Dynamic particle tracking via temporal focusing multiphoton microscopy with astigmatism imaging

Area of Science:

• Biophysics
• Microscopy
• Nanotechnology

Background:

• Accurate 3D tracking of single particles is crucial for understanding molecular dynamics.
• Conventional multiphoton microscopy techniques can suffer from optical trapping interference and limited axial resolution.

Purpose of the Study:

• To develop and validate a 3D single fluorescent particle tracking strategy using temporal focusing multiphoton excitation microscopy (TFMPEM) combined with astigmatism imaging.
• To achieve nanoscale-level axial information for revealing 3D trajectories without z-axis scanning.

Main Methods:

• Utilized temporal focusing multiphoton excitation microscopy (TFMPEM) with astigmatism imaging for particle tracking.
• Implemented a strategy for widefield illumination with minimized optical trapping force.
• Achieved dynamic measurement of fluorosphere motion and diffusion coefficients in glycerol solutions.

Main Results:

• Demonstrated nanoscale-level axial and lateral positioning resolutions of 21 nm and 14 nm (standard deviation), respectively.
• Achieved dynamic measurements at a frame rate up to 100 Hz.
• Showcased the ability to reveal 3D trajectories of single fluorospheres within the multiphoton excitation volume.

Conclusions:

• The proposed TFMPEM with astigmatism imaging provides superior penetration and reduced trapping effects for deep tissue imaging.
• This method offers a fast frame rate and nanoscale positioning, holding promise for exploring dynamic molecular behavior.
• The technique enables precise tracking of particle trajectories, advancing the study of diffusion and molecular motion.