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

Mayandi Sivaguru, Glenn Fried, Barghav S. Sivaguru, Vignesh A. Sivaguru, Xiaochen Lu, Kyung Hwa Choi, M. Taher A. Saif, Brian Lin, Sakthivel Sadayappan

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)295-308
Number of pages14
JournalBioTechniques
Volume59
Issue number5
DOIs
StatePublished - Nov 2015

Keywords

  • Confocal microscopy
  • Heart failure
  • Heart-3-D
  • MYBPC3
  • PAS labeling
  • Two-photon
  • Whole-mount

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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