Non-destructive X-ray tomography of brain tissue ultrastructure

Maps of dense subcellular features in biological tissue are the 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, which strongly challenges anatomical investigations of entire organs such as mammalian brains. As demonstrated for the engineering and physical sciences, X-ray nanotomography represents a promising alternative for ultrastructural 3d imaging without physical sectioning1-15. Leveraging the high brilliance of 4th generation synchrotron X-ray sources, it has the potential to non-destructively image mm3-sized samples at ultrastructural resolution within a few days16. A fundamental barrier to application to the life sciences is that, when irradiated with high-intensity X-rays, biological samples deform and ultimately disintegrate, prohibiting reaching sufficient resolution. Here, we introduce a combination of engineering solutions which defeat this barrier for X-ray ptychography17, a coherent diffractive X-ray imaging technique. The solutions include a cryogenic sample stage with high stability, high-precision interferometric positioners and tailored non-rigid tomographic reconstruction algorithms18. Furthermore, adapting an epoxy resin developed for the nuclear and aerospace industry, we demonstrate radiation resistance to X-ray doses exceeding 1010 Gy. The resulting sub-40 nm isotropic resolution makes it possible to densely resolve axon bundles, boutons, dendrites and reliably identify synapses without physical sectioning. Moreover, we validated the X-ray technique using the current gold standard, namely focused ion beam scanning electron microscopy (FIB-SEM)19,20 to demonstrate intact ultrastructure in tissue volumes first imaged by X-rays. This unlocks the potential of X-ray tomography for high-resolution tissue imaging, coinciding with the transformative advancements of next-generation synchrotrons worldwide21.