Tamiya E, Iwabuchi S, Nagatani N, Murakami Y, Sakaguchi T, Yokoyama K, Chiba N, Muramatsu H
School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan.
Anal Chem. 1997 Sep 15;69(18):3697-701. doi: 10.1021/ac970060w.
A scanning near-field optical/atomic force microscope (SNOAM) system was applied for simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic force microscopy cantilever and as a scanning near-field optical microscopy probe. Optical resolution of this system was about 50-100 nm in fluorescence mode for fluorescent latex beads on a quartz glass plate. Green fluorescent protein (GFP) is a convenient indicator of transformation and should allow cells to be separated by fluorescence-activated cell sorting. The gene coding to GFP was cloned in recombinant Escherichia coli. The SNOAM system used 458- or 488-nm irradiation from a multiline Ar ion laser for excitation of GFP, since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluorescence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. A nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, it can be seen that individual E. coli cells expressed different fluorescence intensities. Fluorescence obtained by SNOAM indicated that GFP oxidation possibly occurred near the cell surface. A SNOAM system also indicated the possibility of precise imaging of native cells in liquid.
一种扫描近场光学/原子力显微镜（SNOAM）系统被用于在空气和液体环境中对生物样品同时进行形貌和荧光成像。SNOAM使用一根弯曲的光纤同时作为动态模式原子力显微镜的悬臂以及扫描近场光学显微镜的探针。对于石英玻璃板上的荧光乳胶珠，该系统在荧光模式下的光学分辨率约为50 - 100纳米。绿色荧光蛋白（GFP）是一种方便的转化指示物，并且应该能够通过荧光激活细胞分选来分离细胞。编码GFP的基因被克隆到重组大肠杆菌中。SNOAM系统使用来自多线氩离子激光器的458纳米或488纳米辐射来激发GFP，因为已知天然GFP在395纳米处有一个最大值并且在500纳米之前有一个宽吸收光谱。重组大肠杆菌的形貌和荧光图像以高空间分辨率同时获得，这明显优于传统的共聚焦显微镜。通过将光纤探针定位在大肠杆菌细胞的明亮区域上方获得了纳米级的GFP荧光光谱。比较形貌和荧光图像，可以看出单个大肠杆菌细胞表达不同的荧光强度。SNOAM获得的荧光表明GFP氧化可能发生在细胞表面附近。一个SNOAM系统还表明了对液体中的天然细胞进行精确成像的可能性。
