Author:
Zickus Vytautas,Wu Ming-Lo,Morimoto Kazuhiro,Kapitany Valentin,Fatima Areeba,Turpin Alex,Insall Robert,Whitelaw Jamie,Machesky Laura,Bruschini Claudio,Faccio Daniele,Charbon Edoardo
Abstract
AbstractFluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 MP resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080—human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 MP.
Funder
Engineering and Physical Sciences Research Council
Canon
The Swiss National Science Foundation
Publisher
Springer Science and Business Media LLC
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