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research article

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

Bucci, Andrea
•
Tortarolo, Giorgio  
•
Held, Marcus Oliver
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July 23, 2024
Nature Communications

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.

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Type
research article
DOI
10.1038/s41467-024-50512-9
Web of Science ID

WOS:001275549300015

PubMed ID

39043637

Author(s)
Bucci, Andrea
•
Tortarolo, Giorgio  
•
Held, Marcus Oliver
•
Bega, Luca
•
Perego, Eleonora
•
Castagnetti, Francesco
•
Bozzoni, Irene
•
Slenders, Eli
•
Vicidomini, Giuseppe
Date Issued

2024-07-23

Publisher

NATURE PORTFOLIO

Published in
Nature Communications
Volume

15

Issue

1

Article Number

6188

Subjects

GREEN FLUORESCENT PROTEIN

•

LIFETIME

•

DYNEIN

•

MOLECULES

•

TRANSPORT

•

MEMBRANE

•

MOBILITY

•

KINESIN

•

Science & Technology

Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
LEB  
FunderFunding(s)Grant NumberGrant URL

European Research Council (ERC)

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

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Available on Infoscience
February 1, 2025
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/246188
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