that ascorbic acid significantly alleviates these phototoxic effects in mitosis. Furthermore, we demonstrate that the addition of ascorbic acid to the imaging media enables fluorescence imaging of mitotic events at very high temporal resolution without obvious photodamage. Thus, this study provides a simple and practical method to effectively reduce the phototoxic effects on mitotic processes in fluorescence live cell imaging.

Harnessing artificial intelligence to reduce phototoxicity in live imaging

“…Considering the varying light energy requirements current microscopy modalities employ, many preventive strategies exist to reduce the effects of phototoxicity, such as limiting light irradiation by reducing the acquisition points or the light dose ( Kiepas et al, 2020 ; Mubaid and Brown, 2017 ; Reynaud et al, 2008 ), using light detectors as an array of 32 GaAsP-PMT detectors or highly sensitive sCMOS cameras ( Huff, 2015 ; Saxena et al, 2015 ) and performing bioluminescence-based assays that reduce the amount of light required ( Suzuki et al, 2016 ). Other strategies focus on controlling oxidative stress effects in biological samples by supplementing antioxidants ( Harada et al, 2022 preprint; Kesari et al, 2020 ) or chemically increasing the oxidative stress resistance of the sample itself ( Kunkel et al, 2018 ). Unfortunately, the degree of photodamage elicited varies based on multiple factors, including sample traits, illumination parameters and imaging modality ( Table 1 ) ( Laissue et al, 2017 ; Reiche et al, 2022 ; Tinevez et al, 2012 ).…”