Smallridge M W, Aktepe T E, Coppo M J C, Vaz P K, Diaz-Méndez A, Murray C M, Segal G, Devlin J M, Hartley C A
Faculty of Science, Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, The University of Melbourne, Melbourne, Victoria, Australia.
Facultad de Ciencias de la Vida, Escuela de Medicina Veterinaria, Universidad Andres Bello, Concepcion, Biobio, Chile.
PLoS One. 2025 Apr 1;20(4):e0320483. doi: 10.1371/journal.pone.0320483. eCollection 2025.
Ultramicroscopy has offered new avenues into the visualisation of tissues within animal models, providing three-dimensional visualisation through the use of light sheet fluorescence microscopy. This study aimed to develop and apply an optical tissue clearing method to investigate the application of light sheet fluorescence microscopy to image late-stage chicken embryos, and compare anatomical visualisation to traditional histological staining. Seventeen-day old specific pathogen free embryos were collected, fixed, and sectioned. Haematoxylin and eosin stained sections were prepared for histology, while light sheet imaging required the tissues to be optically clear. For this, an ethyl cinnamate-based method was utilised, allowing for acquisition of clear, unobstructed three-dimensional images of significant organ structures and systems using only autofluorescence. The use of established histological techniques provided anatomical mapping of structures between familiar histology images and the three-dimensional light sheet images. Rendering of organs using light sheet imaging provided contextual insights into the surrounding tissues and physiological architecture of major organ structures and systems. This was most apparent through the identification of the pulmonary vein and rendering of a volumetric projection of the vasculature branching within the lung and the subsequent merging of vasculature into the left side of the heart. Overall, the visualisation of the chicken embryo was enhanced by combining traditional histology with the information gained by three-dimensional light sheet fluorescence microscopy.
超微显微镜为动物模型中组织的可视化提供了新途径，通过使用光片荧光显微镜实现三维可视化。本研究旨在开发并应用一种光学组织透明化方法，以研究光片荧光显微镜在晚期鸡胚成像中的应用，并将解剖可视化与传统组织学染色进行比较。收集17日龄无特定病原体胚胎，进行固定和切片。制备苏木精和伊红染色切片用于组织学研究，而光片成像则要求组织具有光学透明性。为此，采用了一种基于肉桂酸乙酯的方法，仅利用自发荧光就能获取重要器官结构和系统清晰、无阻碍的三维图像。使用既定的组织学技术可在熟悉的组织学图像和三维光片图像之间提供结构的解剖图谱。利用光片成像对器官进行渲染，可深入了解主要器官结构和系统周围组织及生理结构的背景信息。这一点在识别肺静脉以及对肺内分支血管的体积投影进行渲染并随后将血管合并到心脏左侧时最为明显。总体而言，将传统组织学与三维光片荧光显微镜获得的信息相结合，增强了鸡胚的可视化效果。
