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

Microscopia de folha de luz e limpeza de tecido para imagens cardíacas abrangentes

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.

Autor em destaque: Imagem de folha de luz personalizada para investigar estruturas miocárdicas em corações de roedores

Imagem de folha de luz para revelar estrutura cardíaca em corações de roedores

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.

Configuração de um hardware de sistema de folha de luz varrida axialmente para imagens de amostras de coração de roedores

Para começar, instale uma lente de plataforma giratória elétrica, ou ETL, no plano de Fourier de uma lente cilíndrica. Abra a caixa do driver ETL e remova a tampa. Conecte o driver ETL à porta USB da estação de trabalho.Em seguida, conecte o driver ao ETL usando um cabo Hirose. Em seguida, instale o software do controlador do driver da lente. Altere o modo de operação no software para analógico para controlar o ETL com um gatilho gerado a partir da placa DAC.Mude o Modo de captura para Externo no software da câmera sCMOS. Para sincronizar o sistema, defina o tempo de exposição com base na relação sinal/fundo aceitável. Gere triggers quadrados e triangulares para sincronização no programa LabVIEW, Trigger Generator.vi.Digitalize o feixe de laser ao longo do eixo x e os pixels ativos ao longo do eixo y para gerar uma imagem 2D. As contas mais brilhantes e nítidas ao longo da diagonal na imagem indicam a sincronização precisa. Prossiga para a imagem do coração.

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

37 visualizações.  The University of Texas at Dallas. Para começar, instale uma lente de plataforma giratória elétrica, ou ETL, no plano de Fourier de uma lente cilíndrica. Abra a caixa do driver ETL e remova a tampa. Conecte o driver ETL à porta USB da estação de trabalho.Em seguida, conecte o driver ao ETL usando um cabo Hirose. Em seguida, instale o software do controlador do driver da lente. Altere o modo de operação no software para analógico para controlar o ETL com um gatilho gerado a partir da placa DAC.Mude o Mod...

Vídeo: Configuração de um hardware de sistema de folha de luz varrida axialmente para imagens de amostras de coração de roedores

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 ...