Institute for Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada.
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada.
Rev Sci Instrum. 2022 Nov 1;93(11):113707. doi: 10.1063/5.0111787.
Supercritical angle fluorescence (SAF) microscopy is a novel imaging tool based on the use of distance-dependent fluorophore emission patterns to provide accurate locations of fluorophores relative to a surface. This technique has been extensively used to construct accurate cellular images and to detect surface phenomena in a static environment. However, the capability of SAF microscopy in monitoring dynamic surface phenomena and changes in millisecond intervals is underexplored in the literature. Here, we report on a hardware add-on for a conventional inverted microscope coupled with a post-processing Python module that extends the capability of SAF microscopy to monitor dynamic surface adsorption in sub-second intervals, thereby greatly expanding the potential of this tool to study surface interactions, such as surface fouling and competitive surface adhesion. The Python module enables researchers to automatically extract SAF profiles from each image. We first assessed the performance of the system by probing the specific binding of biotin-fluorescein conjugates to a neutravidin-coated cover glass in the presence of non-binding fluorescein. The SAF emission was observed to increase with the quantity of bound fluorophore on the cover glass. However, a high concentration of unbound fluorophore also contributed to overall SAF emission, leading to over-estimation in surface-bound fluorescence. To expand the applications of SAF in monitoring surface phenomena, we monitored the non-specific surface adsorption of BSA and non-ionic surfactants on a Teflon-AF surface. Solution mixtures of bovine serum albumin (BSA) and nine Pluronic/Tetronic surfactants were exposed to a Teflon-AF surface. No significant BSA adsorption was observed in all BSA-surfactant solution mixtures with negligible SAF intensity. Finally, we monitored the adsorption dynamics of BSA onto the Teflon-AF surface and observed rapid BSA adsorption on Teflon-AF surface within 10 s of addition. The adsorption rate constant (k) and half-life of BSA adsorption on Teflon-AF were determined to be 0.419 ± 0.004 s and 1.65 ± 0.016 s, respectively, using a pseudo-first-order adsorption equation.
超临界角荧光（SAF）显微镜是一种基于使用距离依赖的荧光团发射模式的新型成像工具，可提供荧光团相对于表面的准确位置。该技术已被广泛用于构建准确的细胞图像，并在静态环境中检测表面现象。然而，SAF 显微镜在监测纳秒间隔内的动态表面现象和变化方面的能力在文献中尚未得到充分探索。在这里，我们报告了一种用于传统倒置显微镜的硬件附加组件，该组件与一个后处理 Python 模块相结合，该模块扩展了 SAF 显微镜监测亚秒间隔内动态表面吸附的能力，从而极大地扩展了该工具研究表面相互作用（如表面污垢和竞争表面粘附）的潜力。Python 模块使研究人员能够自动从每个图像中提取 SAF 轮廓。我们首先通过探测生物素-荧光素缀合物在存在非结合荧光素的情况下与中性亲和素涂层盖玻片的特异性结合来评估系统的性能。观察到 SAF 发射随着盖玻片上结合的荧光团数量的增加而增加。然而，高浓度的未结合荧光团也会导致整体 SAF 发射增加，从而导致表面结合荧光的高估。为了扩展 SAF 在监测表面现象中的应用，我们监测了 BSA 和非离子表面活性剂在 Teflon-AF 表面上的非特异性表面吸附。将牛血清白蛋白（BSA）和九种 Pluronic/Tetronic 表面活性剂的溶液混合物暴露于 Teflon-AF 表面。在所有 BSA-表面活性剂溶液混合物中均未观察到明显的 BSA 吸附，SAF 强度可忽略不计。最后，我们监测了 BSA 在 Teflon-AF 表面上的吸附动力学，并在添加后 10 秒内观察到 BSA 在 Teflon-AF 表面上的快速吸附。使用拟一级吸附方程，确定 BSA 在 Teflon-AF 上的吸附速率常数（k）和半衰期分别为 0.419±0.004 s 和 1.65±0.016 s。
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