Wide-field fluorescence lifetime imaging of single molecules with a gated single-photon camera

Nathan Ronceray; Salim Bennani; Marianna Fanouria Mitsioni; Nicole Siegel; Maria J. Marcaida; Claudio Bruschini; Edoardo Charbon; Rahul Roy; Matteo Dal Peraro; Guillermo P. Acuna; Aleksandra Radenovic

doi:10.1038/s41377-025-01901-2

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Wide-field fluorescence lifetime imaging of single molecules with a gated single-photon camera

Original Articles

- Wide-field fluorescence lifetime imaging of single molecules with a gated single-photon camera
- Light: Science & Applications Vol. 14, Issue 9, Pages: 2709-2719(2025)
- Affiliations：
  
  1.Institute of Bioengineering, School of Engineering, EPFL, Lausanne, Switzerland
  2.NCCR Bio-Inspired Materials, EPFL, Lausanne, Switzerland
  3.Department of Physics, University of Fribourg, Fribourg, Switzerland
  4.AQUA, EPFL, Neuchâtel, Switzerland
  5.Department of Chemical Engineering and Department of Bioengineering, Indian Institute of Science Bangalore, Bangalore, India
  6.National Center of Competences in Research Bio-Inspired Materials, University of Fribourg, 1700 Fribourg, Switzerland
- Author bio：
  
  Nathan Ronceray (nathan.ronceray@epfl.ch)
  Aleksandra Radenovic (aleksandra.radenovic@epfl.ch)
- Funds：
- DOI：10.1038/s41377-025-01901-2
  CLC：
- Received：28 November 2024，
  
  Revised：2025-05-07，
  
  Accepted：21 May 2025，
  
  Published Online：04 August 2025，
  
  Published：30 September 2025
- Accepted：
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Ronceray, N. et al. Wide-field fluorescence lifetime imaging of single molecules with a gated single-photon camera. Light: Science & Applications, 14, 2709-2719 (2025).
DOI：

Ronceray, N. et al. Wide-field fluorescence lifetime imaging of single molecules with a gated single-photon camera. Light: Science & Applications, 14, 2709-2719 (2025). DOI： 10.1038/s41377-025-01901-2.

摘要

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment. Traditionally

FLIM uses time-correlated single-photon counting (TCSPC)

which is precise but intrinsically low-throughput due to its dependence on point detectors. Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling

their use in single-molecule microscopy has not been explored extensively. Here

we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera. Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC

while imaging with a large number of pixels (512 × 512) allowing for the spatial multiplexing of over 3000 molecules. With this approach

we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers

and temporal single-molecule Förster resonance energy transfer measurements at 5-25 Hz. This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.

关键词

Keywords

references

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