Introduction: Sarcomere length (SL) is an important indicator of cardiac mechanical function, but current imaging modalities cannot unambiguously measure and characterize SL at the cell level in intact, living tissue(1). We have developed a method for measuring SL and tissue orientation using remote focusing microscopy, an emerging imaging modality that can capture 2-photon fluorescence from oblique planes. Methods: The illumination spot in a microscope can be translated along the z-axis by illuminating the objective lens with a non-parallel light beam, however this introduces significant optical aberrations. To obtain diffraction-limited performance and fast z-scanning, we use an axial scan unit (ASU) consisting of a second, matched objective that compensates for aberrations introduced by the imaging objective and a lightweight mirror. We have demonstrated that rates of up to 2.7 kHz are achievable with our system, due to the low weight of our scanning mirror. The scan speed in the z-direction is therefore comparable with galvanometer scan rates in the xy-plane(2). All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the Animals (Scientific Procedures) Act 1986 (UK). Hearts were isolated from female Sprague-Dawley rats, swiftly perfused via the aorta in Langendorff mode, loaded with membrane dye di-4-ANEPPS, transferred to a custom built cradle and imaged. We measure cell orientation from user selected cells in a field of view by imaging sarcomere structure on two oblique sub-planes that share a common major axis with the cell, at +45° and -45° to the image plane. Results: Our methods captured a wide range of tissue angles, ranging from 0 to 26 degrees over 8 experiments. SL correction using the angle data calculated from the arbitrary planes resulted in an average reduction of SL standard deviation of 7%, and in one experiment the standard deviation for SLs decreases from 0.071 µm to 0.055 µm after correction, a 23% reduction. Discussion: Remote focusing microscopy can unambiguously measure cell orientation in complex two photon data sets without capturing full z-stacks. The techniques presented here will allow rapid assessment of SL in healthy and diseased heart experimental preparations.