Deng Xiangquan, Zhuang Ziwei, Liu Hongji, Qiu Ping, Wang Ke
Opt Express. 2019 Apr 29;27(9):12723-12731. doi: 10.1364/OE.27.012723.
Fluorescent proteins are widely used to visualize structures and dynamics in various biological samples. Multiphoton microscopy is especially suitable for imaging structures expressing fluorescent proteins with subcellular resolution. 3-photon microscopy (3PM) excited at the 1700-nm window has proven to be promising for deep-tissue (such as brain) imaging expressing red fluorescent proteins. However, the 3-photon excitation and emission spectra of fluorescent proteins suitable at this window remain largely unknown, hampering protein selection and detection optimization. Here we demonstrate detailed measurement of 3-photon excitation and emission spectra for selected fluorescent proteins, suitable for 3-photon excitation at the 1700-nm window. The measured 3-photon excitation spectra will provide guidelines for protein and excitation wavelength selection. The measured 3-photon emission spectra and comparison with the 1-photon emission spectra, on one hand proves that the fundamental Kasha's rule is still valid for 3-photon fluorescence in these fluorescent proteins, on the other hand will be helpful for efficient fluorescence signal detection.
荧光蛋白被广泛用于观察各种生物样品中的结构和动态变化。多光子显微镜特别适合对表达荧光蛋白的结构进行亚细胞分辨率成像。在1700纳米窗口激发的三光子显微镜（3PM）已被证明在对表达红色荧光蛋白的深层组织（如大脑）成像方面很有前景。然而，适用于此窗口的荧光蛋白的三光子激发和发射光谱在很大程度上仍然未知，这阻碍了蛋白的选择和检测优化。在此，我们展示了对选定荧光蛋白的三光子激发和发射光谱的详细测量，这些荧光蛋白适用于在1700纳米窗口进行三光子激发。所测量的三光子激发光谱将为蛋白和激发波长的选择提供指导。所测量的三光子发射光谱以及与单光子发射光谱的比较，一方面证明了基本的卡沙规则在这些荧光蛋白的三光子荧光中仍然有效，另一方面将有助于高效的荧光信号检测。
