Rajyaguru J M, Kado M, Richardson M C, Muszynski M J
Department of Pediatric Infectious Diseases Research, Arnold Palmer Hospital for Children and Women, Orlando, Florida 32806, USA.
Biophys J. 1997 Apr;72(4):1521-6. doi: 10.1016/S0006-3495(97)78800-3.
High-resolution x-ray microscopy is a relatively new technique and is performed mostly at a few large synchrotron x-ray sources that use exposure times of seconds. We utilized a bench-top source of single-shot laser (ns) plasma to generate x-rays similar to synchrotron facilities. A 5 microlitres suspension of Escherichia coli ATCC 25922 in 0.9% phosphate buffered saline was placed on polymethylmethyacrylate coated photoresist, covered with a thin (100 nm) SiN window and positioned in a vacuum chamber close to the x-ray source. The emission spectrum was tuned for optimal absorption by carbon-rich material. Atomic force microscope scans provided a surface and topographical image of differential x-ray absorption corresponding to specimen properties. By using this technique we observed a distinct layer around whole cells, possibly representing the Gram-negative envelope, darker stained areas inside the cell corresponding to chromosomal DNA as seen by thin section electron microscopy, and dent(s) midway through one cell, and 1/3- and 2/3-lengths in another cell, possibly representing one or more division septa. This quick and high resolution with depth-of-field microscopy technique is unmatched to image live hydrated ultrastructure, and has much potential for application in the study of fragile biological specimens.
高分辨率X射线显微镜是一种相对较新的技术，主要在少数几个使用数秒曝光时间的大型同步加速器X射线源上进行。我们利用台式单次激光（纳秒）等离子体源来产生与同步加速器设施类似的X射线。将5微升大肠杆菌ATCC 25922悬浮于0.9%磷酸盐缓冲盐水中，置于聚甲基丙烯酸甲酯涂层的光刻胶上，覆盖一层薄（100纳米）的氮化硅窗口，并放置在靠近X射线源的真空室内。发射光谱经过调整，以实现富含碳材料的最佳吸收。原子力显微镜扫描提供了对应于标本特性的差分X射线吸收的表面和形貌图像。通过使用该技术，我们观察到围绕整个细胞的一层明显的层，可能代表革兰氏阴性菌包膜，细胞内染色较深的区域对应于薄切片电子显微镜下所见的染色体DNA，以及一个细胞中途的凹痕，另一个细胞中1/3和2/3长度处的凹痕，可能代表一个或多个分裂隔膜。这种具有景深的快速高分辨率显微镜技术在对活的水合超微结构成像方面无与伦比，在脆弱生物标本的研究中具有很大的应用潜力。
