3D-Imaging of synapses in neuronal tissues with synchrotron X-ray ptychography

Bosch, C.; Diaz, A.; Holler, M.; Guizar-Sicairos, M.; Aidukas, T.; Pacureanu, A.; Mueller, E.; Peddie, C. J.; Collinson, L.; Zhang, Y.; Menzel, A.; Wanner, A.; Schaefer, A.

doi:10.1101/2023.11.16.567403

bioRxiv · 10.1101/2023.11.16.567403

3D-Imaging of synapses in neuronal tissues with synchrotron X-ray ptychography

Bosch, C.·Diaz, A.·Holler, M.·Guizar-Sicairos, M.·Aidukas, T.·Pacureanu, A.·Mueller, E.·Peddie, C. J.·Collinson, L.·Zhang, Y.·Menzel, A.·Wanner, A.·Schaefer, A.

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Abstract

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.

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Bosch, C., Diaz, A., Holler, M., Guizar-Sicairos, M., Aidukas, T., Pacureanu, A., Mueller, E., Peddie, C. J., Collinson, L., Zhang, Y., Menzel, A., Wanner, A., Schaefer, A.. 2023-11-17. 3D-Imaging of synapses in neuronal tissues with synchrotron X-ray ptychography. https://doi.org/10.1101/2023.11.16.567403

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