The lens, a major optical component of the eye, has a gradient refractive index, which is required to provide sufficient refractive power and image quality. The refractive index variations across the lens are dependent on the distributions and concentrations of the varying protein classes. In this study, we present the first measurements of the refractive index in the in situ eye lens from five species using a specially constructed X-ray Talbot grating interferometer. The measurements have been conducted in two planes: the one containing the optic axis (the sagittal plane) and the plane orthogonal to this (the equatorial plane). The results show previously undetected discontinuities and fluctuations in the refractive index profile that vary in different species. These may be linked to growth processes and may be the first optical evidence of discrete developmental stages.

A crucial evolutionary change in vertebrate history was the Palaeozoic (Devonian 419–359 million years ago) water-to-land transition, allowed by key morphological and physiological modifications including the acquisition of lungs. Nonetheless, the origin and early evolution of vertebrate lungs remain highly controversial, particularly whether the ancestral state was paired or unpaired. Due to the rarity of fossil soft tissue preservation, lung evolution can only be traced based on the extant phylogenetic bracket. Here we investigate, for the first time, lung morphology in extensive developmental series of key living lunged osteichthyans using synchrotron x-ray microtomography and histology. Our results shed light on the primitive state of vertebrate lungs as unpaired, evolving to be truly paired in the lineage towards the tetrapods. The water-to-land transition confronted profound physiological challenges and paired lungs were decisive for increasing the surface area and the pulmonary compliance and volume, especially during the air-breathing on land.

An X-ray stereo imaging system with synchrotron radiation was developed at BL20B2, SPring-8. A portion of a wide X-ray beam was Bragg-reflected by a silicon crystal to produce an X-ray beam which intersects with the direct X-ray beam. Samples were placed at the intersection point of the two beam paths. X-ray stereo images were recorded simultaneously by a detector with a large field of view placed close to the sample. A three-dimensional wire-frame model of a sample was created from the depth information that was obtained from the lateral positions in the stereo image. X-ray stereo angiography of a mouse femoral region was performed as a demonstration of real-time stereo imaging. Three-dimensional arrangements of the femur and blood vessels were obtained.

Summary: A phase-contrast X-ray microtomography system using the Talbot imaging has been built at the SPring-8 synchrotron radiation facility. This system has much higher density resolution than absorption-based X-ray microtomography. The tomographic sections of formalin-fixed mouse fetuses obtained with this method clearly depict various organs without any staining at a pixel resolution of up to 5 µm. Since this technique allows us to obtain three-dimensional structural information without sectioning, it will be particularly useful to examine anomalies that take place during development. It can be also used to quantitatively measure volume and mass of organs during development.

Neurons transmit active potentials through axons, which are essential for the brain to function. In this study, the axonal networks of the murine brain were visualized with X-ray tomographic microscopy, also known as X-ray microtomography or micro-CT. Murine brain samples were freeze-dried to reconstitute the intrinsic contrast of tissue constituents and subjected to X-ray visualization. A whole brain hemisphere visualized by absorption contrast illustrated three-dimensional structures including those of the striatum, corpus callosum, and anterior commissure. Axonal tracts observed in the striatum start from the basal surface of the cerebral cortex and end at various positions in the basal ganglia. The distribution of X-ray attenuation coefficients indicated that differences in water and phospholipid content between the myelin sheath and surrounding tissue constituents account for the observed contrast. A rod-shaped cutout of brain tissue was also analyzed with a phase retrieval method, wherein tissue microstructures could be resolved with up to 2.7 μm resolution. Structures of axonal networks of the striatum were reconstructed by tracing axonal tracts. Such an analysis should be able to delineate the functional relationships of the brain regions involved in the observed network.

X-ray phase contrast tomography using a Talbot grating interferometer was applied to biological fresh samples which were not fixed by any fixatives. To achieve a high-throughput measurement for the fresh samples the X-ray phase contrast tomography measurement procedure was improved. The three-dimensional structure of a fresh mouse fetus was clearly depicted as a mass density map using X-ray phase contrast tomography. The mouse fetus measured in the fresh state was then fixed by formalin and measured in the fixed state. The influence of the formalin fixation on soft tissue was quantitatively evaluated by comparing the fresh and fixed samples. X-ray phase contrast tomography was also applied to the dynamic measurement of a biological fresh sample. Morphological changes of a ring-shaped fresh pig aorta were measured tomographically under different degrees of stretching.