Resin-embedded Samples Research Articles

Functional organisation of the endomembrane network in the digestive gland of the Venus flytrap: revisiting an old story with a new microscopy toolbox.

Up-to-date imaging approaches were used to address the spatiotemporal organisation of the endomembrane system in secretory cells of Dionaea muscipula. Different 'slice and view' methodologies were performed on resin-embedded samples to finally achieve a 3D reconstruction of the cell architecture, using ultrastructural tomography, array tomography, serial block face-scanning electron microscopy (SBF-SEM), correlation, and volume rendering at the light microscopy level. Observations of cryo-fixed samples by high-pressure freezing revealed changes of the endomembrane system that occur after trap activation and prey digestion. They provide evidence for an original strategy that adapts the secretory machinery to a specific and unique case of stimulated exocytosis in plant cells. A first secretion peak is part of a rapid response to deliver digestive fluids to the cell surface, which delivers the needed stock of digestive materials 'on site'. The second peak of activity could then be associated with the reconstruction of the Golgi apparatus (GA), endoplasmic reticulum (ER) and vacuolar machinery, in order to prepare for a subsequent round of prey capture. Tubular continuum between ER and Golgi stacks observed on ZIO-impregnated tissues may correspond to an efficient transfer mechanism for lipids and/or proteins, especially for use in rapidly resetting the molecular GA machinery. The occurrence of one vacuolar continuum may permit continuous adjustment of cell homeostasy. The subcellular features of the secretory cells of Dionaea muscipula outline key innovations in the organisation of plant cell compartmentalisation that are used to cope with specific cell needs such as the full use of the GA as a protein factory, and the ability to create protein reservoirs in the periplasmic space. Shape-derived forces of the pleiomorphic vacuole may act as signals to accompany the sorting and entering flows of the cell.

Lignans in Knotwood of Norway Spruce: Localisation with Soft X-ray Microscopy and Scanning Transmission Electron Microscopy with Energy Dispersive X-ray Spectroscopy

Lignans are bioactive compounds that are especially abundant in the Norway spruce (Picea abies L. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and imaging techniques, we were able to quantify, qualify and localise the easily extractable lignans in the xylem tissue. The knotwood samples contained 15 different lignans according to the gas chromatography-mass spectrometry analysis. They comprised 16% of the knotwood dry weight and 82% of the acetone extract. The main lignans were found to be hydroxymatairesinols HMR1 and HMR2. Cryosectioned and resin-embedded ultrathin sections of the knotwood were analysed with scanning transmission X-ray microscopy (STXM). Cryosectioning was found to retain only lignan residues inside the cell lumina. In the resin-embedded samples, lignan was interpreted to be unevenly distributed inside the cell lumina, and partially confined in deposits which were either readily present in the lumina or formed when OsO4 used in staining reacted with the lignans. Furthermore, the multi-technique characterisation enabled us to obtain information on the chemical composition of the structural components of knotwood. A simple spectral analysis of the STXM data gave consistent results with the gas chromatographic methods about the relative amounts of cell wall components (lignin and polysaccharides). The STXM analysis also indicated that a torus of a bordered pit contained aromatic compounds, possibly lignin.

Full-repertoire comparison of the microscopic objects composing the human gut microbiome with sequenced and cultured communities.

The study of the human gut microbiome is essential in microbiology and infectious diseases as specific alterations in the gut microbiome might be associated with various pathologies, such as chronic inflammatory disease, intestinal infection and colorectal cancer. To identify such dysregulations, several strategies are being used to create a repertoire of the microorganisms composing the human gut microbiome. In this study, we used the "microscomics" approach, which consists of creating an ultrastructural repertoire of all the cell-like objects composing stool samples from healthy donors using transmission electron microscopy (TEM). We used TEM to screen ultrathin sections of 8 resin-embedded stool samples. After exploring hundreds of micrographs, we managed to elaborate ultrastructural categories based on morphological criteria or features. This approach explained many inconsistencies observed with other techniques, such as metagenomics and culturomics. We highlighted the value of our culture-independent approach by comparing our microscopic images to those of cultured bacteria and those reported in the literature. This study helped to detect "minimicrobes" Candidate Phyla Radiation (CPR) for the first time in human stool samples. This "microscomics" approach is non-exhaustive but complements already existing approaches and adds important data to the puzzle of the microbiota.

