当电子从激发态进入基态时,会有振动能损失(这一现象的解释见“荧光的分子机理”)。因为波长与辐射能成反比(),能量损失使发射光谱较相应的激发光谱更长。这种现象被称为斯托克斯定律(Stokes’ Law)或斯托克斯位移,由George G. Stokes爵士在十九世纪中期发现。斯托克斯位移越大,就越容易分离激发光和发射光。为了获得最高的荧光强度,最好在荧光染料激发波长的峰值处进行激发,在发射波长的峰值处(或由观察者选择的其他波长下)进行检测。激发波长和发射波长的选择由合适的滤光片控制,从而可限制允许通过的波长。在确定整个光学系统的光谱效果时,要求进行额外的技术校正,需要考虑诸如玻璃的透射率和检测器的灵敏度对应不同波长的差异等因素。
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