School of Chemistry and Photon Science Institute, The University of Manchester, Oxford Road, Manchester, M13 9PL, UK.
Phys Chem Chem Phys. 2018 Jun 27;20(25):16949-16955. doi: 10.1039/c8cp01699b.
LOV-domains are ubiquitous photosensory proteins that are commonly re-engineered to serve as powerful and versatile fluorescent proteins and optogenetic tools. The photoactive, flavin chromophore, however, is excited using short wavelengths of light in the blue and UV regions, which have limited penetration into biological samples and can cause photodamage. Here, we have used non-linear spectroscopy and microscopy of the fluorescent protein, iLOV, to reveal that functional variants of LOV can be activated to great effect by two non-resonant photons of lower energy, near infrared light, not only in solution but also in biological samples. The two photon cross section of iLOV has a significantly blue-shifted S0 → S1 transition compared with the one photon absorption spectrum, suggesting preferential population of excited vibronic states. It is highly likely, therefore, that the two photon absorption wavelength of engineered, LOV-based tools is tuneable. We also demonstrate for the first time two photon imaging using iLOV in human epithelial kidney cells. Consequently, two photon absorption by engineered, flavin-based bio-molecular tools can enable non-invasive activation with high depth resolution and the potential for not only improved image clarity but also enhanced spatiotemporal control for optogenetic applications.
LOV 结构域是普遍存在的光感蛋白，通常被重新设计为强大而多功能的荧光蛋白和光遗传学工具。然而，光活性的黄素发色团使用蓝区和 UV 区的短波长光激发，这些光在生物样本中的穿透深度有限，并且可能导致光损伤。在这里，我们使用非线性光谱学和荧光蛋白 iLOV 的显微镜观察，揭示了 LOV 的功能变体可以通过两个低能量的非共振光子（近红外光）非常有效地激活，不仅在溶液中，而且在生物样本中也是如此。与单光子吸收光谱相比，iLOV 的双光子截面具有明显蓝移的 S0→S1 跃迁，表明激发的振动态优先分布。因此，很可能可以调节工程化 LOV 基工具的双光子吸收波长。我们还首次展示了在人上皮肾细胞中使用 iLOV 进行双光子成像。因此，工程化的基于黄素的生物分子工具的双光子吸收可以实现非侵入性激活，具有高深度分辨率，不仅有可能提高图像清晰度，而且还有可能增强光遗传学应用的时空控制。
