Three‐dimensional motion tracking for high‐resolution optical microscopy, in vivo

When conducting optical imaging experiments, in vivo, the signal to noise ratio and effective spatial and temporal resolution is fundamentally limited by physiological motion of the tissue. A three‐dimensional (3D) motion tracking scheme, using a multiphoton excitation microscope with a resonant galvanometer, (512 × 512 pixels at 33 frames s−1) is described to overcome physiological motion, in vivo. The use of commercially available graphical processing units permitted the rapid 3D cross‐correlation of sequential volumes to detect displacements and adjust tissue position to track motions in near real‐time. Motion phantom tests maintained micron resolution with displacement velocities of up to 200 μm min−1, well within the drift observed in many biological tissues under physiologically relevant conditions. In vivo experiments on mouse skeletal muscle using the capillary vasculature with luminal dye as a displacement reference revealed an effective and robust method of tracking tissue motion to enable (1) signal averaging over time without compromising resolution, and (2) tracking of cellular regions during a physiological perturbation.