Active axial motion compensation in multiphoton-excited fluorescence microscopy.

Area of Science:

• Biomedical Engineering
• Optical Microscopy
• In Vivo Imaging

Background:

• Living organisms exhibit natural motion (heartbeat, breathing, muscle movement) causing tissue deformation.
• This motion displaces the observation plane in intravital microscopy, leading to motion-induced aberrations and limiting observation time.
• Current methods struggle to overcome these limitations for observing dynamic biological processes.

Purpose of the Study:

• To develop a method for active motion compensation in intravital microscopy.
• To overcome limitations imposed by physiological motion on imaging dynamic events.
• To enable stable, long-term observation of biological processes in vivo.

Main Methods:

• Developed a mathematical shape space model to predict periodic motion of cylindrical tissue phantoms (e.g., blood vessels).
• Utilized the model to calculate future positions of the microscope's observation plane.
• Implemented a piezo-actuated objective lens holder for continuous focal plane adjustment to compensate for motion.

Main Results:

• Demonstrated active motion compensation for non-harmonic axial displacements of a vessel phantom.
• Successfully compensated for motion with vertical amplitudes exceeding 100 µm at 0.5 Hz.
• Maintained a stable observation plane within a field of view up to 400 µm × 400 µm.

Conclusions:

• The developed mathematical model and active compensation system effectively counteract physiological tissue motion during intravital microscopy.
• This approach significantly enhances the ability to observe dynamic biological events by stabilizing the imaging plane.
• The method holds promise for improving the duration and quality of in vivo imaging experiments.