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

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

作者机构：Ist Italiano Tecnol Mol Microscopy & Spect Genoa Italy Univ Genoa Dipartimento Informat Bioingn Robot Ingn Sistemi Genoa Italy Ist Italiano Tecnol Non Coding RNAs Physiol & Pathol Genoa Italy Sapienza Univ Dept Biol & Biotechnol Charles Darwin Rome Italy Ecole Polytech Fed Lausanne Lab Expt Biophys Lausanne Switzerland Univ Lausanne Ctr Integrat Genom Lausanne Switzerland 

出 版 物：《NATURE COMMUNICATIONS》 (Nat. Commun.)

年 卷 期：2024年第15卷第1期

页      面：6188页

核心收录：

学科分类：07[理学] 08[工学] 

基　　金：European Research Council [818699, 855923] European Union - Next Generation EU, PNRR MUR - M4C2 - Action 1.4 - Call "Potenziamento strutture di ricerca e creazione di "campioni nazionali di RS" [CUP J33C22001130001] National Center for Gene Therapy and Drugsbased on RNA Technology [CN00000041] Associazione Italiana per la Ricerca Sul Cancro Ministero dell'Istruzione, dell'Universita e Della ricerca (MIUR) [2017P352Z4] European Research Council (ERC) Funding Source: European Research Council (ERC) 

摘      要：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 mu m2/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. Here, the authors upgrade a confocal laser scanning microscope with a single-photon array detector, achieving 40 nm lateral and 60 nm axial localisation precision with 100 photons and a sub-millisecond temporal sampling for real-time single-particle tracking with fluorescence lifetime measurement.