RATIONALE

In vivo microscopy seeks to observe dynamic subcellular processes in a physiologically relevant context. A primary limitation of optical microscopy in vivo is tissue motion, which prevents physiological time course observations or image averaging.

OBJECTIVE

To develop and demonstrate motion compensation methods that can automatically track image planes within biological tissues, including the tissue displacements associated with large changes in blood flow, and to evaluate the effect of global hypoxia on the regional kinetics and steady state levels of mitochondrial NAD(P)H.

METHODS AND RESULTS

A dynamic optical microscope, with real-time prospective tracking and retrospective image processing, was used collect high-resolution images through cellular responses to various perturbations. The subcellular metabolic response to hypoxia was examined in vivo. Mitochondria closest to the capillaries were significantly more oxidized at rest (67+/-3%) than the intrafibrillar mitochondria (83+/-3%; P<0.0001) in the same cell.

CONCLUSIONS

These data are consistent with the hypothesis that a significant oxygen gradient from capillary to muscle core exists at rest, thereby reducing the oxidative load on the muscle cell.