setting up an axially-swept light-sheet system hardware for imaging rodent heart samples

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.

설치류 심장 샘플을 이미징하기 위한 축방향 스윕 Light-Sheet 시스템 하드웨어 설정

시작하려면 원통형 렌즈의 푸리에 평면에 전기 턴테이블 렌즈(ETL)를 설치합니다. ETL 드라이버의 하우징을 열고 덮개를 제거합니다. ETL 드라이버를 워크스테이션의 USB 포트에 연결합니다.그런 다음 Hirose 케이블을 사용하여 드라이버를 ETL에 연결합니다. 그런 다음 렌즈 드라이버 컨트롤러 소프트웨어를 설치합니다. 소프트웨어의 작동 모드를 아날로그로 변경하여 DAC 카드에서 생성된 트리거로 ETL을 제어합니다.sCMOS 카메라의 소프트웨어에서 캡처 모드를 외부로 전환합니다. 시스템을 동기화하려면 허용 가능한 신호 대 배경 비율에 따라 노출 시간을 정의하십시오. LabVIEW 프로그램인 Trigger Generator.vi 에서 동기화를 위해 사각형 및 삼각형 트리거를 모두 생성합니다.x축을 따라 레이저 빔을 스캔하고 y축을 따라 활성 픽셀을 스캔하여 2D 이미지를 생성합니다. 이미지의 대각선을 따라 더 밝고 선명한 구슬은 정확한 동기화를 나타냅니다. 하트 이미지화로 진행합니다.

setting-up-an-axially-swept-light-sheet-system-hardware-for-imaging

Research

JoVE Journal

Bioengineering

Setting Up an Axially-Swept Light-Sheet System Hardware for Imaging Rodent Heart Samples

36 조회수.  The University of Texas at Dallas. 시작하려면 원통형 렌즈의 푸리에 평면에 전기 턴테이블 렌즈(ETL)를 설치합니다. ETL 드라이버의 하우징을 열고 덮개를 제거합니다. ETL 드라이버를 워크스테이션의 USB 포트에 연결합니다.그런 다음 Hirose 케이블을 사용하여 드라이버를 ETL에 연결합니다. 그런 다음 렌즈 드라이버 컨트롤러 소프트웨어를 설치합니다. 소프트웨어의 작동 모드를 아날로그로 변경하여 DAC 카?...

비디오: 설치류 심장 샘플을 이미징하기 위한 축방향 스윕 Light-Sheet 시스템 하드웨어 설정

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 ...