Mareike Töpperwien

Three-dimensional mouse brain cytoarchitecture revealed by laboratory-based x-ray phase-contrast tomography.

Studies of brain cytoarchitecture in mammals are routinely performed by serial sectioning of the specimen and staining of the sections. The procedure is labor-intensive and the 3D architecture can only be determined after aligning individual 2D sections, leading to a reconstructed volume with non-isotropic resolution. Propagation-based x-ray phase-contrast tomography offers a unique potential for high-resolution 3D imaging of intact biological specimen due to the high penetration depth and potential resolution. We here show that even compact laboratory CT at an optimized liquid-metal jet microfocus source combined with suitable phase-retrieval algorithms and a novel tissue preparation can provide cellular and subcellular resolution in millimeter sized samples of mouse brain. We removed water and lipids from entire mouse brains and measured the remaining dry tissue matrix in air, lowering absorption but increasing phase contrast. We present single-cell resolution images of mouse brain cytoarchitecture and show that axons can be revealed in myelinated fiber bundles. In contrast to optical 3D techniques our approach does neither require staining of cells nor tissue clearing, procedures that are increasingly difficult to apply with increasing sample and brain sizes. The approach thus opens a novel route for high-resolution high-throughput studies of brain architecture in mammals.

Propagation-based phase-contrast tomography for high-resolution lung imaging with laboratory sources

We have performed high-resolution phase-contrast tomography on whole mice with a laboratory setup. Enabled by a high-brilliance liquid-metal-jet source, we show the feasibility of propagation-based phase contrast in local tomography even in the presence of strongly absorbing surrounding tissue as it is the case in small animal imaging of the lung. We demonstrate the technique by reconstructions of the mouse lung for two different fields of view, covering the whole organ, and a zoom to the local finer structure of terminal airways and alveoli. With a resolution of a few micrometers and the wide availability of the technique, studies of larger biological samples at the cellular level become possible.

X-ray phase contrast tomography from whole organ down to single cells

We use propagation based hard x-ray phase contrast tomography to explore the three dimensional structure of neuronal tissues from the organ down to sub-cellular level, based on combinations of synchrotron radiation and laboratory sources. To this end a laboratory based microfocus tomography setup has been built in which the geometry was optimized for phase contrast imaging and tomography. By utilizing phase retrieval algorithms, quantitative reconstructions can be obtained that enable automatic renderings without edge artifacts. A high brightness liquid metal microfocus x-ray source in combination with a high resolution detector yielding a resolution down to 1.5 μm. To extend the method to nanoscale resolution we use a divergent x-ray waveguide beam geometry at the synchrotron. Thus, the magnification can be easily tuned by placing the sample at different defocus distances. Due to the small Fresnel numbers in this geometry the measured images are of holographic nature which poses a challenge in phase retrieval.

Laboratory-based x-ray phase-contrast tomography enables 3D virtual histology

Due to the large penetration depth and small wavelength hard x-rays offer a unique potential for 3D biomedical and biological imaging, combining capabilities of high resolution and large sample volume. However, in classical absorption-based computed tomography, soft tissue only shows a weak contrast, limiting the actual resolution. With the advent of phase-contrast methods, the much stronger phase shift induced by the sample can now be exploited. For high resolution, free space propagation behind the sample is particularly well suited to make the phase shift visible. Contrast formation is based on the self-interference of the transmitted beam, resulting in object-induced intensity modulations in the detector plane. As this method requires a sufficiently high degree of spatial coherence, it was since long perceived as a synchrotron-based imaging technique. In this contribution we show that by combination of high brightness liquid-metal jet microfocus sources and suitable sample preparation techniques, as well as optimized geometry, detection and phase retrieval, excellent three-dimensional image quality can be obtained, revealing the anatomy of a cobweb spider in high detail. This opens up new opportunities for 3D virtual histology of small organisms. Importantly, the image quality is finally augmented to a level accessible to automatic 3D segmentation.

Phase-contrast tomography of neuronal tissues: from laboratory- to high resolution synchrotron CT

Assessing the three-dimensional architecture of neuronal tissues with sub-cellular resolution presents a significant analytical challenge. Overcoming the limitations associated with serial slicing, phase-contrast x-ray tomography has the potential to contribute to this goal. Even compact laboratory CT at an optimized liquid-metal jet micro- focus source combined with suitable phase-retrieval algorithms and preparation protocols can yield renderings with single cell sensitivity in millimeter sized brain areas of mouse. Here, we show the capabilities of the setup by imaging a Golgi-Cox impregnated mouse brain. Towards higher resolution we extend these studies at our recently upgraded waveguide-based cone-beam holo-tomography instrument GINIX at DESY. This setup allows high resolution recordings with adjustable field of view and resolution, down to the voxel sizes in the range of a few ten nanometers. The recent results make us confident that important issues of neuronal connectivity can be addressed by these methods, and that 3D (virtual) histology with nanoscale resolution will become an attractive modality for neuroscience research.

