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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

1
Microscopy and Imaging Core Facility, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana Champaign, Urbana.
2
College of Liberal Arts and Sciences, University of Illinois at Urbana Champaign, Urbana.
3
Department of Cell and Developmental Biology and Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana Champaign, Urbana.
4
Department of Mechanical Science and Engineering, University of Illinois at Urbana Champaign, Urbana.
5
Cell and Molecular Physiology, Stritch School of Medicine, Loyola University, Maywood, IL.

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.

KEYWORDS:

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

PMID:
26554507
DOI:
10.2144/000114356
[Indexed for MEDLINE]
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