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.
Highlights
Light microscopy alone does not allow for the generation of such an ultrastructural 3-D map, since it often involves permeabilization and staining of the sample as a prerequisite, which can disrupt the tissue ultrastructure
The main problem for CEMOVIS and CETOVIS is the inability to section tissue to thicker than 500 nm, unrealistic to subsequently image and generate 3-D ultrastructural maps representing tens to a hundred microns in sample thickness. Such methodologies would greatly benefit from incorporating a technology in their workflow that can generate a 3-D map of the ultrastructure of unstained tissue at an intermediate resolution. Such 3-D maps could be immensely useful for cryo-immuno electron microscopy in the case of chemically-fixed, frozen-hydrated tissue samples, and for CEMOVIS/CETOVIS and cryo-FIBSEM when applied to high-pressure frozen samples, since they would help solve a major bottleneck in cryo-FIBSEM and especially CEMOVIS/CETOVIS research: it is challenging and in some cases impossible to pinpoint desired features of interest in unstained tissues prior to imaging using these approaches alone
The current bottleneck for high-resolution imaging, combined with topological biochemistry such as cryo-immuno electron microscopy of tissues[1, 2], is the lack of a critical intermediate imaging step that allows for a fast, non-destructive visualization and scanning of features of interest in unstained tissues that are compatible with such downstream analysis
Summary
Light microscopy alone does not allow for the generation of such an ultrastructural 3-D map, since it often involves permeabilization and staining of the sample as a prerequisite, which can disrupt the tissue ultrastructure. The main problem for CEMOVIS and CETOVIS is the inability to section tissue to thicker than 500 nm, unrealistic to subsequently image and generate 3-D ultrastructural maps representing tens to a hundred microns in sample thickness. Such methodologies would greatly benefit from incorporating a technology in their workflow that can generate a 3-D map of the ultrastructure of unstained tissue at an intermediate resolution. Biological tissue typically exhibits a low electron density contrast and among different X-ray imaging techniques, ptychography has a great potential for imaging such material as it combines high resolution and high sensitivity with quantitative phase contrast[14, 15]
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