Oliver Bunk

Maps of biological tissues at subcellular detail are key for understanding how organs function. X-ray nanotomography is a promising alternative to volume electron microscopy: it has the potential to nondestructively image millimeter-sized samples at ultrastructural resolution within a few days. A fundamental barrier is that the intense X-rays required for imaging also deform and disintegrate the tissue samples. Here we show a combination of solutions that overcome this barrier: We used a cryogenic and stable sample stage, tailored nonrigid tomographic reconstruction algorithms and an epoxy resin developed for the nuclear and aerospace industry. Tissue samples were resistant to radiation doses exceeding 1.15 × 10 ¹⁰ Gy, and sub-40 nm isotropic resolution allowed identifying axon bundles, dendrites and synapses in mouse brain tissue without physical sectioning. Using volume electron microscopy, we demonstrate that tissue ultrastructure remains intact after X-ray imaging. Together, this unlocks the potential of X-ray tomography for high-resolution tissue imaging.

Microcalcifications (MC) are observed in various tissues and in relation to several diseases. For breast cancer, recent studies have reported differences in the nature of the MC and correlations to the degree of malignancy of the neoplasm. Here, investigations of benign, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) breast MC using X-ray fluorescence, X-ray absorption spectroscopy and wide-angle X-ray scattering are reported. While Mg has been observed in all MC, only for the benign MC has a rim of crystalline whitlockite been identified as a minor crystalline phase in addition to the major hydroxyapatite (HAP) one. MC in DCIS and IDC tissue exhibit a higher abundance of a high-crystallinity HAP phase in comparison with the less well ordered MC in the benign tissue. Moreover, the distribution of other trace elements in the MC, such as Na, S, Cl, Sr and Y, is observed. For the quantitative analysis of the elemental maps, the experimentally determined sample thickness in each pixel has been incorporated as an additional parameter in the fitting process to account for sample roughness.

Maps of dense subcellular features in biological tissue are the key to understanding the structural basis of organ function. Electron microscopy provides the necessary resolution, yet - as electrons penetrate samples for only a few 100s of nm - requires physical sectioning or ablation, which strongly challenges anatomical investigations of entire organs such as mammalian brains. As demonstrated for the engineering and physical sciences, X-ray nanotomography represents a promising alternative for ultrastructural 3d imaging without physical sectioning ^1–15 . Leveraging the high brilliance of 4th generation synchrotron X-ray sources, it has the potential to non-destructively image mm³-sized samples at ultrastructural resolution within a few days ¹⁶ . A fundamental barrier to application to the life sciences is that, when irradiated with high-intensity X-rays, biological samples deform and ultimately disintegrate, prohibiting reaching sufficient resolution. Here, we introduce a combination of engineering solutions which defeat this barrier for X-ray ptychography ¹⁷ , a coherent diffractive X-ray imaging technique. The solutions include a cryogenic sample stage with high stability, high-precision interferometric positioners and tailored non-rigid tomographic reconstruction algorithms ¹⁸ . Furthermore, adapting an epoxy resin developed for the nuclear and aerospace industry, we demonstrate radiation resistance to X-ray doses exceeding 10 ¹⁰ Gy. The resulting sub-40 nm isotropic resolution makes it possible to densely resolve axon bundles, boutons, dendrites and reliably identify synapses without physical sectioning. Moreover, we validated the X-ray technique using the current gold standard, namely focused ion beam scanning electron microscopy (FIB-SEM) ^19,20 to demonstrate intact ultrastructure in tissue volumes first imaged by X-rays. This unlocks the potential of X-ray tomography for high-resolution tissue imaging, coinciding with the transformative advancements of next-generation synchrotrons worldwide ²¹ .

Type I collagen physiological scaffold for tissue regeneration is considered one of the widely used biomaterials for tissue engineering and medical applications. It is hierarchically organized: five laterally staggered molecules are packed within fibrils, arranged into fascicles and bundles. The structural organization is correlated to the direction and intensity of the forces which can be loaded onto the tissue. For a tissue-specific regeneration, the required macro- and microstructure of a suitable biomaterial has been largely investigated. Conversely, the function of multiscale structural integrity has been much less explored but is crucial for scaffold design and application. In this work, collagen was extracted from different animal sources with protocols that alter its structure. Collagen of tendon shreds excised from cattle, horse, sheep and pig was structurally investigated by wide- and small-angle X-ray scattering techniques, at both molecular and supramolecular scales, and thermo-mechanically with thermal and load-bearing tests. Tendons were selected because of their resistance to chemical degradation and mechanical stresses. The multiscale structural integrity of tendons’ collagen was studied in relation to the animal source, anatomic location and source for collagen extraction.

