Fluorescence illumination can cause phototoxicity that negatively affects living samples. This study demonstrates that much of the phototoxicity and photobleaching experienced with live-cell fluorescence imaging occurs as a result of 'illumination overhead' (IO). This occurs when a sample is illuminated but fluorescence emission is not being captured by the microscope camera. Several technological advancements have been developed, including fast-switching LED lamps and transistor-transistor logic (TTL) circuits, to diminish phototoxicity caused by IO. These advancements are not standard features on most microscopes and many biologists are unaware of their necessity for live-cell imaging. IO is particularly problematic when imaging rapid processes that require short exposure times. This study presents a workflow to optimize imaging conditions for measuring both slow and dynamic processes while minimizing phototoxicity on any standard microscope. The workflow includes a guide on how to (1) determine the maximum image exposure time for a dynamic process, (2) optimize excitation light intensity and (3) assess cell health with mitochondrial markers.This article has an associated First Person interview with the first author of the paper.
Keywords: Adhesion dynamics; Cell migration; Fluorescence microscopy; Microtubule dynamics; Mitochondrial dynamics; Phototoxicity.
© 2020. Published by The Company of Biologists Ltd.