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.

Figure 1

Overview of the workflow for cryo-PXCT. (a,b) Tissues are processed and prepared for imaging. (c) The cryo-PXCT setup. The sample is scanned on the transverse plane for ptychography and also rotated for tomography measurements. Diffraction patterns are recorded by the detector. (d,e) Quantitative tomograms were reconstructed using ptychography algorithms. (f) The sample is intact after imaging, allowing for further investigation using complementary techniques.

Figure 2

Overview of the sample preparation and cryo-trimming procedures for cryo-PXCT. (a) Gold-coated OMNY pin held by tweezers. Scale bar ≈ 1 mm. (b) Schematic of applying the sample to the shaved surface tip of the OMNY pin, using one leg of a fine electronic-grade tweezer. OMNY specimen-mounting pin with sample is then transferred directly to vitrify in a cryo-ultramicrotomy chamber pre-cooled by gaseous nitrogen to −90 °C. (c) OMNY pin within a cryo-ultramicrotomy chamber, (1) after initial surface trimming using a cryo-diamond knife, and (2) after trimming to a pyramid shape for imaging. Both vitreous ice (clear portion, right side of pyramid) and tissue (pink portion, left side of pyramid) are visible at this pointed tip; scale bars ≈ 350 µm. Bottom right: Radiograph of one of the resulting trimmed pyramids prior to OMNY imaging. Large black dot and inhomogeneous illumination are due to defects in the X-ray wavefront and not part of the sample; scale bar ≈ 100 µm.

Figure 3

Imaging parameters and characteristics of biological samples imaged by cryo-PXCT using OMNY. Representative orthoslices selected from an X-Y plane from each of the 3 reconstructed cryo-PXCT tomographic volumes (top panel), corresponding to distinct and different cryo-trimmed pyramid samples of mouse brainstem (Sample A,B,C). Dimensional “X” value here refers to the approximate measurement of the longest edge of the rectangle (pyramid base) that was measured, and “Y” value refers to the shorter edge that was measured. Orthoslices shown are toward the middle of the pyramid and not at the base. Depth in “Z” refers to the height of the imaged region of the pyramid. Scale bar = 10 µm. Table represents the volume imaged per tomogram, data collection time, projections used for tomographic reconstruction, estimated dose (MGy), and both 2D slice and 3-D resolution, for each of the samples.

Figure 4

Cryo-PXCT and 3-D color rendering of mouse brain tissue. (a) Single orthoslice from a reconstructed 3-D tomogram. Variations of myelin sheath thicknesses of myelinated axons are visible (dark blue arrowheads). Multiple cell nuclei (yellow asterisks) are detected based on size, and contrast differences to the surrounding cellular cytosol. Small and roughly spherical structures (pink arrowheads) are visible. Scale bar = 10 µm. (b) Structure likely corresponding to a nucleus in one cell (yellow arrowheads) in two different orthoslices of the tomogram. Scale bar = 5 µm. (c) Spherical structures (pink dotted circles) most likely representing lysosomal LF or pigmented autophagic vacuoles (PAV) at different orthoslices of the same tomogram. Scale bar = 5 µm. (d) A structure resembling a PAV representing distinct components of lipid and pigment, visible at different z-heights (1–6). Scale bar = 2.5 µm. (e) Semi-automated color-segmentation of the reconstructed tomogram shown in (a), based on the contrast differences within the sample. Yellow = nuclei, Pink = lysosomal LF and putative PAVs, Aqua = Myelinated axons.