In cardiac muscle, intracellular Ca 2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric deformation. Our understanding of the internal relationships between these events has been limited because we can neither 'see' inside the muscle nor precisely track the spatiotemporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca 2+ transient, and an optical coherence tomograph to capture the tissue's geometric changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 µm by 540 µm) and the length of a typical trabecula (>2000 µm). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying imageprocessing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into

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Cardiac trabeculae are widely used as experimental muscle preparations for studying heart muscle. However, their geometry (diameter, length, and shape) can vary not only among samples, but also within a sample, leading to inaccuracies in estimating their stress production, volumetric energy output, and/or oxygen consumption. Hence, it is desirable to have a system that can accurately image each trabecula in vitro during an experiment. To this end, we constructed an optical coherence tomography system and implemented a gated imaging procedure to image actively contracting trabeculae and reconstruct their time-varying geometry. By imaging a single cross section while monitoring the developed force, we found that gated stimulation of the muscle was sufficiently repeatable to allow us to reconstruct multiple contractions to form a four-dimensional representation of a single muscle contraction cycle. The complete muscle was imaged at various lengths and the cross-sectional area along the muscle was quantified during the contraction cycle. The variation of cross-sectional area along the length during a contraction tended to increase as the muscle was contracting, and this increase was greater at longer muscle lengths. To our knowledge, this is the first system that is able to measure the geometric change of cardiac trabeculae in vitro during a contraction, allowing cross-sectional stress and other volume-dependent parameters to be estimated with greater accuracy.