4D Single-Particle Tracking with Asynchronous Read-Out SPAD-Array Detector

Single-particle tracking (SPT) techniques are essential for investigating the complex functions and interactions of individual, specifically labelled particles in biological environments. Many SPT techniques exist, each optimised towards a different balance between spatiotemporal resolution and range, technical complexity, and information content. This bargain is exemplified by the contrast between wide-field camera-based and real-time SPT approaches, with the latter being generally more advanced but at the cost of high complexity. Furthermore, the fluorescence lifetime, a powerful tool for investigating the particle’s interactions and nano-environment, has yet to be measured consistently. To overcome these limitations, we propose a novel real-time three-dimensional SPT technique based on a hybrid approach. In our implementation, we equip a confocal laser-scanning microscope with an asynchronous read-out single-photon avalanche diode (SPAD) array detector and few other optics. Each sensitive detector element acts as a confocal pinhole, and the recorded intensity distribution reflects the particle’s position in three dimensions relative to the excitation volume. This localization is used in a real-time feedback system to keep the particle in the centre of the excitation volume. Importantly, as each pixel is an 1 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2023.08.25.554867; this version posted August 27, 2023. The copyright holder for this preprint independent single-photon detector, SPT is combined with fluorescence lifetime measurement. Our system achieves a localization precision of up to 30nm with 100 photons and microsecond time resolution, while also performing fluorescence lifetime measurements. First, we validated the technique by tracking fluorescent particles in artificial environments. Secondly, as further validation, we investigated the movement of lysosomes in living SK-N-BE cells and measured the fluorescence lifetime of the GFP marker expressed on a membrane protein. We observed an unprecedented correlation between the changes in fluorescence lifetime and the motion state of the lysosomes. Thanks to its simplicity and the great momentum of confocal microscopy based on SPAD array detector, we expect that this implementation will open to many information-rich correlative imaging and tracking studies.