Research Article: Hard X‐Ray Nanoholotomography: Large‐Scale, Label‐Free, 3D Neuroimaging beyond Optical Limit

Date Published: March 30, 2018

Publisher: John Wiley and Sons Inc.

Author(s): Anna Khimchenko, Christos Bikis, Alexandra Pacureanu, Simone E. Hieber, Peter Thalmann, Hans Deyhle, Gabriel Schweighauser, Jürgen Hench, Stephan Frank, Magdalena Müller‐Gerbl, Georg Schulz, Peter Cloetens, Bert Müller.

http://doi.org/10.1002/advs.201700694

Abstract

There have been great efforts on the nanoscale 3D probing of brain tissues to image subcellular morphologies. However, limitations in terms of tissue coverage, anisotropic resolution, stain dependence, and complex sample preparation all hinder achieving a better understanding of the human brain functioning in the subcellular context. Herein, X‐ray nanoholotomography is introduced as an emerging synchrotron radiation‐based technology for large‐scale, label‐free, direct imaging with isotropic voxel sizes down to 25 nm, exhibiting a spatial resolution down to 88 nm. The procedure is nondestructive as it does not require physical slicing. Hence, it allows subsequent imaging by complementary techniques, including histology. The feasibility of this 3D imaging approach is demonstrated on human cerebellum and neocortex specimens derived from paraffin‐embedded tissue blocks. The obtained results are compared to hematoxylin and eosin stained histological sections and showcase the ability for rapid hierarchical neuroimaging and automatic rebuilding of the neuronal architecture at the level of a single cell nucleolus. The findings indicate that nanoholotomography can complement microscopy not only by large isotropic volumetric data but also by morphological details on the sub‐100 nm level, addressing many of the present challenges in brain tissue characterization and probably becoming an important tool in nanoanatomy.

Partial Text

In our aging society, incidence and prevalence of brain disorders are rapidly increasing, with almost one‐third of disabilities being attributed to brain malfunction.1 Micro‐ and nanomorphology of the neuronal network is tightly linked with the brain’s functionality. This has sparked significant interest and efforts aimed at uncovering hierarchically organized neuronal structures.2 Currently available imaging methodologies, however, are limited in their 3D representation of large specimens in a time‐efficient manner with sufficient nanoscale isotropic resolution while preserving the biological context.

Visualization plays an important role in medical research, as there is a direct correlation between abnormalities in size, shape, or topology of neurons and brain disorders. For example, many pathological brain conditions are associated with cell loss,26 abnormal cellular, or dendritic morphology.27 Similarly, changes at the subcellular level have been reported for neurodegenerative disorders, for example, membrane damage inducing curvature adaptation,28 axon demyelination, and abnormal morphology of microglia in Alzheimer’s disease. These pathological (sub)cellular changes are within the resolution range of XNH. The presented segmentations of cellular and subcellular structures can provide quantitative measures, for example, volume or dimension values of subcellular structures, cell number, or shapes. For example, the soma diameter and the envelope curvature of the Purkinje cells were calculated.

We have demonstrated that XNH allows generating 3D images of large portions of brain tissue ex vivo without the need for sectioning, staining, or sample preparation outside clinical practice. Thus, it could find a wide application as an imaging tool in neuroscience research. As a proof‐of‐concept experiment, we showed micro‐ and nanostructural images of human cerebellum and neocortex at multiple resolution scales. This experimental approach paves the way to an automated and objective approach to study brain’s nanoanatomy alterations caused by pathological conditions or medical interventions. XNH provides extraordinary capabilities for 3D imaging of cells, in that subcellular structures can be identified in a label‐free and time‐efficient manner with quantitative values related to biochemical properties. Thus, nanoholotomography bridges the spatial resolution gap between optical and electron microscopy while giving access to nanoscale isotropic resolution 3D data of relatively large tissue volumes. As the acquisition rate can be increased by orders of magnitude and as penetration of hard X‐rays through soft tissue is virtually unlimited, full‐scale subcellular mapping of the human brain is within reach.

Specimen Preparation: Brain specimens were obtained from donated bodies. All donors of the program contributed their bodies or parts of their bodies to education and research purposes. Informed consent for scientific use was obtained in written form and the procedures were conducted in accordance with the Declaration of Helsinki. The brains were fixed in 4% histological‐grade buffered formalin, before samples of the neocortex, adjacent white matter, and cerebellum were excised, dehydrated in ethanol, cleared in xylene, and embedded in a paraffin/plastic polymer mixture (Surgipath Paraplast, Leica Biosystems, Switzerland). Cylindrical specimens with a diameter of 510 µm and a height of ≈1 mm were cut from paraffin blocks using a metal punch.

The authors declare no conflict of interest.

 

Source:

http://doi.org/10.1002/advs.201700694

 

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