Evaluation of secondary ions related to plant tissue using least absolute shrinkage and selection operator.

With regard to life sciences, it is important to understand biological functions such as metabolic reactions at the cellular level. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) that can provide chemical mappings at 100 nm lateral resolutions is useful for obtaining three-dimensional maps of biological molecules in cells and tissues. TOF-SIMS spectra generally contain several hundred to several thousand secondary ion peaks that provide detailed chemical information. In order to manage such a large number of peaks, data analysis methods such as multivariate analysis techniques have been applied to TOF-SIMS data of complex samples. However, the interpretation of the data analysis results is sometimes still difficult, especially for biological samples. In this study, TOF-SIMS data of resin-embedded plant samples were analyzed using one of the sparse modeling methods, least absolute shrinkage and selection operator (LASSO), to directly select secondary ions related to biological structures such as cell walls and nuclei. The same sample was measured by optical microscopy and the same measurement area as TOF-SIMS was extracted in order to prepare a target image for LASSO. The same area of the TOF-SIMS and microscope data were fused to evaluate the influence of the image fusion on the TOF-SIMS spectrum information using principal component analysis. Specifically, the authors examined onion mycorrhizal root colonized with Gigaspora margarita (an arbuscular mycorrhizal fungus). The results showed that by employing this approach using LASSO, important secondary ions from biological samples were effectively selected and could be clearly distinguished from the embedding resin.

Scanning Electron Microscopy of Bone.

This chapter describes methods for preparing samples of bone and bone cells for scanning electron microscopy (SEM). Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. Samples may have 3D detail in a 3D surface, or be topography-free, polished or micromilled, resin-embedded block surfaces, or resin casts of space compartments surrounded by bone matrix. Methods for cells include fixation, drying, looking at undersides of bone cells, and metallic conductive coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralized matrix is described in detail. Attention is given especially to methods for 3D BSE SEM imaging of bone samples. Recommendations are made for the types of resin embedding for BSE SEM imaging. Correlated confocal and SEM imaging of PMMA embedded bone requires the use of glycerol to coverslip. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualizing fluorescent mineralizing front labels such as calcein and tetracyclines. Making spatial casts from PMMA or other resin-embedded samples is an important use of this material. Correlation with other imaging means, including microradiography and microtomography is important. Shipping wet bone samples between labs is best done in glycerol. Control of the vacuum pressure in the SEM sample chamber (now generally available) can be used to eliminate "charging" problems which were common, for example, with large, complex, cancellous bone samples.

Penetration model for chemical reactivation for resin-embedded green fluorescent protein imaging.

.In the so-called surface microscopy, serial block-face imaging is combined with mechanic sectioning to obtain volumetric imaging. While mapping a resin-embedded green fluorescent protein (GFP)-labeled specimen, it has been recently reported that an alkaline buffer is used to chemically reactivate the protonated GFP molecules, and thus improve the signal-to-noise ratio. In such a procedure, the image quality is highly affected by the penetration rate of a solution. We propose a reliable penetration model to describe the penetration process of the solution into the resin. The experimental results are consistent with the parameters predicted using this model. Thus, this model provides a valuable theoretical explanation and aids in optimizing the system parameters for mapping resin-embedded GFP biological samples.

Cuticle matrix imaging by histochemistry, fluorescence, and electron microscopy

Biomineralized structures consist of an organic matrix and mineral constituents. Imaging of the mineralized biological tissues is demanding due to specific requirements for the preservation and visualization of chemically different constituents and due to sectioning difficulties. In this study, a characterization of the cuticular matrix of the crustacean exoskeleton was performed by a combination of microscopic methods, aiming to obtain spatial information on the matrix composition. Histochemical procedures were performed and compared in artificially decalcified and non-decalcified samples, in paraffin and resin sections. Wheat germ agglutinin (WGA) lectin-gold conjugate and a fluorescent chitin-binding probe were used to localize chitin in paraffin and resin sections of samples prepared by different fixations. Calcified regions of the matrix were determined by histochemical staining of aldehyde-fixed, methanol-fixed, and resin-embedded samples and by scanning electron microscopy with energy dispersive X-...

Vertical Bone Augmentation Using Ring Technique with Three Different Materials in the Sheep Mandible Bone.

