荧光光谱Fluorescence spectroscopy

由于激发态先振动弛豫损失部分能量，荧光波长比激发光长（斯托克斯位移）。发射光谱形状与激发波长无关（卡沙规则），且常与吸收光谱呈镜像关系。荧光量子产率 Φ = 发射光子数/吸收光子数，荧光素约 0.95。能产生强荧光的分子一般具有刚性平面共轭结构（如荧光素、罗丹明、芘），给电子取代基增强荧光，重原子（Br、I）和硝基使荧光减弱。荧光可被淬灭剂（O₂、卤离子、重金属）减弱，斯特恩-沃尔默方程 F₀/F = 1 + K_SV[Q] 描述动态淬灭。

Because the excited state loses some energy by vibrational relaxation first, fluorescence lies at longer wavelength than the excitation (the Stokes shift). The emission spectrum's shape is independent of excitation wavelength (Kasha's rule) and often mirrors the absorption spectrum. Quantum yield Φ = photons emitted/photons absorbed, about 0.95 for fluorescein. Strong emitters usually have rigid, planar conjugated structures (fluorescein, rhodamines, pyrene); donor substituents enhance fluorescence while heavy atoms (Br, I) and nitro groups weaken it. Quenchers such as O₂, halide ions and heavy metals reduce it, with dynamic quenching described by the Stern–Volmer equation F₀/F = 1 + K_SV[Q].

1852 年英国物理学家乔治·斯托克斯研究奎宁溶液在紫外光下发出蓝光的现象，发现发射光波长比入射光长，并以萤石（fluorite）命名为 fluorescence。1871 年拜尔合成荧光素。1930 年代雅布隆斯基建立能级图。1962 年下村修发现绿色荧光蛋白（GFP），钱永健等发展了多色荧光蛋白，获 2008 年诺贝尔化学奖；2014 年诺贝尔化学奖授予超分辨荧光显微技术（白兹格、赫尔、莫纳）。

In 1852 the British physicist George Stokes studied the blue glow of quinine solution under ultraviolet light, found the emitted light longer in wavelength than the incident light and named it fluorescence after fluorite. Baeyer synthesised fluorescein in 1871, and Jabłoński drew his diagram in the 1930s. Shimomura discovered green fluorescent protein in 1962, and with Chalfie and Roger Tsien, who developed multicoloured fluorescent proteins, shared the 2008 Nobel Prize in Chemistry; the 2014 prize went to super-resolution fluorescence microscopy (Betzig, Hell, Moerner).

荧光法用于痕量分析（维生素 B₂、奎宁、多环芳烃、黄曲霉毒素）、荧光探针检测金属离子和 pH、免疫荧光和流式细胞术、实时荧光定量 PCR、DNA 测序（荧光标记）、荧光共振能量转移（FRET）测量分子间距离、活细胞成像和超分辨显微成像、OLED 和荧光增白剂。

Fluorescence is used in trace analysis (vitamin B₂, quinine, polycyclic aromatics, aflatoxins), probes for metal ions and pH, immunofluorescence and flow cytometry, real-time quantitative PCR, fluorescently labelled DNA sequencing, Förster resonance energy transfer (FRET) to measure molecular distances, live-cell and super-resolution imaging, OLEDs and optical brighteners.