Journal
JOURNAL OF MICROSCOPY
Volume 246, Issue 2, Pages 160-167Publisher
WILEY
DOI: 10.1111/j.1365-2818.2012.03605.x
Keywords
Digital imaging; light microscopy; live-cell microscopy; photodamage; software
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Funding
- National Science Foundation (NSF) Smart Lighting Engineering Research Center [EEC-0812056]
- School of Engineering at Rensselaer Polytechnic Institute
- Center for Automation Technologies and Systems (CATS) under New York State Empire State Development Division of Science, Technology and Innovation (NYSTAR)
- National Institutes of Health [GM59363]
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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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