Institute of Analytical and Bioanalytical Chemistry, University of Ulm, Germany.
Anal Bioanal Chem. 2011 Mar;399(7):2369-78. doi: 10.1007/s00216-010-4407-z. Epub 2010 Nov 30.
Scanning probe techniques enable direct imaging of morphology changes associated with cellular processes at life specimen. Here, glutaraldehyde-fixed and living alveolar type II (ATII) cells were investigated by atomic force microscopy (AFM), and the obtained topographical data were correlated with results obtained by scanning electron microscopy (SEM) and confocal microscopy (CM). We show that low-force contact mode AFM at glutaraldehyde-fixed cells provides complementary results to SEM and CM. Both AFM and SEM images reveal fine structures at the surface of fixed cells, which indicate microvilli protrusions. If ATII cells were treated with Ca(2+) channel modulators known to induce massive endocytosis, changes of the cell surface topography became evident by the depletion of microvilli. Low force contact mode AFM imaging at fixed ATII cells revealed a significant reduction of the surface roughness for capsazepine and 2-aminoethoxydiphenyl-borate (CPZ/2-APB)-treated cells compared to untreated control cells (Rc of 99.7 ± 6.8 nm vs. Rc of 71.9 ± 4.6 nm for N = 22), which was confirmed via SEM studies. CM of microvilli marker protein Ezrin revealed a cytoplasmic localization of Ezrin in CPZ/2-APB-treated cells, whereas a submembranous Ezrin localization was observed in control cells. Furthermore, in situ AFM investigations at living ATII cells using low force contact mode imaging revealed an apparent decrease in cell height of 17% during stimulation experiments. We conclude that a dynamic reorganization of the microvillous cell surface occurs in ATII cells at conditions of stimulated endocytosis.
扫描探针技术能够直接观察到与生命标本中细胞过程相关的形态变化。在这里，通过原子力显微镜（AFM）研究了戊二醛固定和活的肺泡 II 型（ATII）细胞，并且将获得的形貌数据与扫描电子显微镜（SEM）和共聚焦显微镜（CM）的结果相关联。我们表明，在戊二醛固定细胞中采用低力接触模式 AFM 可提供与 SEM 和 CM 互补的结果。AFM 和 SEM 图像均揭示了固定细胞表面的精细结构，表明微绒毛突起。如果用已知诱导巨胞饮的 Ca(2+)通道调节剂处理 ATII 细胞，则细胞表面形貌的变化可通过微绒毛损耗而变得明显。与未处理的对照细胞相比，用辣椒素和 2-氨基乙氧基二苯硼酸盐（CPZ/2-APB）处理的固定 ATII 细胞的低力接触模式 AFM 成像显示表面粗糙度显著降低（Rc 为 99.7±6.8nm 对 N = 22，而 Rc 为 71.9±4.6nm），这通过 SEM 研究得到了证实。微绒毛标记蛋白 Ezrin 的 CM 显示 CPZ/2-APB 处理的细胞中 Ezrin 发生细胞质定位，而在对照细胞中观察到 Ezrin 的亚膜定位。此外，使用低力接触模式成像在活的 ATII 细胞中的原位 AFM 研究表明，在刺激实验过程中细胞高度明显降低了 17%。我们得出结论，在刺激胞饮的条件下，ATII 细胞中的微绒毛细胞表面发生动态重组。
