EPFL speeds up protein imaging with single-photon detector

13 Aug 2025

Fluorescence lifetime method characterizes thousands of molecules quickly and simultaneously.

A project at Switzerland's EPFL research center has developed a new variant of fluorescence lifetime imaging microscopy (FLIM) that could substantially accelerate the imaging operation.

Described in Light Science & Applications, the technique "marks a first step towards imaging procedures that enable scientists to study the behavior of specific molecules in large samples."

FLIM involves measuring the delay between an excitation laser pulse and the fluorescence duly emitted by a molecule, usually a nanosecond-scale interval. This can reveal details of the electronic structure of the emitter and its surrounding environment.

The measurements typically involve single-photon counting techniques incorporated in a confocal microscope, but do not usually address multiple targets in parallel, so the technique commonly features high localization alongside low throughput.

EPFL tackled this via use of a single-photon avalanche detector (SPAD) array, using a commercial SPAD and adapting its capture rate to match the laser pulses.

Although time-gated cameras have been applied before to FLIM in bright samples with dense labeling, their use in single-molecule microscopy has not been explored extensively, commented the project.

"Our method is slightly less accurate than conventional ones, but it is faster and can detect an unprecedented number of molecules at once," said Aleksandra Radenovic from EPFL Laboratory of Nanoscale Biology (LBEN). "This greater speed can enable rapid analyses of large protein samples."

Precise distances between molecules

In proof-of-concept trials, the project's optimized acquisition scheme achieved single-molecule lifetime measurements with a precision only about three times less than a single-photon counting approach, while imaging over a 512 × 512 pixel area. This allowed for the spatial multiplexing of over 3,000 molecules, according to the project.

Once the new method had proven effective, EPFL began exploring another application: detecting the distance between individual molecules. This involves a phenomenon termed Förster resonance energy transfer (FRET), in which energy is transferred between two light-sensitive molecules, with the fluorescence lifetime of a donor molecule changing if an acceptor molecule is nearby.

"Measuring the fluorescence lifetime of a pair of molecules provides information on the distance between them at a scale of just a few nanometers," commented Nathan Ronceray from LBEN. "The current approach can only be applied to small samples, but our system can expand it to allow for the rapid study of dynamic phenomena on thousands of molecules."

Having established single-photon cameras as a promising tool for the task, EPFL anticipates that its approach could now be valuable in fields such as spatial transcriptomics, which aims to measure gene expression in a tissue while preserving spatial information.

"By enabling the simultaneous reading of many molecular species throughout life, the method could serve as a powerful complement to emerging high-resolution omics tools used to study the different biological layers of an organism in a comprehensive and systematic way," commented EPFL.