Heinze Katrin G, Costantino Santiago, De Koninck Paul, Wiseman Paul W
Department of Physics and Department of Chemistry, McGill University, Montreal, Quebec, Canada.
J Phys Chem B. 2009 Apr 16;113(15):5225-33. doi: 10.1021/jp8060152.
Confocal and two-photon fluorescence microscopy techniques using genetically encoded fluorescent probes are widely used in cell biology. Beyond the common problems of photobleaching and phototoxicity, we present evidence that photounbinding also has the potential to compromise such methods, especially in quantitative studies. We show that laser intensities within excitation regimes typical for imaging approaches such as as fluorescence recovery after photobleaching (FRAP), photolysis, or fluorescence correlation spectroscopy (FCS) experiments can cause the dissociation of antibodies from their ligands. Indeed, both one- and two-photon excitation of a fluorescent anti-GFP antibody caused its dissociation from immobilized GFP in vitro. Importantly, with two-photon excitation, the laser intensity threshold for photobleaching was the same as for photounbinding. By contrast, with single-photon excitation, we found a range of laser intensities where photobleaching can be separated from photounbinding. This photounbinding effect was visualized and measured by rebinding a second fluorescent anti-GFP (Green Fluorescent Protein) antibody, indicating that the GFP remained functional for reassociation following the photoinduced dissociation. Finally, we show that this unbinding effect occurs only when at least one binding partner carries a fluorescent label. Our results show that this photounbinding effect can readily remain masked or be misinterpreted as photobleaching, which can compromise the quantitative interpretation of binding studies made using fluorescence microscopy.
使用基因编码荧光探针的共聚焦和双光子荧光显微镜技术在细胞生物学中被广泛应用。除了光漂白和光毒性等常见问题外，我们还提供证据表明，光解离也有可能影响这些方法，特别是在定量研究中。我们表明，在诸如光漂白后荧光恢复（FRAP）、光解或荧光相关光谱（FCS）实验等成像方法的典型激发范围内的激光强度，可导致抗体与其配体解离。事实上，荧光抗GFP抗体的单光子和双光子激发均导致其在体外与固定化GFP解离。重要的是，对于双光子激发，光漂白的激光强度阈值与光解离的相同。相比之下，对于单光子激发，我们发现了一个激光强度范围，在该范围内光漂白可与光解离区分开来。通过重新结合第二种荧光抗GFP（绿色荧光蛋白）抗体来可视化和测量这种光解离效应，这表明在光诱导解离后，GFP仍具有重新结合的功能。最后，我们表明，只有当至少一个结合伴侣带有荧光标记时，才会发生这种解离效应。我们的结果表明，这种光解离效应很容易被掩盖或被误解为光漂白，这可能会影响使用荧光显微镜进行的结合研究的定量解释。
