Introduction: Cardiac myocytes exhibit cross striations which are formed by alternating segments of thick (A bands) and thin (I bands) protein filaments. Quantitative imaging of the A-I band spacing (i.e., sarcomere length, SL) can provide important information about the relation of interfilament overlap and mechanical contractile performance. SL cannot be measured directly from confocal images as the orientation of cells in the tissue relative to the imaging plane is unknown (Bub et al., 2010). To overcome this limitation, we introduce a novel two-photon microscopy approach, where imaging planes with user defined angles can be scanned at high speed (Botcherby et al. 2008). Methods: Hearts, isolated from Sprague-Dawley rats (~250g), killed by Schedule 1 in accordance to UK Home Office regulations, were swiftly mounted to a Langendorff system for coronary perfusion with normal tyrode, and loaded by coronary perfusion for 5 min with 5 μM di-4-ANEPPS. Hearts were then perfused with zero calcium Tyrode (In mM: 140 NaCl, 5.4 KCl, 1.0 MgSO4, 5.0 HEPES, 1.0 Glucose, pH 7.4, 37°C) to reduce contractile activity, placed on an organ-tailored cradle, stabilized using nylon mesh, and imaged. The tissue orientation information can be obtained by imaging two perpendicular planes if their intersection line is predominantly along the primary (long) axis of the cell (Fig. 1). The cell axis in each of these planes can then be identified from a spatial frequency vector (K) evaluated in three dimensions. Two orthogonal sub-images can be captured within 500 msec allowing unambiguous determination of the angle of the surrounding tissue. Results: Preliminary results demonstrate that cells oriented at a sharp angle relative to the confocal image plane display longer apparent SLs. By correcting for tissue angle, we can reduce measurement variance by as much as 15%. Discussion: This method improves our ability to detect subtle changes in cell structure in diseased models where there is dysregulation in the excitation-contraction coupling mechanism (Li et al., 2012).