Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions
Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions
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DOI：
10.1016/j.saa.2021.120133
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发表时间：
2021-07-07
影响因子：
4.4
通讯作者：
Raicu, Valerica
中科院分区：
文献类型：
--
作者：
Adhikari, Dhruba P.;Biener, Gabriel;Raicu, Valerica
了解荧光蛋白（FP）的生物物理特性，如发射和吸收光谱，分子亮度，光稳定性，和光开关，是至关重要的标准，为他们的选择为标记的荧光为基础的生物应用的发展。虽然双光子激发成像技术已经稳步获得普及-由于相对较深的穿透深度，减少了焦点外的光漂白，以及发射光谱和双光子激发光谱之间的宽间隔-大多数研究报告的FP的光物理性质往往仍然集中在单光子激发。在这里，我们报告了我们的调查的几种常用的荧光蛋白质，使用双光子显微镜与光谱分辨率在激发和发射的光物理性质。我们的测量结果表明，不仅激发（有时发射）光谱的FP可能是显着不同的单光子和双光子激发，而且它们的相对亮度和它们的光稳定性。对FP在双光子激发下的光物理性质的良好理解对于为所需实验选择正确的标签是必不可少的。(c)2021爱思唯尔有限公司版权所有。
Understanding the photophysical properties of fluorescent proteins (FPs), such as emission and absorption spectra, molecular brightness, photostability, and photo-switching, is critical to the development of criteria for their selection as tags for fluorescent-based biological applications. While two-photon excitation imaging techniques have steadily gained popularity - due to comparatively deeper penetration depth, reduced out-of-focus photobleaching, and wide separation between emission spectra and two photon excitation spectra -, most studies reporting on the photophysical properties of FPs tend to remain focused on single-photon excitation. Here, we report our investigation of the photophysical properties of several commonly used fluorescent proteins using two-photon microscopy with spectral resolution in both excitation and emission. Our measurements indicate that not only the excitation (and sometimes emission) spectra of FPs may be markedly different between single-photon and two-photon excitation, but also their relative brightness and their photo-stability. A good understanding of the photophysical properties of FPs under two-photon excitation is essential for choosing the right tag(s) for a desired experiment. (c) 2021 Elsevier B.V. All rights reserved.