Imaging of retina cellular and subcellular structures using ptychographic hard X-ray tomography.

Ptychographic hard X-ray computed tomography (PXCT) is a recent method allowing imaging with quantitative electron-density contrast. Here, we imaged at cryogenic temperature and without sectioning, cellular and subcellular structures of a chemically-fixed and stained wild-type mouse retina, including axons and synapses with complete isotropic 3D information over tens of microns. Comparison with tomograms of degenerative retina from a mouse model of retinitis pigmentosa illustrates the potential of this method for analysing disease processes like neurodegeneration at sub-200 nm resolution. As a non-destructive imaging method, PXCT is very suitable for correlative imaging. Within the outer plexiform layer containing the photoreceptors synapses, we identified somatic synapses. We used a small region inside the X-ray-imaged sample for further high-resolution focused ion beam / scanning electron microscope (FIB/SEM) tomography. The subcellular structures of synapses obtained with the X-ray technique matched the EM data demonstrating that PXCT is a powerful scanning method for tissue volumes of more than 60 cells and sensitive enough for identification of regions as small as 200 nm, remaining available for further structural and biochemical investigations.