2023
bioRxiv

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

Authors:

Andrea Bucci, Giorgio Tortarolo, Marcus Oliver Held, Luca Bega, Eleonora Perego, Francesco Castagnetti, Irene Bozzoni, Eli Slenders, Giuseppe Vicidomini

Keywords:

single-particle tracking, fluorescence lifetime, confocal laser-scanning microscopy

Abstract:

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 com-plexity, 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 distribu-tion 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 par-ticle in the centre of the excitation volume. Importantly, as each pixel is an independent single-photon detector, SPT is combined with fluorescence lifetime measurement.

Our system achieves a localization precision of up to 30 nm 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 move-ment 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.