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Cardiac architecture is inherently three-dimensional, yet most characterizations rely on two-dimensional histological slices or dissociated cells, which remove the native geometry of the heart. We previously developed a method for labeling intact heart sections without dissociation and imaging large volumes while preserving their three-dimensional structure. We further refine this method to permit quantitative analysis of imaged sections. After data acquisition, these sections are assembled using image-processing tools, and qualitative and quantitative information is extracted. By examining the reconstructed cardiac blocks, one can observe end-to-end adjacent cardiac myocytes (cardiac strands) changing cross-sectional geometries, merging and separating from other strands. Quantitatively, representative cross-sectional areas typically used for determining hypertrophy omit the three-dimensional component; we show that taking orientation into account can significantly alter the analysis. Using fast-Fourier transform analysis, we analyze the gross organization of cardiac strands in three dimensions. By characterizing cardiac structure in three dimensions, we are able to determine that the crystallin mutation leads to hypertrophy with cross-sectional area increases, but not necessarily via changes in fiber orientation distribution. [ABSTRACT FROM AUTHOR]

Three-dimensional (3-D) tissue imaging offers substantial benefits to a wide range of biomedical investigations from cardiovascular biology, diabetes, Alzheimer’s disease to cancer. Two-photon tissue cytometry is a novel technique based on high-speed multiphoton microscopy coupled with automated histological sectioning, which can quantify tissue morphology and physiology throughout entire organs with subcellular resolution. Furthermore, two-photon tissue cytometry offers all the benefits of fluorescence-based approaches including high specificity and sensitivity and appropriateness for molecular imaging of gene and protein expression. We use two-photon tissue cytometry to image an entire mouse heart at subcellular resolution to quantify the 3-D morphology of cardiac microvasculature and myocyte morphology spanning almost five orders of magnitude in length scales. [ABSTRACT FROM AUTHOR]