preparation of rodent heart sample for light-sheet microscopy

Kapsamlı kardiyak görüntüleme için ışık tabakası mikroskobu ve doku temizleme

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.

Yazar Spotlight: Kemirgen Kalplerinde Miyokard Yapılarını Araştırmak için Özelleştirilmiş Işık Sayfası Görüntüleme

Kemirgen kalplerinde kalp yapısını ortaya çıkarmak için ışıklı tabaka görüntüleme

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.

Işık Tabakası Mikroskobu için Kemirgen Kalp Numunesinin Hazırlanması

Başlamak için, brüt gömülü bir aynalama kalbi ve bir saat boyunca% 20 metanol çözeltisi yerleştirin. Kalbi susuz bırakmak için sırayla artan metanol konsantrasyonlarına aktarın. Dehidrasyon sırasında çözeltileri kalple birlikte 60 hertz'de bir karıştırıcıya yerleştirin, susuz kalmış kalbi taze% 100 metanol çözeltisine aktarın.Solüsyonu taze% 100 metanol ile değiştirdikten sonra, kalbi 10 dakika boyunca dört santigrat dereceye yerleştirin. Şimdi çözümü aspire edin. Daha sonra kalbi ağartmak için metanole 200 mililitre% 5 hidrojen peroksit ekleyin.Kalbi çözelti içinde gece boyunca dört santigrat derecede inkübe edin. Ertesi gün, inkübasyondan sonra numuneyi %100 metanole aktarın, %66 diklorometan ve %33 metanol içeren bir çözeltiye aktarın. Kalp, şişenin dibine batmalıdır.Kalbin tamamen harap olmasını sağlamak için numuneyi üç saat boyunca bir karıştırıcıya koyun. Son olarak, kırılma indisi eşleşmesi için numuneyi dibenzileter içinde inkübe edin. P1 fare kalbinin dokusu dört günde şeffaf hale getirilirken, sekiz haftalık bir fare yedi günde şeffaf hale getirildi.

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

Research

JoVE Journal

Bioengineering

Preparation of Rodent Heart Sample for Light-Sheet Microscopy

57 Görüntüleme.  The University of Texas at Dallas. Başlamak için, brüt gömülü bir aynalama kalbi ve bir saat boyunca% 20 metanol çözeltisi yerleştirin. Kalbi susuz bırakmak için sırayla artan metanol konsantrasyonlarına aktarın. Dehidrasyon sırasında çözeltileri kalple birlikte 60 hertz'de bir karıştırıcıya yerleştirin, susuz kalmış kalbi taze% 100 metanol çözeltisine aktarın.Solüsyonu taze% 100 metanol ile değiştirdikten sonra, kalbi 10 dakika boyunca dört santigrat dereceye yerleştirin. Şimdi çöz?...

Video: Işık Tabakası Mikroskobu için Kemirgen Kalp Numunesinin Hazırlanması

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 ...