The purpose of this study was to examine the volumetric alterations and osseointegration properties in the augmented area of the ring technique using different types of bone graft material in sheep mandible bone. Three different materials (columnar forms, 7-mm diameter, 3-mm height) were stabilized using dental implants with a turned surface in the mandible bone of Finnish Dorset cross-bred sheep: group A, autogenous bone; group B, bovine bone; group C, biphasic bone substitute. Animals were euthanized after 5 weeks (N = 6). Three-dimensional image data by digital oral scanner were taken at the surgery and sacrifice, and the volume alteration of the material was calculated. The bone samples were fixed in formalin and dehydrated in ethanol. Resin-embedded samples were subjected to non-decalcified ground sectioning, and histologic and histomorphometric analysis (bone and material area alteration, bone-to-implant contact [BIC]) were done. In three-dimensional (3D) image analysis, group A showed a statistically higher percentage of remaining materials compared with groups B and C. The histologic observation showed no new bone formations around the implants in all groups, especially at the maxillary site of the implant in the augmented area. In histomorphometric analysis, group A showed a statistically higher percentage of bone area (BA) compared with groups B and C; however, in all groups, bone-to-implant contact (BIC) showed low values, and there were no statistical differences between groups. The results of this study suggested that the autogenous bone maintained bone volume around the dental implant using the ring technique, and the impact of surface properties was of some importance; osseointegration with the turned surface in the augmented area showed low BIC values in all groups.

Backscattered electron imaging of resin-embedded sections.

Scanning electron microscopes have longer focal depths than transmission electron microscopes and enable visualization of the three-dimensional (3D) surface structures of specimens. While scanning electron microscopy (SEM) in biological research was generally used for the analysis of bulk specimens until around the year 2000, more recent instrumental advances have broadened the application of SEM; for example, backscattered electron (BSE) signals under low accelerating voltages allow block-face and section-face images of tissues embedded in resin to be acquired. This technical breakthrough has led to the development of novel 3D imaging techniques including focused ion beam SEM, serial-block face SEM and serial section SEM. Using these new techniques, the 3D shapes of cells and cell organelles have been revealed clearly through reconstruction of serial tomographic images. In this review, we address two modern SEM techniques: section-face imaging of resin-embedded tissue samples based on BSE observations, and serial section SEM for reconstruction of the 3D structures of cells and organelles from BSE-mode SEM images of consecutive ultrathin sections on solid substrates.

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

Chemical reactivation of fluorescein isothiocyanate immunofluorescence-labeled resin-embedded samples.

Resin embedding is widely used and facilitates microscopic imaging of biological tissues. In contrast, quenching of fluorescence during embedding process hinders the application of resin embedding for imaging of fluorescence-labeled samples. For samples expressing fluorescent proteins, it has been demonstrated that the weakened fluorescence could be recovered by reactivating the fluorophore with alkaline buffer. We extended this idea to immunofluorescence-labeling technology. We showed that the fluorescence of pH-sensitive fluorescein isothiocyanate (FITC) was quenched after resin embedding but reactivated after treating by alkaline buffer. We observed 138.5% fluorescence preservation ratio of reactivated state, sixfold compared with the quenched state in embedding resin, which indicated its application for fluorescence imaging of high signal-to-background ratio. Furthermore, we analyzed the chemical reactivation mechanism of FITC fluorophore. This work would show a way for high-resolution imaging of immunofluorescence-labeled samples embedded in resin.

TriCLEM: Combining high-precision, room temperature CLEM withcryo-fluorescence microscopy to identify very rare events.

Fiducial-based correlation of fluorescence and electron microscopy data from high-pressure frozen and resin-embedded samples allows for high-precision localization of fluorescent signals to subcellular ultrastructure. Here we introduce the triCLEM procedure to facilitate the identification of very rare events for high-precision correlation. We present a detailed protocol to screen high-pressure frozen cell monolayers on sapphire disks for very rare signals by cryo-fluorescence microscopy, relocate the cells of interest after freeze substitution and Lowicryl embedding, and perform fiducial-based correlation of the identified fluorescent signals to high-magnification electron tomograms. We show the applicability of the protocol to localize and image damaged mitochondria marked by the presence of Parkin, a protein involved in initiating mitophagy. We discuss how this extension to previously published fiducial-based correlation procedures has potential to both allow identifying very rare events and assess the quality of preservation in high-pressure frozen samples.

