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

Analyse af zebrafiskhjertesammentrækning med Virtual Reality-interaktion

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.

Forfatter Spotlight: Højopløselig 4D Light-Sheet Imaging og Virtual Reality i zebrafisk til enkeltcelleanalyse af hjertefunktion

4D lys-ark billeddannelse af zebrafisk hjertekontraktion

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.

Zebrafisk larve forberedelse, montering og lys-sheet billeddannelse til hjerteudviklingsundersøgelser

Til at begynde med skal du tænde stereomikroskopet for at registrere zebrafisklarvernes udvikling mellem nul og syv dage efter befrugtning. Forbered 0,8% lavsmeltende agarose med 150 milligram pr. Liter tricain. Når agarosen er afkølet til stuetemperatur, flyttes bedøvet zebrafisk til agaroseen ved hjælp af en overførselspipette.Brug en anden overførselspipette til at montere fisken med agarose i et fluoreret ethylenpropylenrør. Fastgør røret med den monterede zebrafisklarve til prøveprøvetrinnet med seks akser motoriseret lysarkmikroskop. Sænk røret ned i prøvekammeret fyldt med E3-vand.Drej zebrafisklarverne fra den ventrale side for bedre visualisering af hjertet. Tilslut det motoriserede prøvetrin til arbejdsstationen. Og konfigurer alle nødvendige parametre, herunder den ønskede bevægelsestilstand.Opret forbindelse mellem CMOS-kameraet og arbejdsstationen. Indstil trinstørrelsen til 1 mikrometer, det samlede antal billeder til 300 og eksponeringstiden til 5 millisekunder. Definer derefter det ønskede interesseområde.Tænd laseren og start lysmikroskopmikroskopbilleddannelse af larverne. Optag 300 billeder som en 2D-billedsekvens for at dække tre til fem hjertecyklusser af larverne. Optag en anden billedsekvens af den nye prøveskive, indtil hele hjertet er dækket.

Research

JoVE Journal

Bioengineering

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

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 ...

4D Imaging Reconstruction of Zebrafish Larva, 3D Cell Segmentation, and Cardiac Contractility Analysis in Virtual Reality

4D-billedrekonstruktion af zebrafisklarve, 3D-cellesegmentering og hjertekontraktilitetsanalyse i virtual reality