• Zhang, Yuxin
• Guizar-Sicairos, Manuel
• Schaefer, Andreas
• Wanner, Adrian
• Pacureanu, Alexandra
• Cook, Phil K.
• Aeppli, Meret
• Bunk, Oliver
• Müller, Elisabeth
• Peddie, Christopher
• Aidukas, Tomas
• Bosch, Carles
• Holler, Mirko
• Menzel, Andreas
• Collinson, Lucy
• Diaz, Ana

Abstract

<jats:title>Abstract</jats:title><jats:p>Densely resolving subcellular features in biological tissue is key to understanding the structural basis of organ function. Electron microscopy provides the necessary resolution, yet - as electrons penetrate samples for only a few 100s of nm - requires physical sectioning or ablation. This provides a significant challenge when aiming to scale up anatomical investigations to entire organs such as mammalian brains. X-ray nanotomography has been suggested as a promising alternative for ultrastructural 3d imaging without physical sectioning(<jats:italic>1–15</jats:italic>). Leveraging the high brilliance of 4th generation synchrotron X-ray sources, it has the theoretical potential to non-destructively acquire mm³-sized samples at ultrastructural resolution within a few days(<jats:italic>16</jats:italic>). A fundamental barrier to the practical implementation has been that, when irradiated with high-intensity X-rays, biological samples deform and ultimately disintegrate, prohibiting reaching sufficient resolution. Here, we demonstrate that X-ray ptychography(<jats:italic>17</jats:italic>), a coherent diffractive X-ray imaging technique, can faithfully acquire 3-dimensional images of metal-stained mouse neuronal tissue. Achieving high imaging quality requires minimization of the radiation damage to the sample, which we achieve by imaging at cryogenic temperatures and using tailored non-rigid tomographic reconstruction algorithms(<jats:italic>18</jats:italic>). Adapting an epoxy resin developed for the nuclear and aerospace industry, we demonstrate radiation resistance to X-ray doses exceeding 10<jats:sup>10</jats:sup>Gy. Sub-40 nm isotropic resolution makes it possible to densely resolve axon bundles, boutons, dendrites and reliably identify synapses without physical sectioning. Moreover, the tissue volumes exposed to X-rays can subsequently be imaged in 3D using high-resolution focused ion beam scanning electron microscopy (FIB-SEM)(<jats:italic>19, 20</jats:italic>) showing intact ultrastructure and demonstrating that metal-stained neuronal tissue can be highly radiation-stable. Thus, our work removes the fundamental limitations to employing X-ray tomography for high-resolution tissue imaging. Ongoing improvements in synchrotron, X-ray and detector physics(<jats:italic>21</jats:italic>), as well as further optimization of sample preparation and staining procedures(<jats:italic>22–27</jats:italic>), could lead to substantial improvements in acquisition speed(<jats:italic>16</jats:italic>), whilst complementary X-ray techniques such as laminography(<jats:italic>28–31</jats:italic>) and nano-holotomography(<jats:italic>2, 32</jats:italic>) could allow for non-destructive X-ray imaging of synapses and neural circuits in volumes of increasing size.</jats:p>

Topics

• impedance spectroscopy
• scanning electron microscopy
• tomography
• focused ion beam
• isotropic
• resin
• sectioning