4D Single-particle tracking with asynchronous read-out single-photon avalanche diode array detector

Andrea Bucci, Giorgio Tortarolo, Marcus Oliver Held, Luca Bega, Eleonora Perego, Francesco Castagnetti, Irene Bozzoni, Eli Slenders and Giuseppe Vicidomini ()
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Andrea Bucci: Istituto Italiano di Tecnologia
Giorgio Tortarolo: Istituto Italiano di Tecnologia
Marcus Oliver Held: Istituto Italiano di Tecnologia
Luca Bega: Istituto Italiano di Tecnologia
Eleonora Perego: Istituto Italiano di Tecnologia
Francesco Castagnetti: Istituto Italiano di Tecnologia
Irene Bozzoni: Istituto Italiano di Tecnologia
Eli Slenders: Istituto Italiano di Tecnologia
Giuseppe Vicidomini: Istituto Italiano di Tecnologia

Nature Communications, 2024, vol. 15, issue 1, 1-12

Abstract: Abstract Single-particle tracking techniques enable investigation of the complex functions and interactions of individual particles in biological environments. Many such techniques exist, each demonstrating trade-offs between spatiotemporal resolution, spatial and temporal range, technical complexity, and information content. To mitigate these trade-offs, we enhanced a confocal laser scanning microscope with an asynchronous read-out single-photon avalanche diode array detector. This detector provides an image of the particle’s emission, precisely reflecting its position within the excitation volume. This localization is utilized in a real-time feedback system to drive the microscope scanning mechanism and ensure the particle remains centered inside the excitation volume. As each pixel is an independent single-photon detector, single-particle tracking is combined with fluorescence lifetime measurement. Our system achieves 40 nm lateral and 60 nm axial localization precision with 100 photons and sub-millisecond temporal sampling for real-time tracking. Offline tracking can refine this precision to the microsecond scale. We validated the system’s spatiotemporal resolution by tracking fluorescent beads with diffusion coefficients up to 10 μm2/s. Additionally, we investigated the movement of lysosomes in living SK-N-BE cells and measured the fluorescence lifetime of the marker expressed on a membrane protein. We expect that this implementation will open other correlative imaging and tracking studies.

Date: 2024
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DOI: 10.1038/s41467-024-50512-9

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