Kong Cihang, Bobe Stefanie, Pilger Christian, Lachetta Mario, Øie Cristina Ionica, Kirschnick Nils, Mönkemöller Viola, Hübner Wolfgang, Förster Christine, Schüttpelz Mark, Kiefer Friedemann, Huser Thomas, Schulte Am Esch Jan
Department of Physics, Bielefeld University, Bielefeld, Germany.
European Institute for Molecular Imaging, University of Münster, Münster, Germany.
Front Physiol. 2021 Feb 17;12:637136. doi: 10.3389/fphys.2021.637136. eCollection 2021.
The liver as the largest organ in the human body is composed of a complex macroscopic and microscopic architecture that supports its indispensable function to maintain physiological homeostasis. Optical imaging of the human liver is particularly challenging because of the need to cover length scales across 7 orders of magnitude (from the centimeter scale to the nanometer scale) in order to fully assess the ultrastructure of the entire organ down to the subcellular scale and probe its physiological function. This task becomes even more challenging the deeper within the organ one hopes to image, because of the strong absorption and scattering of visible light by the liver. Here, we demonstrate how optical imaging methods utilizing highly specific fluorescent labels, as well as label-free optical methods can seamlessly cover this entire size range in excised, fixed human liver tissue and we exemplify this by reconstructing the biliary tree in three-dimensional space. Imaging of tissue beyond approximately 0.5 mm length requires optical clearing of the human liver. We present the successful use of optical projection tomography and light-sheet fluorescence microscopy to derive information about the liver architecture on the millimeter scale. The intermediate size range is covered using label-free structural and chemically sensitive methods, such as second harmonic generation and coherent anti-Stokes Raman scattering microscopy. Laser-scanning confocal microscopy extends the resolution to the nanoscale, allowing us to ultimately image individual liver sinusoidal endothelial cells and their fenestrations by super-resolution structured illumination microscopy. This allowed us to visualize the human hepatobiliary system in 3D down to the cellular level, which indicates that reticular biliary networks communicate with portal bile ducts via single or a few ductuli. Non-linear optical microscopy enabled us to identify fibrotic regions extending from the portal field to the parenchyma, along with microvesicular steatosis in liver biopsies from an older patient. Lastly, super-resolution microscopy allowed us to visualize and determine the size distribution of fenestrations in human liver sinusoidal endothelial cells for the first time under aqueous conditions. Thus, this proof-of-concept study allows us to demonstrate, how, in combination, these techniques open up a new chapter in liver biopsy analysis.
肝脏作为人体最大的器官，由复杂的宏观和微观结构组成，这些结构支持其维持生理稳态的不可或缺的功能。对人体肝脏进行光学成像极具挑战性，因为需要覆盖跨越7个数量级的长度尺度（从厘米尺度到纳米尺度），以便全面评估整个器官直至亚细胞尺度的超微结构并探究其生理功能。由于肝脏对可见光的强烈吸收和散射，在器官内部越深的位置进行成像，这项任务就变得越具挑战性。在这里，我们展示了利用高度特异性荧光标记的光学成像方法以及无标记光学方法如何能够无缝覆盖切除并固定的人体肝脏组织中的整个尺寸范围，并且我们通过在三维空间中重建胆管树来举例说明这一点。对长度超过约0.5毫米的组织进行成像需要对人体肝脏进行光学透明处理。我们展示了成功使用光学投影断层扫描和光片荧光显微镜来获取关于毫米尺度肝脏结构的信息。中间尺寸范围使用无标记的结构和化学敏感方法进行覆盖，例如二次谐波产生和相干反斯托克斯拉曼散射显微镜。激光扫描共聚焦显微镜将分辨率扩展到纳米尺度，使我们最终能够通过超分辨率结构照明显微镜对单个肝窦内皮细胞及其窗孔进行成像。这使我们能够在三维空间中可视化人体肝胆系统直至细胞水平，这表明网状胆管网络通过单个或少数小胆管与门静脉胆管相通。非线性光学显微镜使我们能够识别从门静脉区域延伸至实质的纤维化区域，以及来自一位老年患者肝脏活检中的微泡性脂肪变性。最后，超分辨率显微镜首次使我们能够在水性条件下可视化并确定人体肝窦内皮细胞窗孔的大小分布。因此，这项概念验证研究使我们能够证明，这些技术相结合如何开启了肝脏活检分析的新篇章。