Investigation of the structure of a functionally intact xenopericardial bioconduit after long-term implantation

to investigate the cellular composition of a functionally intact xenopericardial valve in a recipient with acquired mitral defect after long-term implantation. A Uniline bioconduit (BC) ('Neocor', Kemerovo) removed from the heart in the mitral position at 7.2 years after implantation was investigated. Heart valve leaflets were fixed in a buffered 4% paraformaldehyde solution and imbedded in paraffin or epoxy resin. Slices made from the paraffin samples were stained with hematoxylin and eosin or underwent immunohistochemical (IHC) examination for typing endothelial cells, smooth muscle cells, macrophages, fibroblasts, and T and B lymphocytes. The epoxy resin-embedded samples were examined using light and scanning electron microscopy according to the original procedure. For this, the samples were ground and polished, then stained with toluidine blue and basic fuchsin or contrasted with uranyl acetate and lead citrate. Different cell types were found in the outer layers of heart valve leaflets. IHC showed that endothelial cells, macrophages, smooth muscle cells, and fibroblasts were present in the samples. A relationship was found between the degree of degenerative changes in the BC surface and the magnitude of cellular infiltration in xenotissue. This paper debates whether impaired integrity of the surface leaflet layers plays a trigger role in structural dysfunctions of the implanted valves and whether BC endothelialization has a protective effect, which can considerably reduce the immunogenicity of xenotussie and prevent the penetration of recipient cells. The paper shows that it is expedient to modify the surface of the heart valve leaflets in order to create favorable conditions for the attachment and function of endothelial progenitor cells.

3-D EM exploration of the hepatic microarchitecture - lessons learned from large-volume in situ serial sectioning.

To-date serial block-face scanning electron microscopy (SBF-SEM) dominates as the premier technique for generating three-dimensional (3-D) data of resin-embedded biological samples at an unprecedented depth volume. Given the infancy of the technique, limited literature is currently available regarding the applicability of SBF-SEM for the ultrastructural investigation of tissues. Herein, we provide a comprehensive and rigorous appraisal of five different SBF-SEM sample preparation protocols for the large-volume exploration of the hepatic microarchitecture at an unparalleled X, Y and Z resolution. In so doing, we qualitatively and quantitatively validate the use of a comprehensive SBF-SEM sample preparation protocol, based on the application of heavy metal fixatives, stains and mordanting agents. Employing the best-tested SBF-SEM approach, enabled us to assess large-volume morphometric data on murine parenchymal cells, sinusoids and bile canaliculi. Finally, we integrated the validated SBF-SEM protocol with a correlative light and electron microscopy (CLEM) approach. The combination of confocal scanning laser microscopy and SBF-SEM provided a novel way to picture subcellular detail. We appreciate that this multidimensional approach will aid the subsequent research of liver tissue under relevant experimental and disease conditions.

Basal body structure in Trichonympha.

Trichonympha is a symbiotic flagellate of many species of termites and of the wood-feeding cockroach. Remarkably, this unicellular organism harbors up to over ten thousand flagella on its surface, which serve to propel it through the viscous environment of the host hindgut. In the 1960s, analysis of resin-embedded Trichonympha samples by electron microscopy revealed that the basal bodies that give rise to these flagella are exceptionally long, with a proximal, cartwheel-bearing, region some 50 times longer than that of regular centrioles. In recent years, this salient feature has prompted the analysis of the 3D architecture of Trichonympha basal bodies in the native state using cryo-electron tomography. The resulting ~40 Å resolution map of the basal body proximal region revealed a number of novel features that may be conserved in centrioles of other systems. These include proximal–distal polarity of the pinhead structure that links the cartwheel to centriolar microtubules, as well as of the linker between the A and the C microtubules. Moreover, this work demonstrated that the cartwheel is made of stacked ring-like structures that likely each comprise 18 molecules of SAS-6 proteins.

High-resolution line-scan analysis of resin-embedded sediments using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS)

