Rationale: Sarcomere length (SL) is a key indicator of cardiac mechanical function, but current imaging technologies are limited in their ability to unambiguously measure and characterize SL at the cell level in intact, living tissue.
Objective: We develop a method for measuring SL and regional cell orientation, using remote focusing microscopy, an emerging imaging modality that can capture light from arbitrary oblique planes within a sample.
Methods and Results: We present a protocol that unambiguously and quickly determines cell orientation from user-selected areas in a field of view, by imaging two oblique planes that share a common major axis with the cell. We demonstrate the effectiveness of this technique in establishing single cell SL in Langendorff perfused hearts, loaded with the membrane dye di-4-ANEPPS.
Conclusions: Remote focusing microscopy can measure cell orientation in complex two photon data sets without capturing full z-stacks. The technique allows rapid assessment of SL in healthy and diseased heart experimental preparations.
