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 language | English (US) |
|---|---|
| Pages (from-to) | 295-308 |
| Number of pages | 14 |
| Journal | BioTechniques |
| Volume | 59 |
| Issue number | 5 |
| DOIs | |
| State | Published - Nov 2015 |
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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)
Cite this
Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using twophoton fluorescence and confocal microscopy. / Sivaguru, Mayandi; Fried, Glenn Allen; Sivaguru, Barghav S.; Sivaguru, Vignesh A.; Lu, Xiaochen; Choi, Kyung Hwa; Saif, M Taher A; Lin, Brian; Sadayappan, Sakthivel.
In: BioTechniques, Vol. 59, No. 5, 11.2015, p. 295-308.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using twophoton fluorescence and confocal microscopy
AU - Sivaguru, Mayandi
AU - Fried, Glenn Allen
AU - Sivaguru, Barghav S.
AU - Sivaguru, Vignesh A.
AU - Lu, Xiaochen
AU - Choi, Kyung Hwa
AU - Saif, M Taher A
AU - Lin, Brian
AU - Sadayappan, Sakthivel
PY - 2015/11
Y1 - 2015/11
N2 - 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.
AB - 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.
KW - Confocal microscopy
KW - Heart failure
KW - Heart-3-D
KW - MYBPC3
KW - PAS labeling
KW - Two-photon
KW - Whole-mount
UR - http://www.scopus.com/inward/record.url?scp=84946916011&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84946916011&partnerID=8YFLogxK
U2 - 10.2144/000114356
DO - 10.2144/000114356
M3 - Article
C2 - 26554507
AN - SCOPUS:84946916011
VL - 59
SP - 295
EP - 308
JO - BioTechniques
JF - BioTechniques
SN - 0736-6205
IS - 5
ER -