Simultaneous 3D single-particle tracking and fluorescence lifetime measurement with a single-photon detector array

Single-particle tracking (SPT) techniques are a fundamental tool for providing information on the complex functions and interactions of individual, specifically labelled particles in biological environments. The main concern about following single particles resides mostly in how to make the registered photons informative. The solution is not unique and, given a certain photon budget, it is usually optimized only to better extrapolate either the temporal or the spatial information. This tradeoff ultimately reflects into the contraposition of wide-field camera-based techniques and real-time SPT, with the latter representing the state of the art, but at the cost of high technical complexity. Furthermore, fluorescence lifetime information, despite being accessible in many cases, has yet to be measured consistently. Within the context of the BrightEyes ERC-2018-COG project, we present a real-time three-dimensional SPT implementation based on a confocal laser-scanning microscope featuring a 5×5 single-photon avalanche diode (SPAD) array detector and few other optics capable of overcoming the above limitations due to a hybrid approach. In brief, each sensitive element of the detector acts as a confocal pinhole, and the recorded intensity distribution mirrors the position of the particle in the three dimensions with respect to the center of the excitation volume. Any displacement of the particle leads to a variation of the distribution of the recorded signal, thus allowing the estimation of the position. This localization is then used as input for a real-time feedback tracking system with the aim to move the illumination and keep the particle always in the center of the field-of-view. Notably, each pixel is an independent photon counting detector, allowing for concurrent measurement of the fluorescence lifetime. The system is capable of experimentally tracking single particles with a localization precision up to 30 nm with 100 photons and microsecond time resolution while performing fluorescence lifetime using the same photon stream. The technique has been validated by tracking 20 nm fluorescent beads moved along a predetermined path and by measuring the diffusion coefficient when freely diffusing in different water-glycerol solutions. As application, the movement of lysosomes in living cells has been investigated while assessing the lifetime of the GFP marker expressed on their membrane.