How to make mapping images of biological specimens--data collection and image processing.

To make high-quality elemental mapping images of biological specimens, the conditions of data collection were optimized, and image-processing methods were examined. The most important step was to obtain a sufficient number of electrons to make the images. The exposure time was limited by the characteristics of the CCD camera. Obtaining a long exposure time exceeded the limitations of the camera, so exposures of the same area were performed many times and recorded in many images. The divided images were merged after observation. To merge images, a new software, 'Rotate & Merge' (R&M), was developed. Because of the characteristics of biological specimens, R&M must have several functions. Picture-zoom and image rotation are necessary because of shrinkage of the specimens due to irradiation during exposure, since even cryo techniques, including low-dose techniques, do not prevent shrinkage of specimens. Merged phosphorus-mapping images of ultra-thin slices of yeast cells were made. In these images, ribosome particles and DNA in the nucleus were observed clearly. The merging was very useful for improving the quality of mapping images.