Scientists have developed a novel X-ray imaging technique that provides unprecedented three-dimensional views of thick biological tissues without requiring them to be dehydrated, stained with heavy metals, or physically sectioned. This new method, known as cryogenic X-ray ptychography (cryo-PXCT), offers a label-free way to visualize the intricate internal structures of tissues, opening new avenues for understanding cellular processes and disease. By preserving samples in a frozen, hydrated state, the technique allows for subsequent analysis with other imaging methods, a significant advantage over existing high-resolution techniques that are often destructive.
The breakthrough addresses a major challenge in biological imaging: how to obtain high-resolution, 3D views of tissues that are thicker than a single cell while keeping them in a near-native state. Traditional methods like cryo-electron microscopy often require samples to be sliced into extremely thin sections, losing the broader three-dimensional context. Cryo-PXCT, however, can penetrate tissues approaching 100 microns in thickness—roughly the width of a human hair—and achieve a spatial resolution of around 100 nanometers. This capability allows researchers to generate detailed 3D maps of complex structures within tissues, such as the brain, providing valuable context for more focused, higher-resolution studies.
Overcoming Existing Imaging Limitations
For decades, researchers have sought methods to bridge the gap between light microscopy, which can image living cells but with limited resolution, and electron microscopy, which offers high resolution but requires harsh sample preparation that can alter tissue structure. High-resolution cryogenic techniques have been developed, but they come with significant drawbacks. Techniques such as cryo-focused ion beam milling-scanning electron microscopy (cryo-FIBSEM) and cryo-electron tomography of vitreous sections (CEMOVIS/CETOVIS) provide remarkable detail but are inherently destructive, as they rely on sequentially milling away or physically sectioning the specimen to create a 3D image. This process prevents any further analysis of the imaged volume.
Cryo-PXCT circumvents these issues by using hard X-rays, which can penetrate thick samples without the need for physical sectioning. The “ptychography” aspect of the technique involves a lens-free imaging approach where diffraction patterns are recorded from overlapping areas of the sample. Sophisticated algorithms then reconstruct these patterns into a high-resolution image with quantitative phase contrast, which is particularly effective for imaging the subtle density variations in biological tissues that have low electron density contrast. This non-destructive approach preserves the sample, making it available for correlative studies with other imaging modalities.
A New Instrument for Cryogenic Imaging
The OMNY System
The successful application of cryo-PXCT to biological tissues was made possible by a newly developed instrument called OMNY (tOMography Nano crYo), located at the Paul Scherrer Institut’s Swiss Light Source. The OMNY system is specifically designed for high-resolution 3D imaging of cryogenically fixed specimens in an ultra-high vacuum. One of the key innovations of the OMNY instrument is its use of differential laser interferometry to ensure highly stable and accurate positioning of the sample during scanning, which is crucial for achieving nanometer-scale resolution in three dimensions.
Sample Preparation and Workflow
The workflow for cryo-PXCT begins with a sample preparation process similar to that used for cryo-immuno electron microscopy. In the demonstration of this technique, brain tissue from mice was chemically fixed and then infused with a cryoprotectant solution to prevent the formation of damaging ice crystals during freezing. The tissue was then mounted on a specialized pin and trimmed into a pyramid shape using a cryo-ultramicrotome. This prepared sample was then transferred to the OMNY instrument, where it was maintained at a cryogenic temperature of -183°C during the imaging process.
Detailed Views of Brain Tissue
In its inaugural application, cryo-PXCT was used to image blocks of mouse brainstem tissue. The resulting 3D tomograms, with a resolution down to 115 nanometers, revealed a wealth of subcellular details. Researchers were able to clearly visualize myelinated axons, the nerve fibers responsible for transmitting electrical signals, and even variations in the thickness of their myelin sheaths. The high contrast and signal-to-noise ratio of the images also allowed for the 3D segmentation of other cellular features, including cell nuclei and small, spherical structures believed to be lysosomal lipofuscin and pigmented autophagic vacuoles, which are associated with aging.
The ability to distinguish these features without the use of stains is a significant advantage of the technique. The quantitative nature of ptychography also allows for the creation of accurate 3D maps of the electron density of the specimen, which can be converted into mass densities. This provides another layer of information for researchers studying the composition and organization of cellular structures.
Future Directions and Potential Applications
The successful demonstration of cryo-PXCT on brain tissue opens the door to a wide range of applications in biology and medicine. One of the most immediate benefits is the potential to create detailed 3D ultrastructural maps of tissues prior to more targeted investigations. For example, a researcher studying a particular protein in a diseased tissue could first use cryo-PXCT to get a broad overview of the tissue architecture and identify regions of interest. They could then use the precise 3D coordinates from the cryo-PXCT map to guide them in preparing thin sections from those specific regions for higher-resolution imaging with cryo-electron microscopy or for molecular analysis with mass spectrometry.
Future developments of the technique will focus on improving the resolution and increasing the volume of tissue that can be imaged. This will involve the development of new data analysis methods, such as multi-slice algorithms that can account for the complex interaction of X-rays with thick samples. The researchers also anticipate that next-generation synchrotron sources will provide the increased X-ray flux needed to achieve resolutions below 50 nanometers.
Expanding to Other Tissues and Diseases
Beyond the brain, cryo-PXCT could be applied to a variety of other biological tissues to study a wide range of diseases. For instance, it could be used to investigate the tumor microenvironment in cancer models or to study axonal remodeling in demyelinating diseases like multiple sclerosis. The ability to image tissues in a near-native, unstained state makes this technique particularly valuable for studying human tissues obtained postmortem, where genetic labeling is not an option. As the technology matures, cryo-PXCT is poised to become an indispensable tool for researchers seeking to unravel the complex three-dimensional organization of biological tissues in both health and disease.
