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Proceedings Paper

Fluorescence lifetime imaging of high-speed particles with single-photon image sensors
Author(s): Istvan Gyongy; Andrew Green; Sam W. Hutchings; Amy Davies; Neale A. W. Dutton; Rory R. Duncan; Colin Rickman; Robert K. Henderson; Paul A. Dalgarno
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Paper Abstract

The capability of Single-Photon Avalanche Diodes (SPADs) to detect photons with picosecond timing precision and shotnoise limited performance has given rise to a range of biological and biomedical applications, from Fluorescence Lifetime Imaging Microscopy (FLIM) to Raman Spectroscopy and Positron Emission Tomography (PET). The use of SPAD sensors has also been successfully demonstrated in Single-Molecule Localisation Microscopy. Traditionally implemented as point detectors, recent advances in SPAD technology, such as compact, binary pixels and back-side illuminated, 3D-stacked architectures, have led to 2-D imaging arrays of increasing resolution and fill factor. Combined with high frame rates (in the kFPS range), and negligible read noise, the sensors offer an exciting prospect for capturing fast temporal dynamics in life science cellular imaging. The work in this paper considers the application of SPAD imaging arrays to widefield fluorescence lifetime imaging of high-speed particles in microscopy. We demonstrate, using a time-gated binary SPAD array, that by tracking particles, and spatially re-assigning the underlying photon counts accordingly, lifetime estimates for fast-moving objects are possible. Moreover, we give the first demonstration of FLIM using a SPAD imaging array with on-chip histogramming of photon arrival time, with potential frame rates of several 100FPS. Both FLIM techniques are illustrated using experimental results based on fluorescent microspheres undergoing Brownian motion. The results pave the way towards applications in live-cell microscopy, such as the monitoring of the fluorescence lifetime of highly mobile cell structures, with a view, for example, to study molecular interactions using Förster Resonance Energy Transfer (FRET) measurements.

Paper Details

Date Published: 4 March 2019
PDF: 8 pages
Proc. SPIE 10889, High-Speed Biomedical Imaging and Spectroscopy IV, 108890O (4 March 2019); doi: 10.1117/12.2510773
Show Author Affiliations
Istvan Gyongy, The Univ. of Edinburgh (United Kingdom)
Andrew Green, Heriot-Watt Univ. (United Kingdom)
Sam W. Hutchings, The Univ. of Edinburgh (United Kingdom)
Amy Davies, Heriot-Watt Univ. (United Kingdom)
Neale A. W. Dutton, STMicroelectronics Ltd. (United Kingdom)
Rory R. Duncan, Heriot-Watt Univ. (United Kingdom)
Colin Rickman, Heriot-Watt Univ. (United Kingdom)
Robert K. Henderson, The Univ. of Edinburgh (United Kingdom)
Paul A. Dalgarno, Heriot-Watt Univ. (United Kingdom)

Published in SPIE Proceedings Vol. 10889:
High-Speed Biomedical Imaging and Spectroscopy IV
Kevin K. Tsia; Keisuke Goda, Editor(s)

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