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Biotechniques. 2015 Nov 1;59(5):295-308. doi: 10.2144/000114356. eCollection 2015 Nov.

Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy.

Author information

Microscopy and Imaging Core Facility, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana Champaign, Urbana.
College of Liberal Arts and Sciences, University of Illinois at Urbana Champaign, Urbana.
Department of Cell and Developmental Biology and Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana Champaign, Urbana.
Department of Mechanical Science and Engineering, University of Illinois at Urbana Champaign, Urbana.
Cell and Molecular Physiology, Stritch School of Medicine, Loyola University, Maywood, IL.


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.


MYBPC3; PAS labeling; confocal microscopy; heart failure; heart-3-D; two-photon; whole-mount

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