Department of Medical Biosciences, Faculty of Natural Sciences, University of The Western Cape, Cape Town, South Africa.
Department of Physics and Astronomy, Electron Microscopy Unit, University of The Western Cape, Cape Town, South Africa.
PLoS One. 2022 Jul 8;17(7):e0266943. doi: 10.1371/journal.pone.0266943. eCollection 2022.
Scanning electron microscopy (SEM) provides a technical platform for nanoscopic mapping of biological structures. Correct preparation of SEM samples can provide an unprecedented understanding of the nexus between cellular morphology and topography. This comparative study critically examines two coating methods for preparing biological samples for scanning electron microscopy, while also providing novel advice on how to prepare in vitro epithelial or endothelial samples for high-resolution scanning-electron microscopy (HR-SEM). Two obstacles often confront the biologist when investigating cellular structures grown under tissue culture conditions, namely., how to prepare and present the biological samples to the HR-SEM microscope without affecting topographical membrane and cellular structural alterations. Firstly, our use of the Millicell cellulose inserts on which to grow our cellular samples in preparation for HR-SEM is both novel and advantageous to comparing the permeability function of cells to their morphological function. Secondly, biological material is often non-conducting, thermally sensitive and fragile and, therefore, needs to be fixed correctly and coated with thin conducting metal to ensure high-resolution detail of samples. Immortalized mouse brain endothelial cells (bEnd5) was used as a basis for describing the preferences in the use of the protocol. We compare two biological sample coating modalities for the visualizing and analysis of texturized, topographical, membranous ultrastructures of brain endothelial cell (BEC) confluent monolayers, namely, carbon and gold:palladium (Au:Pd) sputter coating in preparation for HR-SEM. BEC monolayers sputter-coated with these two modalities produced three-dimensional micrographs which have distinctly different topographical detail from which the nanostructural cellular data can be examined. The two coating methods display differences in the amount of nanoscopic detail that could be resolved in the nanosized membrane cytoarchitecture of BEC monolayers. The micrographical data clearly showed that Au:Pd sputter-coated samples generate descript imagery, providing useful information for profiling membrane nanostructures compared to carbon-coated samples. The recommendations regarding the contrast in two modalities would provide the necessary guidance to biological microscopists in preparing tissue culture samples for HR-SEM.
扫描电子显微镜（SEM）为生物结构的纳米级测绘提供了技术平台。正确的 SEM 样品制备可以提供对细胞形态和形貌之间关系的前所未有的理解。本研究对比分析了两种用于准备 SEM 样品的涂层方法，同时还提供了有关如何为高分辨率扫描电子显微镜（HR-SEM）准备体外上皮或内皮样品的新建议。当研究在组织培养条件下生长的细胞结构时，生物学家通常会遇到两个障碍，即如何准备和呈现生物样品而不会影响拓扑膜和细胞结构的改变。首先，我们在准备 HR-SEM 时使用 Millicell 纤维素插入物来培养细胞样品，这既新颖又有利于比较细胞对其形态功能的通透性功能。其次，生物材料通常是非导电的、热敏的和脆弱的，因此需要正确固定并用薄的导电金属涂层来确保样品的高分辨率细节。永生小鼠脑内皮细胞（bEnd5）被用作描述该方案使用偏好的基础。我们比较了两种生物样品涂层方法，用于可视化和分析脑内皮细胞（BEC）紧密单层的纹理化、拓扑和膜超微结构，即用于 HR-SEM 的碳和金钯（Au：Pd）溅射涂层。用这两种方法溅射涂层的 BEC 单层产生了具有明显不同拓扑细节的三维显微照片，从中可以检查纳米级细胞数据。这两种涂层方法在 BEC 单层的纳米级膜细胞结构中可以分辨的纳米级细节量上存在差异。显微数据清楚地表明，与碳涂层样品相比，Au：Pd 溅射涂层样品生成描述性图像，为分析膜纳米结构提供了有用信息。关于两种模式对比度的建议将为准备 HR-SEM 组织培养样品的生物显微镜提供必要的指导。
