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

Author

Listed:
  • Andrea Bucci

    (Istituto Italiano di Tecnologia
    University of Genoa)

  • Giorgio Tortarolo

    (Istituto Italiano di Tecnologia
    EPFL)

  • Marcus Oliver Held

    (Istituto Italiano di Tecnologia)

  • Luca Bega

    (Istituto Italiano di Tecnologia)

  • Eleonora Perego

    (Istituto Italiano di Tecnologia
    Université de Lausanne)

  • Francesco Castagnetti

    (Istituto Italiano di Tecnologia)

  • Irene Bozzoni

    (Istituto Italiano di Tecnologia
    Sapienza University)

  • Eli Slenders

    (Istituto Italiano di Tecnologia)

  • Giuseppe Vicidomini

    (Istituto Italiano di Tecnologia)

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.

Suggested Citation

  • Andrea Bucci & Giorgio Tortarolo & Marcus Oliver Held & Luca Bega & Eleonora Perego & Francesco Castagnetti & Irene Bozzoni & Eli Slenders & Giuseppe Vicidomini, 2024. "4D Single-particle tracking with asynchronous read-out single-photon avalanche diode array detector," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-50512-9
    DOI: 10.1038/s41467-024-50512-9
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    References listed on IDEAS

    as
    1. Giorgio Tortarolo & Alessandro Zunino & Francesco Fersini & Marco Castello & Simonluca Piazza & Colin J. R. Sheppard & Paolo Bianchini & Alberto Diaspro & Sami Koho & Giuseppe Vicidomini, 2022. "Focus image scanning microscopy for sharp and gentle super-resolved microscopy," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    2. Alessandro Rossetta & Eli Slenders & Mattia Donato & Sabrina Zappone & Francesco Fersini & Martina Bruno & Francesco Diotalevi & Luca Lanzanò & Sami Koho & Giorgio Tortarolo & Andrea Barberis & Marco , 2022. "The BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
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    4. Eleonora Perego & Sabrina Zappone & Francesco Castagnetti & Davide Mariani & Erika Vitiello & Jakob Rupert & Elsa Zacco & Gian Gaetano Tartaglia & Irene Bozzoni & Eli Slenders & Giuseppe Vicidomini, 2023. "Single-photon microscopy to study biomolecular condensates," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
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    6. Roman Schmidt & Tobias Weihs & Christian A. Wurm & Isabelle Jansen & Jasmin Rehman & Steffen J. Sahl & Stefan W. Hell, 2021. "MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
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