Real-Time Axial Motion Compensation for Intravital Two-Photon Imaging of Mechanically Loaded Bone

Area of Science:

• Biomedical Engineering
• Cellular and Molecular Imaging
• Orthopedics

Background:

• Motion artifacts, particularly axial shifts, are a significant challenge in intravital imaging of dynamic tissues.
• These artifacts reduce data reproducibility and reliability, especially during mechanical loading experiments.
• Current post-acquisition methods are insufficient for real-time correction in mechanically loaded bone.

Purpose of the Study:

• To develop a novel method for real-time correction of axial motion during mechanical loading of bone.
• To improve the accuracy and reliability of intravital imaging data in mechanically stimulated bone tissue.
• To provide a user-friendly and adaptable framework for other mechanically loaded tissues.

Main Methods:

• Developed a system synchronizing objective piezo motor movement with the actuator piezo motor.
• Implemented a user-refined reduction via potentiometer for precise synchronization.
• Validated the system using static fluorescent markers and dynamic calcium indicators (GCaMP6f) in osteocytes.

Main Results:

• Effectively removed artificial changes in fluorescent intensity caused by axial motion up to 3000με.
• Demonstrated improved accuracy in capturing dynamic calcium signals (GCaMP6f) in osteocytes.
• Quantified improvements using similarity and average intensity metrics before and during loading.

Conclusions:

• The developed system provides robust, real-time axial motion correction during mechanical loading of bone.
• This approach enhances data quality and expands the applicability of two-photon imaging in mechanobiology.
• The framework is adaptable for studying other mechanically loaded biological tissues.