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 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.

Traditional paradigms for imaging rely on the use of a spatial structure, either in the detector (pixels arrays) or in the illumination (patterned light). Removal of the spatial structure in the detector or illumination, i.e., imaging with just a single-point sensor, would require solving a very strongly ill-posed inverse retrieval problem that to date has not been solved. Here, we demonstrate a data-driven approach in which full 3D information is obtained with just a single-point, single-photon avalanche diode that records the arrival time of photons reflected from a scene that is illuminated with short pulses of light. Imaging with single-point time-of-flight (temporal) data opens new routes in terms of speed, size, and functionality. As an example, we show how the training based on an optical time-of-flight camera enables a compact radio-frequency impulse radio detection and ranging transceiver to provide 3D images. Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation, and DOI.

No abstract

Fluorescence lifetime imaging is an important tool in bioimaging that allows one to detect subtle changes in cell dynamics and their environment. Most time-domain approaches currently involve scanning a single illumination point across the sample, which can make imaging dynamic scenes challenging, while single-shot “rapid lifetime determination” can suffer from large uncertainties when the lifetime is not appropriately sampled. Here, we propose a time-folded fluorescence lifetime imaging microscopy (TFFLIM) approach, whereby a time-folding cavity provides multiple spatially sheared replicas of the lifetime, each shifted temporally with respect to a fixed time gate. This provides a robust, single-shot FLIM approach that we experimentally validate across a broad lifetime range on fluorescent beads and Convallaria samples.

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.