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© 2015 SPIE. In cardiac imaging, the spacing between sub-cellular sarcomere structures is of great importance to physiologists in understanding muscle design and performance. Making accurate measurements of the sarcomere length (SL) presents a significant imaging challenge owing to the size of the SL (∼2μm) and its naturally low variability (<6%), requiring a high level of precision to determine subtle changes between heart disease models. Moreover, measurements of SL from traditional two-photon imaging have so far been ambiguous to within a factor of cos(α), where α is the inclination of the tissue with respect to the focal plane. By remotely focussing a customised two-photon microscope, it is possible to image heart cells at two oblique angles within 200ms. The oblique images uniquely resolve the tissue inclination ambiguity and reduce the variance of SL measures by as much as 23%. This improved precision is crucial in discerning between pathological models of chronic hypertension. As well as improving measurement precision, the distribution of α across the field of view provides additional structural information which can be related to disease morphology. To validate this new imaging protocol, the value ofα calculated from the oblique planes provided the input to a rigid model cell which was used to predict the appearance of the cell in the conventional focal plane. The comparison of the model to the image data provided a confidence metric for our measurements. Finally, by considering the optical transfer function, the range of cell orientations for which the method is valid could be calculated.

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