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.
authors.affiliation: Microscopy and Imaging Core Facility, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana Champaign, Urbana.
authors.forename: Mayandi
authors.initials: M
authors.lastname: Sivaguru
authors.affiliation: Microscopy and Imaging Core Facility, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana Champaign, Urbana.
authors.forename: Glenn
authors.initials: G
authors.lastname: Fried
authors.affiliation: College of Liberal Arts and Sciences, University of Illinois at Urbana Champaign, Urbana.
authors.forename: Barghav S
authors.initials: BS
authors.lastname: Sivaguru
authors.affiliation: College of Liberal Arts and Sciences, University of Illinois at Urbana Champaign, Urbana.
authors.forename: Vignesh A
authors.initials: VA
authors.lastname: Sivaguru
authors.affiliation: Department of Cell and Developmental Biology and Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana Champaign, Urbana.
authors.forename: Xiaochen
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Mechanical Science and Engineering, University of Illinois at Urbana Champaign, Urbana.
authors.forename: Kyung Hwa
authors.initials: KH
authors.lastname: Choi
authors.affiliation: Department of Mechanical Science and Engineering, University of Illinois at Urbana Champaign, Urbana.
authors.forename: M Taher A
authors.initials: MT
authors.lastname: Saif
authors.affiliation: Cell and Molecular Physiology, Stritch School of Medicine, Loyola University, Maywood, IL.
authors.forename: Brian
authors.initials: B
authors.lastname: Lin
authors.affiliation: Cell and Molecular Physiology, Stritch School of Medicine, Loyola University, Maywood, IL.
authors.forename: Sakthivel
authors.initials: S
authors.lastname: Sadayappan
mesh_terms: D016475:3T3 Cells; D000818:Animals; D006321:Heart / Q000033:anatomy & histology* / Q000503:physiopathology*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009206:Myocardium / Q000648:ultrastructure*
keywords: MYBPC3; PAS labeling; confocal microscopy; heart failure; heart-3-D; two-photon; whole-mount