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.
Highlights
There have been great efforts on the nanoscale 3D probing of brain tissues being attributed to brain malfunction.[1]
The 3D analysis of subcellular structures based on 2D histological sections in combination with logical details on the sub-100 nm level, addressing many of the present chalmicroscopy is limited by sectioning- or lenges in brain tissue characterization and probably becoming an important staining-related artefacts, and may lead to tool in nanoanatomy
First a fast overview scan with lower spatial resolution was performed
Summary
As limitations given by wavelength are shifted to subnanometers, application of X-ray optics can extend spatial resolution far into the nanometer range allowing visualization down to the subcellular level (Figure 1a,e). First a fast overview (reconstructed volume is a cylinder of 400 μm height and 400 μm in diameter) scan with lower spatial resolution (effective detector pixel length of 200 nm and a voxel size of the reconstructed data set of 200 × 200 × 200 nm3) was performed This prescan enabled us to select regions to image in a holotomography configuration at four propagation distances with voxel lengths ranging from 130 to 25 nm (Figure 1b). For the formalin-fixed paraffin-embedded neocortex specimen, the data recorded with a pixel size of 50 nm and binned by a factor of two (Figure 1e), in order to increase the density contrast at the expense of spatial resolution,[21] exhibit a contrast-to-noise ratio (CNR) with respect to the background of 0.806 [0.795 0.818], whereas the CNR of the data with 100 nm voxels corresponds to 0.734 [0.731 0.762].
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