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Updated: Jan 10, 2026

A Protocol for Real-time 3D Single Particle Tracking

A Protocol for Real-time 3D Single Particle Tracking

Published on: January 3, 2018

Single-Photon Single-Particle Tracking.

Lance W Q Xu^1,2, Nathan Ronceray³, Marianna Fanouria Mitsioni^3,4

¹Center for Biological Physics, Arizona State University, Tempe, AZ, USA.

Biorxiv : the Preprint Server for Biology

|November 24, 2025

Summary

This summary is machine-generated.

We developed single-photon single-particle tracking (SP2T) to track fast-moving biological particles using 1-bit images. This method bypasses traditional tracking limitations, enabling accurate motion analysis at microsecond scales.

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3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

Published on: October 1, 2014

Area of Science:

Biophysics
Imaging Science
Computational Biology

Background:

Fast-moving biological particles (biomolecules, viral capsids) exceed conventional imaging speeds, causing motion blur.
Existing tracking methods fail with 1-bit imaging due to reliance on intensity-based localization.
High-speed imaging requires microsecond exposures, challenging traditional tracking algorithms.

Purpose of the Study:

Introduce a novel framework, single-photon single-particle tracking (SP2T), for unbiased particle tracking.
Enable accurate trajectory estimation from 1-bit image stacks, overcoming limitations of conventional methods.
Provide a robust solution for analyzing fast-moving biological particles at microsecond temporal resolutions.

Main Methods:

Developed SP2T to estimate particle numbers and trajectories directly from 1-bit image stacks, bypassing localization and linking.
Utilized single-photon avalanche diode (SPAD) arrays for microsecond-scale exposures over large fields of view.
Accounted for detector-specific artifacts like hot and cold pixels within the SP2T framework.

Main Results:

SP2T successfully tracks programmed fluorescent bead trajectories and biological systems (aerolysin, ganglioside).
Simulations and experiments show longer exposures bias diffusion coefficient estimates by up to 70% for fast-diffusing particles.
SP2T achieves over a 50-fold GPU speedup compared to CPU-based methods, demonstrating computational efficiency.

Conclusions:

SP2T is a robust and data-efficient solution for unbiased particle tracking in fast-diffusion regimes.
The framework enables accurate analysis of particle dynamics at millisecond-to-microsecond temporal resolutions.
Photon-by-photon tracking is essential for unbiased diffusion coefficient estimation and accurate jump-distance distribution analysis.