Wouters C H, Koerten H K, Bonnet J, Daems W T, Ploem J S
J Microsc. 1986 Jan;141(Pt 1):41-53. doi: 10.1111/j.1365-2818.1986.tb02699.x.
An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.
本文描述了一种将光度计单元与扫描电子显微镜（SEM）和光学显微镜（LM）相结合的仪器。扫描电子显微镜真空腔中的一个特殊载物台集成了用于透射和落射荧光光学显微镜的光学显微镜光学元件（物镜和聚光镜）。一个光学桥将这些光学元件连接到一台没有物镜和聚光镜的光学显微镜上。使用鸡红细胞、人淋巴细胞或小鼠肝细胞的标本测试了在这种组合显微镜（LM/SEM）中进行DNA定量测量的可能性。细胞经固定、置于盖玻片上、进行DNA定量染色、脱水并临界点干燥（CPD）。细胞固定后进行金镀膜。将标本放入组合显微镜的真空腔中，用扫描电子显微镜和光学显微镜对单个细胞进行研究。同时，通过连接到显微镜上的光度计单元进行DNA测量。本研究表明，与使用传统荧光显微镜对包埋细胞进行的DNA测量相比，组合显微镜中细胞的DNA测量结果相似。此外，研究表明，尽管覆盖LM/SEM标本的金层会减弱荧光信号，但它不会干扰DNA测量。
