Fluorescence lifetime imaging of high-speed particles with single-photon image sensors

Istvan Gyongy*, Andrew Green, Sam W. Hutchings, Amy Davies, Neale A. W. Dutton, Rory R. Duncan, Colin Rickman, Robert K. Henderson, Paul A. Dalgarno

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingConference contribution

5 Citations (Scopus)


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.

Original languageEnglish
Title of host publicationHigh-Speed Biomedical Imaging and Spectroscopy IV
EditorsKevin K. Tsia, Keisuke Goda
ISBN (Electronic)9781510624214
ISBN (Print)9781510624207
Publication statusPublished - 4 Mar 2019
EventSPIE BiOS 2019 - Moscone Center, San Francisco, United States
Duration: 2 Feb 20193 Feb 2019

Publication series

NameProceedings of SPIE
ISSN (Print)0277-786X
ISSN (Electronic)1996-756X


ConferenceSPIE BiOS 2019
Country/TerritoryUnited States
CitySan Francisco


  • Flourescence lifetime microscopy
  • Fluorescence microscopy
  • Particle tracking
  • Photon counting image sensor
  • Single-photon avalanche diode

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics
  • Biomaterials
  • Radiology Nuclear Medicine and imaging


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