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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.