preparation of rodent heart sample for light-sheet microscopy

Light-sheet microscopie en weefselopruiming voor uitgebreide cardiale beeldvorming

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.

Auteur in de schijnwerpers: Aangepaste lichtbladbeeldvorming voor het onderzoeken van myocardiale structuren in knaagdierharten

Light-sheet beeldvorming om de hartstructuur in knaagdierharten te onthullen

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.

Voorbereiding van een knaagdierhartmonster voor light-sheet-microscopie

Plaats om te beginnen een agross ingebed spiegelend hart en een oplossing van 20% methanol gedurende een uur. Breng het hart achtereenvolgens over naar toenemende concentraties methanol om het uit te drogen. Plaats de oplossingen met het hart op een mixer op 60 hertz tijdens uitdroging, breng het gedehydrateerde hart over in een verse 100%methanol oplossing.Nadat u de oplossing hebt vervangen door verse 100% methanol, plaatst u het hart gedurende 10 minuten op vier graden Celsius. Zuig nu de oplossing op. Voeg vervolgens 200 milliliter 5% waterstofperoxide in methanol toe om het hart te bleken.Incubeer het hart een nacht in de oplossing bij vier graden Celsius. Breng het monster de volgende dag na incubatie over in 100% methanol en breng het over in een oplossing met 66% dichloormethaan en 33% methanol. Het hart moet naar de bodem van de injectieflacon zinken.Plaats het monster gedurende drie uur op een mixer om volledige verval van het hart te garanderen. Incubeer ten slotte het monster in dibenzylether voor het matchen van de brekingsindex. Het weefsel van het P1-muizenhart werd in vier dagen transparant gemaakt, terwijl een acht weken oude muis in zeven dagen transparant werd gemaakt.

Research

JoVE Journal

Bioengineering

Preparation of Rodent Heart Sample for Light-Sheet Microscopy

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 ...

Light-Sheet Imaging of Rodent Hearts

Light-sheet beeldvorming van knaagdierharten

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

Het opzetten van een axiaal geveegd light-sheet-systeem Hardware voor het afbeelden van hartmonsters van knaagdieren