Four dimensional material movies : High speed phase-contrast tomography by backprojection along dynamically curved paths

We present an approach towards four dimensional (4d) movies of materials, showing dynamic processes within the entire 3d structure. The method is based on tomographic reconstruction on dynamically curved paths using a motion model estimated by optical flow techniques, considerably reducing the typical motion artefacts of dynamic tomography. At the same time we exploit x-ray phase contrast based on free propagation to enhance the signal from micron scale structure recorded with illumination times down to a millisecond (ms). The concept is demonstrated by observing the burning process of a match stick in 4d, using high speed synchrotron phase contrast x-ray tomography recordings. The resulting movies reveal the structural changes of the wood cells during the combustion.

Axonal ensheathment in the nervous system of lamprey : implications for the evolution of myelinating glia

In the nervous system, myelination of axons enables rapid impulse conduction and is a specialized function of glial cells. Myelinating glia are the last cell type to emerge in the evolution of vertebrate nervous systems, presumably in ancient jawed vertebrates (gnathostomata) because jawless vertebrates (agnathans) lack myelin. We have hypothesized that, in these unmyelinated species, evolutionary progenitors of myelinating cells must have existed that should still be present in contemporary agnathan species. Here, we used advanced electron microscopic techniques to reveal axon–glia interactions in the sea lamprey Petromyzon marinus. By quantitative assessment of the spinal cord and the peripheral lateral line nerve, we observed a marked maturation-dependent growth of axonal calibers. In peripheral nerves, all axons are ensheathed by glial cells either in bundles or, when larger than the threshold caliber of 3 μm, individually. The ensheathing glia are covered by a basal lamina and express SoxE-transcription factors, features of mammalian Remak-type Schwann cells. In larval lamprey, the ensheathment of peripheral axons leaves gaps that are closed in adults. CNS axons are also covered to a considerable extent by glial processes, which contain a high density of intermediate filaments, glycogen particles, large lipid droplets, and desmosomes, similar to mammalian astrocytes. Indeed, by in situ hybridization, these glial cells express the astrocyte marker Aldh1l1. Specimens were of unknown sex. Our observations imply that radial sorting, ensheathment, and presumably also metabolic support of axons are ancient functions of glial cells that predate the evolutionary emergence of myelin in jawed vertebrates. SIGNIFICANCE STATEMENT We used current electron microscopy techniques to examine axon–glia units in a nonmyelinated vertebrate species, the sea lamprey. In the PNS, lamprey axons are fully ensheathed either individually or in bundles by cells ortholog to Schwann cells. In the CNS, axons associate with astrocyte orthologs, which contain glycogen and lipid droplets. We suggest that ensheathment, radial sorting, and metabolic support of axons by glial cells predate the evolutionary emergence of myelin in ancient jawed vertebrates.

Propagation-based phase-contrast x-ray tomography of cochlea using a compact synchrotron source

We demonstrate that phase retrieval and tomographic imaging at the organ level of small animals can be advantageously carried out using the monochromatic radiation emitted by a compact x-ray light source, without further optical elements apart from source and detector. This approach allows to carry out microtomography experiments which - due to the large performance gap with respect to conventional laboratory instruments - so far were usually limited to synchrotron sources. We demonstrate the potential by mapping the functional soft tissue within the guinea pig and marmoset cochlea, including in the latter case an electrical cochlear implant. We show how 3d microanatomical studies without dissection or microscopic imaging can enhance future research on cochlear implants.

Publisher Correction: Four dimensional material movies: High speed phase-contrast tomography by backprojection along dynamically curved paths

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Three-dimensional virtual histology of human cerebellum by X-ray phase-contrast tomography

Significance The complex cytoarchitecture of human brain tissue is traditionally studied by histology, providing structural information in 2D planes. This can be partly extended to 3D by inspecting many parallel slices, however, at nonisotropic resolution. This work shows that propagation-based X-ray phase-contrast tomography, both at the synchrotron and even at a compact laboratory source, can be used to perform noninvasive 3D virtual histology on unstained paraffin-embedded human cerebellum at isotropic subcellular resolution. The resulting data quality is high enough to visualize and automatically locate ∼106 neurons within the different layers of the cerebellum, providing unprecedented data on its 3D cytoarchitecture and spatial organization. To quantitatively evaluate brain tissue and its corresponding function, knowledge of the 3D cellular distribution is essential. The gold standard to obtain this information is histology, a destructive and labor-intensive technique where the specimen is sliced and examined under a light microscope, providing 3D information at nonisotropic resolution. To overcome the limitations of conventional histology, we use phase-contrast X-ray tomography with optimized optics, reconstruction, and image analysis, both at a dedicated synchrotron radiation endstation, which we have equipped with X-ray waveguide optics for coherence and wavefront filtering, and at a compact laboratory source. As a proof-of-concept demonstration we probe the 3D cytoarchitecture in millimeter-sized punches of unstained human cerebellum embedded in paraffin and show that isotropic subcellular resolution can be reached at both setups throughout the specimen. To enable a quantitative analysis of the reconstructed data, we demonstrate automatic cell segmentation and localization of over 1 million neurons within the cerebellar cortex. This allows for the analysis of the spatial organization and correlation of cells in all dimensions by borrowing concepts from condensed-matter physics, indicating a strong short-range order and local clustering of the cells in the granular layer. By quantification of 3D neuronal “packing,” we can hence shed light on how the human cerebellum accommodates 80% of the total neurons in the brain in only 10% of its volume. In addition, we show that the distribution of neighboring neurons in the granular layer is anisotropic with respect to the Purkinje cell dendrites.