Three-Dimensional Imaging of Biological Tissue by Cryo X-Ray Ptychography

Abstract

High-throughput three-dimensional cryogenic imaging of thick biological specimens is valuable for identifying biologically- or pathologically-relevant features of interest, especially for subsequent correlative studies. Unfortunately, high-resolution imaging techniques at cryogenic conditions often require sample reduction through sequential physical milling or sectioning for sufficient penetration to generate each image of the 3-D stack. This study represents the first demonstration of using ptychographic hard X-ray tomography at cryogenic temperatures for imaging thick biological tissue in a chemically-fixed, frozen-hydrated state without heavy metal staining and organic solvents. Applied to mammalian brain, this label-free cryogenic imaging method allows visualization of myelinated axons and sub-cellular features such as age-related pigmented cellular inclusions at a spatial resolution of ~100 nanometers and thicknesses approaching 100 microns. Because our approach does not require dehydration, staining or reduction of the sample, we introduce the possibility for subsequent analysis of the same tissue using orthogonal approaches that are expected to yield direct complementary insight to the biological features of interest.

Correlations between lignin content and structural robustness in plants revealed by X-ray ptychography

“…Iterative phase retrieval algorithms are applied to reconstruct the complex-valued transmissivity of the specimen 26 . By combining the data acquisition at different angles, we obtained tomographic reconstructions that provide three-dimensional reconstructions with nanometric precision 27,[30][31][32] .…”

“…A real space image of the electron density distribution is retrieved through iterative algorithms 28 , applied to precise measurements of coherent X-rays scattered by the specimen, ultimately reaching spatial resolutions on the order of 10 nm in the case of strongly scattering materials 29,30 . PXCT was applied to unveil the nanometric features of stain-free biological samples, on single cells and within tissues, resolving organelles and macromolecules 31,32 . Here, we obtained a three-dimensional image of plant tissues corresponding to volumes of 25 ×50 ×50 μm 3 of Arabidopsis thaliana, which is considered a model organism for plants.…”

Dense neuronal reconstruction through X-ray holographic nano-tomography

“…Previously, attenuation-based X-ray microtomography with a synchrotron source enabled visualization of metal stained, sparse wiring in the Drosophila brain (Mizutani et al, 2013). The advent of Xray phase-contrast techniques enabled imaging of both stained and unstained brain tissue at micrometer and submicrometer scales (Cedola et al, 2017;Dyer et al, 2017;Fonseca et al, 2018;Khimchenko et al, 2018;Massimi et al, 2019;Schulz et al, 2010;Shahmoradian et al, 2017;Töpperwien et al, 2018). However, until now, X-ray microscopy has not achieved the necessary resolving power to enable dense reconstruction of neuronal morphologies.…”

Quantitative ptychographic bio-imaging in the water window

“…X-ray ptychography is a scanning variant of CXDI to obtain large field of view representations of objects with high spatial resolution [4,5]. Biological samples have been measured with CXDI in three dimensions (3D) [6] and by ptychography in a cryogenic environment with hard X-rays (8 keV) while the contrast between sample and ice is rather low [7,8]. In order to gain element specific sensitivity it was suggested to combine the high-resolution imaging capabilities of ptychography with X-ray fluorescence microscopy using hard X-rays [9,10].…”