Predictive-focus illumination for reducing photodamage in live-cell microscopy

Due to photobleaching and phototoxicity induced by high-intensity excitation light, the number of fluorescence images that can be obtained in live cells is always limited. This limitation becomes particularly prominent in multidimensional recordings when multiple Z-planes are captured at every time point. Here we present a simple technique, termed predictive-focus illumination (PFI), which helps to minimize cells' exposure to light by decreasing the number of Z-planes that need to be captured in live-cell 3D time-lapse recordings. PFI utilizes computer tracking to predict positions of objects of interest (OOIs) and restricts image acquisition to small dynamic Z-regions centred on each OOI. Importantly, PFI does not require hardware modifications and it can be easily implemented on standard wide-field and spinning-disc confocal microscopes.