Summary
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.
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