preparation of rodent heart sample for light-sheet microscopy

포괄적인 심장 이미징을 위한 Light-Sheet Microscopy and Tissue Clearing (광시트 현미경 검사) 및 Tissue Clearing(조직 투명화)

Heart failure remains the leading cause of mortality worldwide, primarily due to the lack of regenerative capacity of mature cardiomyocytes1. The intricate architecture of the heart plays a crucial role in its function and provides insights into developmental processes. A profound understanding of cardiac structure is essential for elucidating the fundamental processes of cardiac morphogenesis and remodeling in response to myocardial infarction. Recent progress has demonstrated that neonatal mice can restore cardiac function following injury, while adult mice lack such regenerative capacity2. This establishes a foundation for investigating cues associated with structural and functional abnormalities in mouse models. Traditional imaging methods, such as confocal microscopy, have technical limitations, including restricted penetration depth, slow point-scanning scheme, and photo damage from prolonged exposure to laser light. These hinder comprehensive three-dimensional (3D) imaging of the intact heart. In this context, light-sheet microscopy (LSM) emerges as a powerful solution, offering the advantages of high-speed imaging, reduced photo damage, and exceptional optical sectioning capabilities3,4,5. The unique features of LSM position it as a promising method to overcome the limitations of conventional techniques, providing unprecedented insights into cardiac development and remodeling processes6,7,8.
In this protocol, we introduce an imaging strategy that combines advanced LSM with adapted tissue clearing approaches9, allowing for the imaging of entire mouse hearts without the need for specific labeling and mechanical sectioning. We further propose that conventional LSM imaging can be enhanced through multiview deconvolution10 or axially swept light-sheet microscopy (ASLM) techniques11,12,13,14,15 to improve axial resolution. Additionally, the integration of image stitching with either of these methods can effectively overcome the trade-off between spatial resolution and field of view (FOV), thereby advancing the imaging of adult mouse hearts. The incorporation of numerous tissue clearing approaches, including hydrophobic, hydrophilic, and hydrogel-based methods, enables deeper light penetration for capturing the morphology of the entire heart16,17,18,19.
While multiple clearing methods are compatible with current LSM systems, the goal is to minimize photon scattering and enhance light penetration in tissues, like the heart, by replacing lipids with a medium that closely matches its refractive index. iDISCO was chosen as the representative20,21 and adapted for autofluorescence imaging in this protocol due to its rapid processing and high transparency (Figure 1A). Collectively, the integration of the advanced LSM approach with tissue clearing techniques offers a promising framework to unravel intricate cardiac anatomy in rodent hearts, holding significant potential for advancing our understanding of cardiac morphogenesis and pathogenesis.

저자 스포트라이트: 설치류 심장의 심근 구조를 조사하기 위한 맞춤형 광시트 이미징

설치류 심장의 심장 구조를 밝히기 위한 Light-Sheet Imaging

Our research aims at customizing a light-sheet imaging system to investigate the myocardial structures of the intact rodent heart at a cellular level. We have developed a workflow to explore the intact myocardium with isotropic resolution at the micron level from neonatal to adult rodent hearts. Our study fills a gap in investigating the intricate myocardial microstructures, such as trabeculation, inside the intact ventricles, with high spatial resolution.This imaging strategy provides an entry point to assess microstructure without physical slicing, leading to an enhanced understanding of cardiac development and remodeling. We will concentrate on exploring the structural changes of the myocardium in response to cardiac injury and remodeling.

Light-Sheet Microscopy를 위한 설치류 심장 샘플 준비

시작하려면 agross embedded 미러링 심장과 20% 메탄올 용액을 1시간 동안 배치합니다. 심장을 탈수시키기 위해 메탄올 농도를 증가시키는 곳으로 순차적으로 전달합니다. 탈수 중에 심장과 함께 용액을 60Hz의 믹서에 놓고 탈수된 심장을 신선한 100% 메탄올 용액으로 옮깁니다.용액을 신선한 100% 메탄올로 교체한 후 심장을 섭씨 4도에서 10분 동안 둡니다. 이제 용액을 흡인합니다. 그런 다음 메탄올에 200ml의 5% 과산화수소를 첨가하여 심장을 표백합니다.섭씨 4도에서 밤새 용액에 심장을 배양합니다. 다음날 배양 후 샘플을 100% 메탄올로 옮기고 66% 디클로로메탄 및 33% 메탄올을 포함하는 용액으로 옮깁니다. 심장은 바이알 바닥으로 가라앉아야 합니다.심장이 완전히 황폐화되도록 샘플을 믹서에 3시간 동안 올려 놓습니다. 마지막으로, 굴절률 일치를 위해 샘플을 디벤질에테르에 배양합니다. P1 쥐의 심장 조직은 4일 만에 투명하게 렌더링되었고, 8주 된 마우스는 7일 만에 투명하게 렌더링되었습니다.

preparation-of-rodent-heart-sample-for-light-sheet-microscopy

Research

JoVE Journal

Bioengineering

Preparation of Rodent Heart Sample for Light-Sheet Microscopy

57 조회수.  The University of Texas at Dallas. 시작하려면 agross embedded 미러링 심장과 20% 메탄올 용액을 1시간 동안 배치합니다. 심장을 탈수시키기 위해 메탄올 농도를 증가시키는 곳으로 순차적으로 전달합니다. 탈수 중에 심장과 함께 용액을 60Hz의 믹서에 놓고 탈수된 심장을 신선한 100% 메탄올 용액으로 옮깁니다.용액을 신선한 100% 메탄올로 교체한 후 심장을 섭씨 4도에서 10분 동안 둡니다. 이제 용액을 흡인합니...

비디오: Light-Sheet Microscopy를 위한 설치류 심장 샘플 준비

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...