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

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Authors: Sivaguru, M.; Fried, G.; Sivaguru, B. S.; Sivaguru, V. A.; Lu, X; Choi, K. H.; Saif, M. T.; Lin, B.; Sadayappan, S
Article Title: 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.
Journal Title: BioTechniques
Volume: 59
Issue: 5
ISSN: 1940-9818; 0736-6205
Publisher: Unknown  
Journal Place: England
Date Published: 2015
Start Page: 295
End Page: 308
Language: eng
DOI/URL:

10.2144/000114356
Notes: JID: 8306785; OTO: NOTNLM; 2015 [ecollection]; 2015/05/05 [received]; 2015/09/03 [accepted]; epublish