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 bio-specimens. 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.

Evaluation of noise limits to improve image processing in soft X-ray projection microscopy.

Soft X-ray microscopy has been developed for high resolution imaging of hydrated biological specimens due to the availability of water window region. In particular, a projection type microscopy has advantages in wide viewing area, easy zooming function and easy extensibility to computed tomography (CT). The blur of projection image due to the Fresnel diffraction of X-rays, which eventually reduces spatial resolution, could be corrected by an iteration procedure, i.e., repetition of Fresnel and inverse Fresnel transformations. However, it was found that the correction is not enough to be effective for all images, especially for images with low contrast. In order to improve the effectiveness of image correction by computer processing, we in this study evaluated the influence of background noise in the iteration procedure through a simulation study. In the study, images of model specimen with known morphology were used as a substitute for the chromosome images, one of the targets of our microscope. Under the condition that artificial noise was distributed on the images randomly, we introduced two different parameters to evaluate noise effects according to each situation where the iteration procedure was not successful, and proposed an upper limit of the noise within which the effective iteration procedure for the chromosome images was possible. The study indicated that applying the new simulation and noise evaluation method was useful for image processing where background noises cannot be ignored compared with specimen images.