Breast microcalcifications are a common mammographic finding. Microcalcifications are considered suspicious signs of breast cancer and a breast biopsy is required, however, cancer is diagnosed in only a few patients. Reducing unnecessary biopsies and rapid characterization of breast microcalcifications are unmet clinical needs. In this study, 473 microcalcifications detected on breast biopsy specimens from 56 patients were characterized entirely by Raman mapping and confirmed by X-ray scattering. Microcalcifications from malignant samples were generally more homogeneous, more crystalline, and characterized by a less substituted crystal lattice compared with benign samples. There were significant differences in Raman features corresponding to the phosphate and carbonate bands between the benign and malignant groups. In addition to the heterogeneous composition, the presence of whitlockite specifically emerged as marker of benignity in benign microcalcifications. The whole Raman signature of each microcalcification was then used to build a classification model that distinguishes microcalcifications according to their overall biochemical composition. After validation, microcalcifications found in benign and malignant samples were correctly recognized with 93.5% sensitivity and 80.6% specificity. Finally, microcalcifications identified in malignant biopsies, but located outside the lesion, reported malignant features in 65% of in situ and 98% of invasive cancer cases, respectively, suggesting that the local microenvironment influences microcalcification features. This study confirms that the composition and structural features of microcalcifications correlate with breast pathology and indicates new diagnostic potentialities based on microcalcifications assessment.Significance:Raman spectroscopy could be a quick and accurate diagnostic tool to precisely characterize and distinguish benign from malignant breast microcalcifications detected on mammography.

Breast microcalcifications are a common mammographic finding. Microcalcifications are considered suspicious signs of breast cancer and a breast biopsy is required, however, cancer is diagnosed in only a few patients. Reducing unnecessary biopsies and rapid characterization of breast microcalcifications are unmet clinical needs. In this study, 473 microcalcifications detected on breast biopsy specimens from 56 patients were characterized entirely by Raman mapping and confirmed by X-ray scattering. Microcalcifications from malignant samples were generally more homogeneous, more crystalline, and characterized by a less substituted crystal lattice compared with benign samples. There were significant differences in Raman features corresponding to the phosphate and carbonate bands between the benign and malignant groups. In addition to the heterogeneous composition, the presence of whitlockite specifically emerged as marker of benignity in benign microcalcifications. The whole Raman signature of each microcalcification was then used to build a classification model that distinguishes microcalcifications according to their overall biochemical composition. After validation, microcalcifications found in benign and malignant samples were correctly recognized with 93.5% sensitivity and 80.6% specificity. Finally, microcalcifications identified in malignant biopsies, but located outside the lesion, reported malignant features in 65% of in situ and 98% of invasive cancer cases, respectively, suggesting that the local microenvironment influences microcalcification features. This study confirms that the composition and structural features of microcalcifications correlate with breast pathology and indicates new diagnostic potentialities based on microcalcifications assessment.Significance:Raman spectroscopy could be a quick and accurate diagnostic tool to precisely characterize and distinguish benign from malignant breast microcalcifications detected on mammography.

The supplementary data file contains detailed patient's characteristics including the number of MC detected for each sample, statistical significance calculations, classification and validation results, wide angle X-ray scattering (WAXS) diffractograms, WAXS and small angle X-ray scattering (SAXS) images, mean Raman spectra of different diagnostic categories, box-plot reporting phosphate peak position shift among diagnostic categories, loading of the first principal components emerging from PCA and correlation between Raman variables and a) age, b) BIRADS classification and c) MC size.

The supplementary data file contains detailed patient's characteristics including the number of MC detected for each sample, statistical significance calculations, classification and validation results, wide angle X-ray scattering (WAXS) diffractograms, WAXS and small angle X-ray scattering (SAXS) images, mean Raman spectra of different diagnostic categories, box-plot reporting phosphate peak position shift among diagnostic categories, loading of the first principal components emerging from PCA and correlation between Raman variables and a) age, b) BIRADS classification and c) MC size.

Glycosylation is the process of combining one or more glucose molecules (or other monosaccharides) with molecules of a different nature (which are therefore glycosylated). In biochemistry, glycosylation is catalyzed by several specific enzymes, and assumes considerable importance since it occurs mainly at the expense of proteins and phospholipids which are thus transformed into glycoproteins and glycolipids. Conversely, in diabetes and aging, glycation of proteins is a phenomenon of non-enzymatic nature and thus not easily controlled. Glycation of collagen distorts its structure, renders the extracellular matrix stiff and brittle and at the same time lowers the degradation susceptibility thereby preventing renewal. Based on models detailed in this paper and with parameters determined from experimental data, we describe the glycation of type 1 collagen in bovine pericardium derived bio-tissues, upon incubation in glucose and ribose. With arginine and lysine/hydroxylysine amino acids as the primary sites of glycation and assuming that the topological polar surface area of the sugar molecules determines the glycation rates, we modelled the glycation as a function of time and determined the glycation rate and thus the progression of glycation as well as the resulting volume increase.