Imagine you could watch a single molecule inside a living cell respond to its environment in real time – measuring how viscous the fluid around it is, or detecting a fleeting interaction with a neighbouring protein. Techniques like fluorescence lifetime imaging microscopy (FLIM) can do exactly that. By measuring how long a fluorescent molecule stays “lit up” after being excited by a laser pulse, scientists can read out a remarkable range of biological information without ever needing to count the molecules directly.
Nonetheless, nature rarely reveals its secrets easily! Some of the most interesting fluorescent molecules decay so rapidly – in just one to ten trillionths of a second (picoseconds) – that conventional detectors simply can’t keep up. This is a fundamental timing wall set by the electronics involved, and it has left whole families of biologically relevant fluorophores out of reach.
A few years ago, our group proposed a way around this wall using an idea borrowed from quantum optics: the Hong–Ou–Mandel (HOM) effect. In a technique we called FL-HOM, fluorescent photons from the sample are brought together with reference photons at a beamsplitter, and the way they “bunch” together – a deeply quantum phenomenon – encodes the lifetime of the fluorescence with a time resolution far beyond what conventional detectors can achieve, down to the sub-picosecond scale.
The challenge until now was that FL-HOM required a rather specialised (and expensive) dual-output laser system to produce both the excitation light and the reference light at the right wavelengths. In our new paper, published in APL Photonics, we show how to achieve the same measurements using just a single, more affordable femtosecond laser.
We explored two different strategies for generating the reference photons from that single source. In the first, we use the laser to produce what’s known as a supercontinuum – a broad rainbow of light generated by firing a powerful pulse through a special optical fibre. In the second, we use a secondary sample of a different fluorescent dye as the reference, letting one dye “time” the other. Both approaches successfully recovered the ultrashort lifetimes of our test dyes, including 4-DASPI, a “molecular rotor” whose lifetime changes depending on the viscosity of its surroundings and which holds real promise for probing the physical properties of living cells.
Alongside the practical demonstrations, we take a close look at how the photon statistics of each reference source — essentially, how regularly and predictably photons arrive – affects the quality of the measurements. This matters because the HOM effect is sensitive to these statistical properties, and understanding the trade-offs helps anyone wanting to build their own FL-HOM system choose the right approach for their needs.
This work is a collaboration between our group at the University of Glasgow (Vytautas Zickus, Euan Millar, Ilya Starshynov and Ashley Lyons) and colleagues at the Center for Physical Sciences and Technology in Vilnius, Lithuania (Evita Spalinskaitė, Povilas Jurkšaitis and Zigmas Balevičius). By lowering both the cost and complexity of FL-HOM, we hope to put this technique within reach of a much wider range of researchers – opening up new possibilities for exploring biology at timescales that have, until now, been invisible.
The full paper is open access and can be read here: The role of photon statistics in fluorescence lifetime sensing with Hong–Ou–Mandel effect, APL Photonics 11, 106102 (2026).