Cardiac Muscle Organization Revealed in 3-D by Imaging Whole-Mount Mouse Hearts using Two-Photon Fluorescence and Confocal Microscopy

Abstract

The ability to image the entire adult mouse heart at high resolution in 3-D would provide enormous advantages in the study of heart disease. However, a technique for imaging nuclear/cellular detail as well as the overall structure of the entire heart in 3-D with minimal effort is lacking. To solve this problem, we modified the benzyl alcohol:benzyl benzoate (BABB) clearing technique by labeling mouse hearts with periodic acid Schiff (PAS) stain. We then imaged the hearts with a combination of two-photon fluorescence microscopy and automated tile-scan imaging/stitching. Utilizing the differential spectral properties of PAS, we could identify muscle and nuclear compartments in the heart. We were also able to visualize the differences between a 3-month-old normal mouse heart and a mouse heart that had undergone heart failure due to the expression of cardiac myosin binding protein-C (cMyBP-C) gene mutation (t/t). Using 2-D and 3-D morphometric analysis, we found that the t/t heart had anomalous ventricular shape, volume, and wall thickness, as well as a disrupted sarcomere pattern. We further validated our approach using decellularized hearts that had been cultured with 3T3 fibroblasts, which were tracked using a nuclear label. We were able to detect the 3T3 cells inside the decellularized intact heart tissue, achieving nuclear/cellular resolution in 3-D. The combination of labeling, clearing, and two-photon microscopy together with tiling eliminates laborious and time-consuming physical sectioning, alignment, and 3-D reconstruction.

3D imaging in CUBIC-cleared mouse heart tissue: going deeper

“…Confocal microscopy is a well-established imaging technique that plays an important role in studying tissue with high magnification. However, this technology is limited by the reduced light penetration depth (around ~100-200 µm) due to changes in the refractive indices in the biological tissue (opacity of the tissue) and the resulting light scattering effects [4,5]. Furthermore, reduced antibody penetration represents an additional limitation when performing confocal microscopy in immunohistochemical studies using thick tissue sections.…”

Advances in Ex Situ Tissue Optical Clearing

“…BABB might be also used to clear and image >2.0 mm thick regions of adult mouse heart (Figure c,g), as presented by Smith et al . and Sivaguru et al . However, as BABB clearing does not elute heme from tissue, special attention must be paid during the perfusion step to remove as much hemoglobin as possible.…”

“…Perhaps suboptimal perfusion of hearts studied by Smith and colleagues resulted in high sample absorbance related to uncleared blood clots and heme, which contributed to significant background noise during the image acquisition and, therefore, could impede or even prevent its further analysis. In contrast, addition of cardioplegic solution during the perfusion step, followed by periodic acid Shiff staining allowed Sivaguru et al . to successfully clear and image the entire heart of 3‐month‐old mouse.…”

Application of an advanced maximum likelihood estimation restoration method for enhanced‐resolution and contrast in second‐harmonic generation microscopy

“…Wild type mouse hearts in the FVB/N (WT) background were euthanised in a carbon dioxide (CO 2 ) chamber by slow flow of CO 2 (10-30% of chamber volume per min) as described previously (Lynch et al, 2015;Sivaguru et al, 2015). This treatment was continued for 15-30 min after breathing stops.…”