Wide-field Fluorescence Lifetime Imaging Microscopy with a High-Speed Mega-pixel SPAD Camera

Zickus, V.; Wu, M. L.; Morimoto, K.; Kapitany, V.; Farima, A.; Turpin, A.; Insall, R.; Whitelaw, J.; Machesky, L.; Bruschini, C.; Faccio, D.; Charbon, E.

doi:10.1101/2020.06.07.138685

bioRxiv · 10.1101/2020.06.07.138685

Wide-field Fluorescence Lifetime Imaging Microscopy with a High-Speed Mega-pixel SPAD Camera

Zickus, V.·Wu, M. L.·Morimoto, K.·Kapitany, V.·Farima, A.·Turpin, A.·Insall, R.·Whitelaw, J.·Machesky, L.·Bruschini, C.·Faccio, D.·Charbon, E.

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Abstract

Fluorescence 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 Megapixel 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 megapixels.

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Zickus, V., Wu, M. L., Morimoto, K., Kapitany, V., Farima, A., Turpin, A., Insall, R., Whitelaw, J., Machesky, L., Bruschini, C., Faccio, D., Charbon, E.. 2020-06-08. Wide-field Fluorescence Lifetime Imaging Microscopy with a High-Speed Mega-pixel SPAD Camera. https://doi.org/10.1101/2020.06.07.138685

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