Mora-Antoinette, M. | bioRxiv Science

bioRxiv2026

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

Motion artifacts present a major challenge for intravital imaging of tissues undergoing physiological movement or mechanical loading. Blurring or artificial changes in image intensity due to shifting on the z-axis reduce data reproducibility and reliability. Existing post-acquisition approaches can partially compensate for motion, but they increase experimental complexity and are not well suited for use in mechanically loaded bone. Therefore, we developed a novel method for real-time correction of axial motion during mechanical loading of bone by synchronizing the movement of the objective to the actuator. Synchronization was achieved by linking the position of the actuator piezo motor to the objective piezo motor with a user refined reduction via potentiometer. Applying axial motion correction effectively removed artificial changes in fluorescent intensity in a static fluorescent marker up to 3000{varepsilon} in bone as measured by similarity and average intensity before and during loading. This improvement was reflected in the improved accuracy in capture of a dynamic fluorescent calcium indicator (GCaMP6f) in osteocytes. Our system provides a user-friendly, robust framework that can be easily adapted to other mechanically loaded tissues, improving data collection and expanding the utility of two-photon imaging across a variety of biological applications.

bioengineering↗

bioRxiv2023

Nicotinic Acetylcholine Receptors on Osteocytes Impact Bone Mechanoadaptation in a Sexually Dimorphic Manner

Recent evidence suggests acetylcholine has a positive influence on bone mechanotransduction. Osteocytes express components for nicotinic acetylcholine receptors (nAChRs), which are known for mediating calcium signaling and may impact mechanosensitivity. Here, we use novel fluorescent imaging approaches to provide the first evidence of direct interaction between osteocytes and cholinergic nerve fibers in cortical bone in vivo. Moreover, we show that osteocytes are functional targets of cholinergic signaling for bone mechanoadaptation. We report sexually dimorphic patterns in bone structure and mechanobiology based on nAChR function. In females, osteocyte mechanosensitivity was decreased at small force magnitudes and tissue level deficits were recovered with anabolic loading. In males, osteocyte mechanosensitivity was increased in some groups and anabolic loading had very little effect on overall tissue architecture. This work establishes a new signaling paradigm wherein osteocytes interface with cholinergic nerves and bone mechanotransduction is regulated by osteocyte cholinergic signaling in a sexually dimorphic way.

physiology↗