A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos

• Published in: Scientific reports Vol. 14; no. 1; pp. 7247 - 17
• Main Authors: Nedbal, Jakub, Mattioli Della Rocca, Francesco, Ivanova, Iveta T., Allan, Andrew, Graham, Jeremy, Walker, Richard, Henderson, Robert K., Suhling, Klaus
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 27.03.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 631/57/2267; 639/624/1107/328/2236; 639/624/1107/328/2237; Algae; Algorithms; Cameras; Data processing; Fluorescence; Fluorescence lifetime imaging (FLIM); Giant unilamellar vesicle (GUV); Humanities and Social Sciences; Illumination; Microscopy, Fluorescence - methods; multidisciplinary; Nonlinearity correction; Science; Science (multidisciplinary); Selective-plane illumination microscopy; Single photon avalanche diode (SPAD); Single photon avalanche diode (SPAD); Giant unilamellar vesicle (GUV); FLIM video; Fluorescence lifetime imaging (FLIM); Algae; Selective-plane illumination microscopy; Nonlinearity correction
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-024-56122-1

A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of 192 × 128 pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with ≈ 38 ps accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples.