Zhang Daibo, Lindsey Stephanie E
Mechanical and Aerospace Engineering, University of California San Diego.
Mechanical and Aerospace Engineering, University of California San Diego;
J Vis Exp. 2025 May 23(219). doi: 10.3791/68206.
In small animal models of cardiovascular development and diseases, subject-specific computational simulations of blood flow enable quantitative assessments of hemodynamic metrics that are difficult to measure experimentally. Computational fluid dynamic simulations shed light on the critical roles of mechanics in cardiovascular function and disease progression. Acquiring high-quality volumetric images of the vessels of interest is central to the accuracy and reproducibility of morphological measurement and flow quantitation results. This study proposes a rapid, cost-effective, and accessible method for whole-mount high-resolution imaging of small animal vasculature using light-sheet fluorescence microscopy. The modified iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) light-sheet sample preparation protocol involves (1) labeling vasculature with a fluorescent agent, (2) preserving the sample, and (3) rendering the sample transparent. Unlike classical iDISCO+, which uses immunohistochemical staining, the authors label vascular endothelium with FITC-tagged poly-L-lysine, an affordable non-specific fluorescent dye that is highly resistant to photo-bleaching, in a process termed "endo-painting." The rapid labeling reduces sample preparation time from approximately four weeks to less than 3 days. Furthermore, the use of minimally hazardous solvent ethyl cinnamate (ECi) as the clearing agent and imaging solution makes the samples safer to handle and compliant with a wider range of imaging facilities. The proposed protocol is applied to obtain highly resolved light-sheet fluorescence microscopy image stacks of the cardiovascular system in chick embryos ranging from day 3 (HH18) to day 8 (HH34). This study further demonstrates the suitability of this method for vascular quantitation through 3D reconstruction and computational hemodynamic modeling of a day 5 (HH 26) chick embryo.
在心血管发育和疾病的小动物模型中，针对特定个体的血流计算模拟能够对难以通过实验测量的血流动力学指标进行定量评估。计算流体动力学模拟揭示了力学在心血管功能和疾病进展中的关键作用。获取感兴趣血管的高质量容积图像对于形态测量和流量定量结果的准确性和可重复性至关重要。本研究提出了一种使用光片荧光显微镜对小动物脉管系统进行整体高分辨率成像的快速、经济高效且易于操作的方法。改良的iDISCO+（免疫标记的溶剂清除器官三维成像）光片样品制备方案包括：（1）用荧光剂标记脉管系统；（2）保存样品；（3）使样品透明。与使用免疫组织化学染色的经典iDISCO+不同，作者在一个称为“内皮染色”的过程中，用异硫氰酸荧光素标记的聚-L-赖氨酸（一种价格低廉、抗光漂白性强的非特异性荧光染料）标记血管内皮。这种快速标记将样品制备时间从大约四周减少到不到3天。此外，使用危害最小的溶剂肉桂酸乙酯（ECi）作为清除剂和成像溶液，使样品更易于操作，并且适用于更广泛的成像设备。所提出的方案被应用于获取第3天（HH18）至第8天（HH34）鸡胚心血管系统的高分辨率光片荧光显微镜图像堆栈。本研究进一步证明了该方法通过对第5天（HH26）鸡胚进行三维重建和计算血流动力学建模来进行血管定量的适用性。
