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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
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Fluorescence lifetime imaging with a single-photon SPAD array using long overlapping gates: an experimental and

Andrei Ardelean1, Arin Can Ulku1, Xavier Michalet2

  • 1AQUA Laboratory, EPFL, 71b Rue de la Maladière, Neuchâtel, Switzerland.

Proceedings of Spie--The International Society for Optical Engineering
|April 9, 2021
PubMed
Summary

This study shows that using long, overlapping time gates in single-photon avalanche diode (SPAD) arrays effectively extracts fluorescence lifetimes. This method improves signal-to-noise ratio without compromising lifetime resolution, even for complex samples.

Keywords:
FLIMSPADgated FLIMlong gatephasor

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Area of Science:

  • Photonics and advanced optical imaging techniques.
  • Development of novel detector arrays for fluorescence lifetime imaging microscopy (FLIM).

Background:

  • Developing large single-photon avalanche diode (SPAD) arrays with on-chip time-correlated single-photon counting (TCSPC) is challenging due to space and complexity constraints.
  • Time-gated architectures offer an alternative to TCSPC for fluorescence lifetime measurements.
  • Previous limitations in gate length (nanoseconds) exceeded typical fluorophore lifetimes, but recent findings highlight gate step and rise/fall times as critical for resolution.

Purpose of the Study:

  • To investigate the impact of long, overlapping time gates on fluorescence lifetime extraction using phasor analysis.
  • To evaluate the performance of a new 512x512 time-gated SPAD array for lifetime measurements.
  • To assess the capability of this method for resolving multi-exponential decays.

Main Methods:

  • Utilized a 512x512 time-gated SPAD array for fluorescence lifetime measurements.
  • Employed phasor analysis for lifetime extraction with varying gate window lengths (11.3-23 ns) and gate steps (17.86 ps - 3 ns).
  • Validated results against a standard time-correlated single-photon counting (TCSPC) setup and used simulations for multi-exponential samples.

Main Results:

  • Lifetime extraction accuracy was maintained despite the use of longer time gates.
  • The signal-to-noise ratio (SNR) was enhanced by capturing more photons within longer gates.
  • The ability to resolve multi-exponential fluorescence decays was not negatively affected by the long gate approach.

Conclusions:

  • Long, overlapping time gates are a viable and effective strategy for fluorescence lifetime measurements with SPAD arrays.
  • This approach overcomes limitations of traditional short-gate time-gated methods and enhances SNR.
  • The developed method shows promise for advanced FLIM applications, including the analysis of complex biological samples.