Department of Biomedical Engineering, University of California - Davis, Davis, CA, 95616, USA.
Department of Electrical Engineering, University of California - Santa Cruz, Santa Cruz, CA, 95064, USA.
Sci Rep. 2017 Mar 27;7(1):427. doi: 10.1038/s41598-017-00447-7.
Quantitative analysis of fluorescence signals from cells reacted with fluorescently labeled probes is a widely-used method for assessing cell biology. This method has become especially powerful for screening novel nanostructured materials for their influence on cell behavior. However, the effect of nanostructured surface on fluorescence intensity has largely been ignored, which likely leads to erroneous conclusions about cell behavior. This paper investigates this possibility by using fibroblasts cultured on nanoporous gold (np-Au) as a model nanostructured material system. We found that fibroblasts stained for f-actin using phalloidin conjugated with common fluorophores display different levels of fluorescence on np-Au, planar gold, and glass, suggesting different levels of f-actin composition. However, direct quantification via western blots indicates that the actin expression is the same across all conditions. We further investigated whether the fluorescence intensity depended on np-Au feature size, complementing the findings with reflection dark field measurements from different np-Au surfaces. Overall, our experimental measurements in agreement with our electrodynamic simulations suggest that nanostructured surfaces alter the fluorescence intensity of fluorophores by modulating both the excitation and light emission processes. We conclude that comparison of fluorescence on materials with different nanostructures should be done with a quantification method decoupled from the nanostructure's influence.
用荧光标记探针与细胞反应后的荧光信号进行定量分析是评估细胞生物学的一种广泛使用的方法。这种方法对于筛选新型纳米结构材料对细胞行为的影响变得尤为强大。然而，纳米结构表面对荧光强度的影响在很大程度上被忽视了，这可能导致对细胞行为的错误结论。本文通过使用在纳米多孔金(np-Au)上培养的成纤维细胞作为模型纳米结构材料系统来研究这种可能性。我们发现，使用与常见荧光团偶联的鬼笔环肽对肌动蛋白进行染色的成纤维细胞在 np-Au、平面金和玻璃上显示出不同水平的荧光，表明肌动蛋白组成水平不同。然而，通过 Western blot 进行的直接定量分析表明，所有条件下的肌动蛋白表达都是相同的。我们进一步研究了荧光强度是否取决于 np-Au 的特征尺寸，通过来自不同 np-Au 表面的反射暗场测量补充了这些发现。总的来说，我们的实验测量结果与我们的电动力学模拟一致，表明纳米结构表面通过调节激发和光发射过程来改变荧光团的荧光强度。我们得出结论，应该使用与纳米结构影响解耦的定量方法来比较具有不同纳米结构的材料上的荧光。