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) line-scanning is a promising technique for producing high-resolution (μm-scale) geochemical records on resin-embedded sediments. However, this approach has not yet been thoroughly tested on sediment samples of known elemental compositions. Here, we address this through the analysis of resin-embedded quartz, calcite, and clay (montmorillonite) sediments spiked with Al, V, Mo, and Ba across a range of concentrations. LA-ICP-MS spot analyses on these samples were compared to data independently obtained by conventional techniques: solution nebulization-ICP-optical emission spectroscopy (SN-ICP-OES), SN-ICP-MS, and X-Ray Fluorescence on fused glass beads. Data were reported as (log-)ratios of the analyte elements normalized to Ca, Si, and Al for calcite, quartz and clay respectively to correct for varying ablation yields. Our LA-ICP-MS data demonstrate close agreement to within 6% of the reference values determined by the conventional techniques, with high correlation coefficients (R2≈1.0) across the full range of concentrations, and high precision (<3% relative standard deviations after three repeated analyses). Barium was hosted in both aluminosilicates and carbonates in the clay matrix, giving different yields (elemental fractionation), and leading to variable accuracy (mean deviation of 15%). A selection of the spiked sediments was used to produce artificial laminated sediments to further test the effects of line scanning on LA-ICP-MS accuracy and precision in resin-embedded samples. It appears that LA-ICP-MS line-scan analyses have good accuracy (deviations from the reference values generally <5%) and high precision (relative standard deviations of repeated analyses on the same sample generally <5%) for our target elements. Moreover, the LA-ICP-MS line-scanning closely records the alternating geochemical profiles of our artificial laminations. However, calcium showed a clear tailing signal at the transition from a calcite to a quartz layer, indicating that geochemical signals can be smeared at transitions to sediment layers devoid of analyte element(s). We also analyzed resin-embedded natural sediments for comparison of LA-ICP-MS line-scan data to parallel sub-samples measured by SN-ICP-OES and SN-ICP-MS. In general, the results from LA-ICP-MS line-scans of natural sediments correspond well with reference values and with high reproducibility, corroborating the results of the artificial laminations. The results for Ca, V, Mn, Sr, Mo, Ba, and U (in log-ratios to Al) suggest that these elements can be analyzed semi-quantitatively, showing correlation coefficients (R2) ranging from 0.59 to 0.85 compared to conventional analytical techniques. However, we show that using non-matrix matched calibration standards such as NIST SRM 610 may induce deviations from the LA-ICP-MS values to the reference values. Consequently, we recommend a simple protocol for further data correction in which binned-mean LA-ICP-MS values are calibrated to a parallel series of discrete samples analyzed by these conventional techniques.

Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy.

Block-face imaging is a scanning electron microscopic technique which enables easier acquisition of serial ultrastructural images directly from the surface of resin-embedded biological samples with a similar quality to transmission electron micrographs. In the present study, we analyzed the three-dimensional architecture of podocytes using serial block-face imaging. It was previously believed that podocytes are divided into three kinds of subcellular compartment: cell body, primary process, and foot process, which are simply aligned in this order. When the reconstructed podocytes were viewed from their basal side, the foot processes were branched from a ridge-like prominence, which was formed on the basal surface of the primary process and was similar to the usual foot processes in structure. Moreover, from the cell body, the foot processes were also emerged via the ridge-like prominence, as found in the primary process. The ridge-like prominence anchored the cell body and primary process to the glomerular basement membrane, and connected the foot processes to the cell body and primary process. In conclusion, serial block-face imaging is a powerful tool for clear understanding the three-dimensional architecture of podocytes through its ability to reveal novel structures which were difficult to determine by conventional transmission and scanning electron microscopes alone.

Fixation-resistant photoactivatable fluorescent proteins for CLEM.

Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they cease to function following heavy fixation, hindering advanced applications such as correlative light and electron microscopy. Here we report engineered variants of the photoconvertible Eos fluorescent protein that function normally in heavily fixed (0.5–1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

Isolation, cryotomography, and three-dimensional reconstruction of centrioles.

Centrioles and basal bodies (referred to hereafter as centrioles for simplicity) are microtubule-based cylindrical organelles that are typically ∼450-nm long and ∼250nm in diameter. The centriole is composed of three distinct regions: the distal part characterized by microtubule doublets, the central core that harbors microtubule triplets, which are also present in the proximal part that also contains the cartwheel, a structure crucial for centriole assembly. The cartwheel was initially revealed by conventional electron microscopy of resin-embedded samples and is thought to impart the near universal ninefold symmetry of centrioles. Deciphering the native architecture of the cartwheel has proven challenging owing to its small dimensions and the difficulties in isolating it. Here, we present a method to purify and analyze the structure of the exceptionally long Trichonympha centriole by cryotomography and subtomogram averaging. Using this method, we revealed the native architecture of the proximal cartwheel-containing region at ∼40Å-resolution. This method can be applied as a general strategy for uncovering the structure of centrioles in other species.