Improvement of imaging and image correction methods for the soft X-ray projection microscopy

BACKGROUND: The soft X-ray projection microscope has been developed for high resolution imaging of hydrated biospecimens. Image blurring due to X-ray diffraction can be corrected by an iteration procedure. The correction is not efficient enough for all images, especially for low contrast chromosome images. OBJECTIVE: The purpose of this study is to improve X-ray imaging techniques using a finer pinhole and reducing capture time, as well as to improve image correction methods. A method of specimen staining prior to the imaging was tested in order to capture images with high contrasts. The efficiency of the iteration procedure and its combined version with an image enhancement method was also assessed. METHODS: In image correction, we used the iteration procedure and its combined version with an image enhancement technique. To capture higher contrast images, we stained chromosome specimens with the Platinum blue (Pt-blue) prior to the imaging. RESULTS: The iteration procedure combined with image enhancement corrected the chromosome images with 329 or lower magnification effectively. Using the Pt-blue staining for the chromosome, images with high contrast have been captured and successfully corrected. CONCLUSIONS: The image enhancement technique combining contrast enhancement and noise removal together was effective to obtain higher contrast images. As a result, the chromosome images with 329 or lower times magnification were corrected effectively. With Pt-blue staining, chromosome images with contrasts of 2.5 times higher than unstained case could be captured and corrected by the iteration procedure.