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.
Publisher
Cold Spring Harbor Laboratory
Reference44 articles.
1. Lakowicz, J. R. Time-Domain Lifetime Measurements. In Lakowicz, J. R. (ed.) Principles of Fluorescence Spec-troscopy, 97–155 (Springer US, Boston, MA, 2006).
2. Fluorescence lifetime imaging - techniques and applications
3. Suhling, K. et al. Fluorescence Lifetime Imaging. In Ho, A. H.-P. , Kim, D. & Somekh, M. G. (eds.) Handbook of Photonics for Biomedical Engineering, 1–50 (Springer Netherlands, Dordrecht, 2014).
4. van Munster, E. B. & Gadella, T. W. J. Fluorescence Lifetime Imaging Microscopy (FLIM). In Rietdorf, J. (ed.) Microscopy Techniques, 143–175 (Springer, Berlin, Heidelberg, 2005).
5. Fluorescence lifetime-resolved pH imaging of living cells;Cytometry A,2003
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