Nanoscale Biophotonics Laboratory, School of Chemistry , National University of Ireland , University Road , Galway H91 CF50 , Ireland.
Langmuir. 2018 Sep 18;34(37):10913-10923. doi: 10.1021/acs.langmuir.8b02509. Epub 2018 Sep 10.
Total internal reflection fluorescence microscopy (TIRFM) is an important method in surface science and for the analysis of surface-bound macromolecules. Here, we developed and explored the use of a novel fluorescein isothiocyanate isomer I (FITC)-adsorbed monolayer for alignment and validation of TIRFM measurements and configurations. Aqueous solutions of FITC exist as several different protolytic forms (dianionic, anionic, neutral, and cationic) with each form having different emission characteristics. However, the emission behavior of FITC adsorbed on hydrophilic, hydrophobic, and unmodified glass surfaces at different pH was unknown. TIRFM imaging and spectroscopy were used to study FITC and FITC-labeled bovine serum albumin (BSA-FITC) monolayers generated on three different glass surfaces. Monolayer emission intensity, spectra, and the photobleaching profiles were all dependent on pH and the surface properties of the glass. Very strangely, however, at pH 5.0 on hydrophobic surfaces, the FITC monolayers produced were both bright and apparently unbleachable over ∼20 min of imaging (60 s total exposure). During monolayer formation at pH 5.0, we saw clear evidence for concentration-based quenching, indicating high surface coverage. When the monolayer had been rinsed with buffer to remove unbound FITC, we observed an increase in emission intensity during illumination indicative of some form of photoactivated species being present. Eventually, the fluorescence emission stabilized and remained constant for extended periods of time with no evidence of photobleaching. We hypothesize that during the adsorption process (a hydrophobic-hydrophobic interaction) there was conversion to the fluorescent quinoid form of FITC. In contrast, at pH 7.4 and 9.6 on hydrophobic surfaces, FITC monolayers had well-defined, fast photobleaching kinetics (decay to ∼50% intensity in 5-10 s). The equivalent BSA-FITC monolayers were slightly brighter, with similar photobleaching kinetics. While the precise mechanism for this unusual behavior is still unknown, all these low-cost monolayers were easily prepared, were reproducible, and can serve as convenient test samples for TIRFM alignment, calibration, and validation prior to undertaking measurements with more sensitive biogenic or biological specimens.
全内反射荧光显微镜（TIRFM）是表面科学和表面结合大分子分析的重要方法。在这里，我们开发并探索了使用新型异硫氰酸荧光素（FITC）吸附单层来对齐和验证 TIRFM 测量和配置。FITC 在水溶液中存在几种不同的质子化形式（二阴离子、阴离子、中性和阳离子），每种形式都具有不同的发射特性。然而，在不同 pH 值下，FITC 吸附在亲水、疏水和未修饰玻璃表面上的发射行为是未知的。使用 TIRFM 成像和光谱学研究了在三种不同玻璃表面上生成的 FITC 和 FITC 标记牛血清白蛋白（BSA-FITC）单层。单层发射强度、光谱和光漂白曲线都依赖于 pH 值和玻璃的表面性质。然而，非常奇怪的是，在 pH 5.0 下，在疏水表面上，FITC 单层既明亮又明显在成像（60 秒总曝光）的 20 分钟内不可漂白。在 pH 5.0 下进行单层形成时，我们看到了基于浓度的淬灭的明显证据，表明表面覆盖率很高。当用缓冲液冲洗单层以去除未结合的 FITC 时，我们观察到在照射期间发射强度增加，表明存在某种形式的光激活物质。最终，荧光发射稳定并保持长时间不变，没有光漂白的迹象。我们假设，在吸附过程中（疏水性-疏水性相互作用），FITC 转化为荧光醌型。相比之下，在 pH 7.4 和 9.6 下，疏水表面上的 FITC 单层具有明确定义的快速光漂白动力学（在 5-10 秒内衰减至约 50%强度）。等效的 BSA-FITC 单层稍亮，具有相似的光漂白动力学。虽然这种异常行为的确切机制仍不清楚，但所有这些低成本的单层都很容易制备，具有可重复性，并且可以在使用更敏感的生物或生物样本进行测量之前，作为 TIRFM 对齐、校准和验证的方便测试样本。
