High-speed, high-precision, deep-tissue three-dimensional single-particle tracking

The invention enhances particle tracking in biological research using spatiotemporal offset light beams with two-photon microscopy. It achieves high-speed, precise, deep tissue tracking, offering improved penetration depth and temporal resolution for studying molecular processes.

Background

Single-particle tracking (SPT) is a powerful tool for observing the dynamic behaviors of particles within complex biological systems, offering insights into processes such as motor protein kinetics, cellular membrane dynamics, and virus internalization. However, existing SPT techniques face significant challenges, including limited penetration depth due to one-photon excitation, restricted z-tracking range, poor temporal resolution from frame-by-frame analysis, and low information content without fluorescence lifetime data.

While two-photon microscopy has advanced deep tissue imaging, traditional methods for 3D tracking using two-photon excitation are hindered by slow temporal resolution and limited working depth in scattering samples. Additionally, multifocal setups often require multiple detectors, complicating the system and reducing signal efficiency. These limitations hinder the comprehensive study of rapid molecular transport dynamics in highly scattering environments and at significant depths, which are essential for understanding complex biological processes and disease mechanisms.

Technology description

The technology is an advanced system for tracking particles using spatiotemporal offset light beams, designed to work with conventional two-photon microscopy equipment. This system allows for high-speed, high-precision, and deep-tissue three-dimensional single-particle tracking. It employs a unique spatiotemporally multiplexed point-spread function (PSF) created by four temporally offset beams that illuminate a tetrahedral pattern in the image space.

The PSF is generated by a passive optical system positioned before the scanning optics of a two-photon microscope. The tracking algorithm uses Time-Correlated-Single-Photon Counting (TCSPC) electronics to demultiplex the fluorescent decay from the excitation beams, enabling sub-diffraction localization of single particles. This approach significantly enhances penetration depth and temporal resolution, allowing particle tracking at depths up to 200 micrometers with spatial localization precision of up to 35 nanometers and temporal resolution as fine as 50 microseconds.

This technology stands out due to its ability to achieve deep-tissue imaging with high temporal and spatial precision, which is a significant improvement over traditional methods. The use of a spatiotemporally multiplexed PSF allows for the simultaneous excitation of multiple points in space, enhancing the system's ability to track particles in three dimensions. The integration of TCSPC electronics for demultiplexing fluorescence signals further differentiates this system by enabling precise localization of particles beyond the diffraction limit of light. Additionally, the system's compatibility with conventional two-photon microscopy equipment makes it accessible for existing setups, facilitating its adoption in various research applications. The ability to track particles at significant depths and with high precision opens new possibilities for studying complex biological processes, such as molecular diffusion and transport, in their native environments.

Benefits

High-speed and high-precision tracking of particles
Deep-tissue three-dimensional single-particle tracking up to 200 micrometers
Sub-diffraction localization precision up to 35 nanometers
Temporal resolution as fine as 50 microseconds
Compatible with conventional two-photon microscopy equipment
Facilitates single-molecule studies of biological diffusion and transport processes
Requires only one detector for 3D particle tracking
Allows for multi-color two-photon imaging
Utilizes a unique spatiotemporally multiplexed point-spread function
Enables simultaneous fluorescence lifetime measurements on tracked particles