Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montréal, Québec H3A 2K6, Canada.
Biointerphases. 2010 Dec;5(4):139-48. doi: 10.1116/1.3523470.
Fluorescence microscopy methods including total internal reflection fluorescence and confocal laser scanning microscopy have played a major role in modern cell biology research by permitting imaging of fluorescently tagged macromolecules in living cells. These methods are often used to examine the initial events in signal transduction, which involve interactions occurring between membrane receptors and ligands such as antibodies and growth factors. Most quantitative biophysical applications using these fluorescence imaging methods, including ligand binding assays, are based on the assumption that the fluorophore label of interest has equal access to all areas of the membrane on the cell. Our findings suggest that there is limited accessibility of fluorophores (25±2%)(-) under the basal membrane of adherent CHO-K1 cells expressing epidermal growth factor receptor plated on a bare glass in standard two-dimensional tissue cultures. The authors present a detailed study of the extent to which a small fluorescent dye molecule (Alexa 647) is able to propagate under the basal membrane of cells plated on a variety of biologically compatible substrates: fibronectin, bovine serum albumin, poly-d-lysine, collagen I, collagen IV, Geltrex™, and fibronectin such as binding polymer. For nonspecific dye propagation the best overall accessibility was achieved using a thin layer preparation of a commercially available basement membrane matrix, Geltrex™ (67±8%). Coupling of a specific high affinity ligand (epidermal growth factor) to the dye did result in a moderate increase in propagation for most substrates examined. Despite the overall increase in propagation for most substrates (60%-80%), large areas under the central regions of the adherent cells still remained inaccessible to the fluorescently labeled ligand. More importantly, the presence of the specific ligand did not result in consistent increase in ligand propagation. Taken together these results suggest that the reduced accessibility is not exclusively due to steric effects, and the chemistry of both the ligand and the substrate may be important when working under conditions of reduced dimensionality.
荧光显微镜方法，包括全内反射荧光显微镜和共聚焦激光扫描显微镜，通过对活细胞中荧光标记的大分子进行成像，在现代细胞生物学研究中发挥了重要作用。这些方法常用于研究信号转导的初始事件，其中涉及到膜受体和配体（如抗体和生长因子）之间的相互作用。使用这些荧光成像方法进行的大多数定量生物物理应用，包括配体结合测定，都基于这样的假设，即感兴趣的荧光标记物能够平等地进入细胞膜的所有区域。我们的研究结果表明，在标准二维组织培养中，在表达表皮生长因子受体的贴壁 CHO-K1 细胞的基底膜下，荧光团（25±2%）（-）的可接近性有限。作者详细研究了在各种生物相容的基底上种植细胞时，一个小荧光染料分子（Alexa 647）能够在基底膜下传播的程度：纤连蛋白、牛血清白蛋白、聚-d-赖氨酸、I 型胶原、IV 型胶原、Geltrex™和纤连蛋白等结合聚合物。对于非特异性染料传播，使用商业可得的基底膜基质 Geltrex™的薄层制剂获得了最佳的总体可接近性（67±8%）。将特定的高亲和力配体（表皮生长因子）与染料偶联确实导致大多数检查的底物的传播适度增加。尽管大多数底物的传播总体上增加了（60%-80%），但粘附细胞的中央区域下的大部分区域仍然无法接近荧光标记的配体。更重要的是，特定配体的存在并没有导致配体传播的一致增加。总之，这些结果表明，可接近性的降低不仅是由于空间位阻效应，配体和底物的化学性质在降低维度的条件下工作时可能很重要。
