Computed Tomography using projection X-ray microscope for Biological materials

投影X射线显微镜利用由扫描电子显微镜的聚焦电子束激发的薄(0.10-3μm)靶金属薄膜产生的非常小的X射线源。如果将对象放置在目标金属膜的正下方，发散的X射线会放大对象的阴影。由于X射线光源的尺寸小于100 nm，该显微镜的分辨率约为0.1μm，优于光学显微镜的分辨率。我们已将该显微镜应用于锥束计算机层析成像的三维结构分析。用步进电机控制样品旋转器，在全方向(360°)每1°记录一次小昆虫的投影图像。用临界点干燥法制备了Omiscus Porcellio，并将其固定在样品旋转器的薄胶带(厚度为0.4μm)上。图像记录采用直接探测X射线的冷却CCD摄像机(Hamamatsu C4880)。利用反投影法从玉米射束投影重建三维图像。重建的三维图像(256x256x256)的分辨率约为3μm。消化器官在3D中清晰可见。我们将分析包含更详细结构的较小对象，如神经网络。

The projection X-ray microscope makes use of a very small X-ray source from a thin (0.1-3 μm) target metal film that is excited by the focused electron beam of a scanning electron microscope (SEM). If an object is placed just below the target metal film, the diverging X-rays enlarge the shadow of the object. Because the size of X-ray light source is less than 100nm, the resolution of this microscope is about 0.1μm which is better than that of optical microscopes. We have applied this microscope for three-dimensional (3-D) structure analysis by means of cone-beam computerized tomography. The projection images of small insect, Omiscus Porcellio, were recorded in every 1° for whole direction (360°) with a stepping motor controlled sample rotator. An Omiscus Porcellio was prepared by critical point drying method and fixed on a thin adhesive tape (0.4μm thick) of the sample rotator. The images were recorded with cooled CCD camera (HAMAMATSU C4880) which detect X-ray directly. The 3-D image was reconstructed from corn-beam projections using back-projection algorithm. The resolution of the reconstructed 3-D image (256 x 256 x 256) was about 3 μm. The digestive organs were clearly visualized in 3-D.We are going to analyze smaller objects such as nerve networks which contains more detailed structure.