3D Light Sheet Imaging of the Human Hypertrophied Septum

Introduction:: Hypertrophic cardiomyopathy (HCM) is often caused by genetic mutations, resulting in abnormal thickening of ventricular muscle, particularly the septum, and causing left ventricular outflow tract (LVOT) obstruction. Hypertrophic cardiomyopathy can be microscopically assessed and diagnosed by enlarged cell size of the hypertrophic cardiomyocytes, disarray of those cells, and a dense abnormal fibrotic microenvironment [1]. The thickening of the ventricular wall and septum can cause poor cardiac outcomes, which consequently lead to heart failure and mortality [2]. The extent of cell hypertrophy and fibrosis can directly impact the mechanical behavior of septal tissues. Determining the microstructural abnormalities is hence essential to better understand the biomechanical properties of the hypertrophic septal tissues. In this study, our goal is to develop a light sheet imaging protocol to assess the 3D microstructure of human hypertrophic septal tissues and compare with the healthy septum.

Materials and Methods:: Hypertrophic septal tissues were obtained as the surgical waste following septal myectomy surgery performed by Dr. Pietro Bajona at the Allegheny Healthy Network (AHN IRB Approval Number: 2020-240). The iDISCO method employs a three-step process comprising dehydration, dilapidation, and refractive index matching. The method involves the complete removal of lipids and dehydration of the tissue, which largely minimizes light scattering. The organic solvent solution is then used to replace the dehydrated and delipidated tissue with refractive index matching. The light sheet is created with a cylindrical lens, and ETL moves the focal region of the Gaussian beam which is synchronized with the active sensor on the camera. The auto-fluorescence emitting from the heart is captured by the detection objective lens and sCMOS camera. A five-axis stage is used to translate and rotate the heart along the detection axis while the light sheet is stationary, enabling us to capture a 3D image stack of the heart. A customized LabVIEW control algorithm is used to synchronize the propagation of the laser, the stage movement, the sCMOS camera rolling shutter, and the electrically tunable lens (ETL). During the image acquisition process, the inherent trigger and rolling shutter on the sCMOS are employed to capture sequential uniform 2D images, while the stage continuously translates the heart across the light sheet at a constant velocity that is determined by the step size and exposure time [3]. Volume rendering of the acquired images was done using the Amira software.


Results, Conclusions, and Discussions:: Our results demonstrated that the light sheet imaging was able to capture the microstructural differences in muscle fiber orientations, fiber alignment, fiber density and size (Figure 1). As shown by both 2D section image and 3D reconstructed image (Figure 1-A,C), the human hypertrophied septum showed much denser and larger size heart muscle fibers, as well as abrupt switching of major fiber bundle orientations (~90°). One the other hand, the healthy septum showed normal heart muscle fiber morphology (Figure 1-B,D). In short, we have successfully developed a tissue clearance and light sheet imaging protocol to assess the 3D microstructure of human hypertrophic septal tissues and healthy septal tissues. More detailed imaging and analyses efforts are currently ongoing, with a goal to capture more 3D anatomic details in large volume.

Acknowledgements (Optional): : NIH R15HL159599, R01HL157050

References (Optional): :

1. A. Varnava, et al. Heart 84(5) (2000) 476-482.


2. R. Hinojar, et al. International journal of cardiology 249 (2017) 467-472.


3. O. Sodimu et al., J. Biophotonics (2023) doi:10.1002/JBIO.202200278.