Fine visualization of biological cells using X-ray micro-CT with the slow freezing contrast improved method.

X-ray computed tomography (CT) is widely used in various fields for the non-destructive three-dimensional (3D) observation of internal structures within objects. However, biological cells are primarily composed of light elements such as oxygen and carbon, which have high X-ray transmittance. Consequently, conventional absorption contrast X-ray CT is unable to achieve fine 3D observations of such specimens. We hereby present a technique of novel contrast improvement, the slow freezing contrast improvement method. This method utilizes the aggregation of solutes during slow freezing of aqueous solutions to increase contrast. As plant cells are slowly cooled, intracellular fluid crystallizes, concentrating sugars in specific areas. This process allows for micron-scale visualization of cell structures without staining, using conventional absorption contrast X-ray CT. Experiments on slowly frozen fruits and formalin-fixed mouse organs using synchrotron-based cryo micro-X-ray CT produced high-resolution images of cellular structures. The ice crystal patterns formed within cells varied based on sugar concentration, suggesting potential for detecting sugar levels in individual cells. This method shows promise as a third approach for fine 3D observation of biological cells, complementing contrast agent and phase-contrast imaging techniques.