Hough P V, McKinney W R, Ledbeter M C, Pollack R E, Moos H W
Proc Natl Acad Sci U S A. 1976 Feb;73(2):317-21. doi: 10.1073/pnas.73.2.317.
Proteins, nucleic acids, and fluorescein-conjugated antibody are shown to be identifidable in situ via the fluorescence excited by the focused electron beam of a canning electron microscope. A molecular species is identified by its characteristic fluorescence spectrum and by a characteristic alteration of the spectrum with time under the electron beam. Primary protein fluorescence is relatively rapidly destroyed by the beam, but protein photoproduct fluorescence is more rugged and will in some cases permit detection of small numbers of protein molecules. Nucleic acid fluorescence is extremely long-lived and will permit detection of small numbers of nucleic acid residues. The theoretical resolution limit for localization of a particular molecular species -- about 20 A--is determined by the known maximum distance for molecular excitation by fast electrons. Drect extapolation from an observed resolution of 900 A in the localization of nucleic acid using a low-efficiency detector leads to an experimental resolution limit of less than 60 A. Fluorescence is strongly quenched by residual water in the specimen. Similar quenching is produced by some macromolecular associations and so may serve to localize such associations.
通过扫描电子显微镜聚焦电子束激发的荧光，蛋白质、核酸和荧光素偶联抗体可在原位被识别。一种分子种类通过其特征荧光光谱以及在电子束下光谱随时间的特征变化来识别。初级蛋白质荧光相对较快地被电子束破坏，但蛋白质光产物荧光更稳定，在某些情况下能检测到少量蛋白质分子。核酸荧光寿命极长，能检测到少量核酸残基。特定分子种类定位的理论分辨率极限约为20埃，由快速电子激发分子的已知最大距离决定。使用低效探测器在核酸定位中观察到900埃的分辨率直接外推，得出实验分辨率极限小于60埃。荧光会被标本中的残留水强烈淬灭。一些大分子缔合也会产生类似的淬灭，因此可用于定位此类缔合。
