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 developed 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 2 oblique planes that share a common major axis with the cell. We demonstrate the effectiveness of the 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 2-photon data sets without capturing full z stacks. The technique allows rapid assessment of SL in healthy and diseased heart experimental preparations.