zebrafish larva preparation, mounting, and light-sheet imaging for cardiac development studies

가상 현실 상호 작용을 통한 제브라피시 심장 수축 분석

The zebrafish (Danio rerio) is a widely used model organism for studying cardiac development, physiology, and repair due to its optical transparency, genetic tractability, and regenerative capacity1,2,3,4. Upon myocardial infarction, while structural and functional changes impact the cardiac ejection and hemodynamics, technical limitations continue to hinder the ability to investigate the dynamic process during cardiac regeneration with the high spatiotemporal resolution. For example, conventional imaging methods, such as confocal microscopy, have limitations in terms of imaging depth, temporal resolution, or phototoxicity for capturing the dynamic changes and assessing cardiac contractile function during multiple cardiac cycles5.
Light-sheet microscopy represents a state-of-the-art imaging method that successfully addresses these issues by quickly sweeping the laser across the heart&#39;s ventricle and atrium, achieving detailed images with enhanced spatiotemporal resolution and negligible photo-bleaching and photo-toxic effects6,7,8,9,10,11.
This protocol introduces a comprehensive imaging strategy that includes LSM system construction, 4D image reconstruction, 3D cell tracking, and interactive analysis to capture and analyze the dynamics of cardiomyocytes across the entire heart during multiple cardiac cycles12. The customized imaging system and computational methodology allow&#160;one to track the myocardial microstructure and contractile function at the single-cell level in transgenic Tg(myl7:nucGFP) zebrafish larvae. Furthermore, small molecule compounds were delivered into the embryos using microinjection to assess drug-induced cardiac injury and subsequent regeneration. This holistic strategy provides an entry point to in vivo investigate structural, functional, and mechanical properties of myocardium at the single-cell level during cardiac development and regeneration.

저자 스포트라이트: 심장 기능의 단일 세포 분석을 위한 제브라피시의 고해상도 4D 광시트 이미징 및 가상 현실

제브라피시 심장 수축의 4D 광시트 이미징

Our research uses 4D light-sheet imaging to explore cardiac contractile function in zebrafish, aiming to understand the dynamics of cardiac contraction at the single-cell level. One of the foremost experimental challenges is achieving high spatial terminal resolution, dynamic imaging of cardiac function while observing natural heart rhythms. Our study fills the gap in analyzing intricate 4D cardiac dynamic at single-cell resolution.We utilize GPU-based parallel computation, virtual reality, and advance cell tracking to interpret light-sheet imaging data. Our novel virtual reality platform offers an interactive approach to assess regional cardiac contractions, providing manipulative functionalities for more in-depth analysis. By facilitating user-directed analysis of cardiac contraction, our protocol could lead to an enhanced understanding of cardiac development and disease.

심장 발달 연구를 위한 제브라피시 유충 준비, 장착 및 광시트 이미징

시작하려면 실체 현미경을 켜서 수정 후 0일에서 7일 사이의 제브라피시 유충 발달을 기록합니다. 0.8 % 저융점 아가로스를 리터 당 150 밀리그램으로 준비합니다. 아가로스가 실온으로 냉각되면 이송 피펫을 사용하여 마취된 제브라피시를 아가로스로 옮깁니다.다른 이송 피펫을 사용하여 플루오르화 에틸렌 프로필렌 튜브에 아가로스가 있는 물고기를 장착합니다. 제브라피시 유충이 장착된 튜브를 6축 전동 광시트 현미경 샘플 스테이지에 부착합니다. 튜브를 E3 물로 채워진 샘플 챔버에 담그십시오.제브라피시 유충을 복부 쪽에서 회전시켜 심장을 더 잘 시각화합니다. 전동식 샘플 스테이지를 워크스테이션에 연결합니다. 그리고 원하는 이동 모드를 포함하여 필요한 모든 매개변수를 구성합니다.CMOS 카메라와 워크스테이션을 연결합니다. 단계 크기를 1마이크로미터로, 총 프레임 수를 300으로, 노출 시간을 5밀리초로 설정합니다. 그런 다음 원하는 관심 영역을 정의합니다.레이저의 전원을 켜고 유충의 광시트 현미경 이미징을 시작합니다. 300개의 프레임을 2D 이미지 시퀀스로 기록하여 유충의 3-5회 심장 주기를 다룹니다. 심장 전체가 덮일 때까지 새 샘플 슬라이스의 다른 이미지 시퀀스를 기록합니다.

zebrafish-larva-preparation-mounting-light-sheet-imaging-for-cardiac

Research

JoVE Journal

Bioengineering

Zebrafish Larva Preparation, Mounting, and Light-Sheet Imaging for Cardiac Development Studies

100 조회수.  The University of Texas at Dallas. 시작하려면 실체 현미경을 켜서 수정 후 0일에서 7일 사이의 제브라피시 유충 발달을 기록합니다. 0.8 % 저융점 아가로스를 리터 당 150 밀리그램으로 준비합니다. 아가로스가 실온으로 냉각되면 이송 피펫을 사용하여 마취된 제브라피시를 아가로스로 옮깁니다.다른 이송 피펫을 사용하여 플루오르화 에틸렌 프로필렌 튜브에 아가로스가 있는 물고기를 장착합니다. 제브?...

비디오: 심장 발달 연구를 위한 제브라피시 유충 준비, 장착 및 광시트 이미징

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...