A cryo-fixation protocol to study the structure of the synaptonemal complex.

Genetic variability in sexually reproducing organisms results from an exchange of genetic material between homologous chromosomes. The genetic exchange mechanism is dependent on the synaptonemal complex (SC), a protein structure localized between the homologous chromosomes. The current structural models of the mammalian SC are based on electron microscopy, superresolution, and expansion microscopy studies using chemical fixatives and sample dehydration of gonads, which are methodologies known to produce structural artifacts. To further analyze the structure of the SC, without chemical fixation, we have adapted a cryo-fixation method for electron microscopy where pachytene cells are isolated from mouse testis by FACS, followed by cryo-fixation, cryo-substitution, and electron tomography. In parallel, we performed conventional chemical fixation and electron tomography on mouse seminiferous tubules to compare the SC structure obtained with the two fixation methods. We found several differences in the structure and organization of the SC in cryo-fixed samples when compared to chemically preserved samples. We found the central region of the SC to be wider and the transverse filaments to be more densely packed in the central region of the SC.

Immuno-gold Techniques in Biomedical Sciences.

Since their development in the 1960s, immuno-gold techniques have been steadily used in biomedical science, because these techniques are applicable to all kinds of antigens, from viruses to animal tissues. Immuno-gold staining exploits antigen-antibody reactions and is used to investigate locations and interactions of components in the ultrastructure of tissues, cells, and particles. These methods are increasingly used with advanced technologies, such as correlative light and electron microscopy and cryo-techniques. In this protocol, we introduce the principles and technical details of recent advances in this area and discuss their advantages and limitations.

X-ray elemental mapping techniques for elucidating the ecophysiology of hyperaccumulator plants.

Contents Summary 432 I. Introduction 433 II. Preparation of plant samples for X-ray micro-analysis 433 III. X-ray elemental mapping techniques 438 IV. X-ray data analysis 442 V. Case studies 443 VI. Conclusions 446 Acknowledgements 449 Author contributions 449 References 449 SUMMARY: Hyperaccumulators are attractive models for studying metal(loid) homeostasis, and probing the spatial distribution and coordination chemistry of metal(loid)s in their tissues is important for advancing our understanding of their ecophysiology. X-ray elemental mapping techniques are unique in providing in situ information, and with appropriate sample preparation offer results true to biological conditions of the living plant. The common platform of these techniques is a reliance on characteristic X-rays of elements present in a sample, excited either by electrons (scanning/transmission electron microscopy), protons (proton-induced X-ray emission) or X-rays (X-ray fluorescence microscopy). Elucidating the cellular and tissue-level distribution of metal(loid)s is inherently challenging and accurate X-ray analysis places strict demands on sample collection, preparation and analytical conditions, to avoid elemental redistribution, chemical modification or ultrastructural alterations. We compare the merits and limitations of the individual techniques, and focus on the optimal field of applications for inferring ecophysiological processes in hyperaccumulator plants. X-ray elemental mapping techniques can play a key role in answering questions at every level of metal(loid) homeostasis in plants, from the rhizosphere interface, to uptake pathways in the roots and shoots. Further improvements in technological capabilities offer exciting perspectives for the study of hyperaccumulator plants into the future.

Correlative microscopy.

In recent years correlative microscopy, combining the power and advantages of different imaging system, e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all the possible combinations of techniques, light and electron microscopy, have made an especially big step forward and are being implemented in more and more research labs. Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultrastructure and identification of the organelles. It, however, has two severe limitations: the restricted field of view and the fact that no live imaging can be done. On the other hand light microscopy has the advantage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications the large field of view facilitates the identification and location of sparse individual cells in a large context, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to dissect biological events at a submicrometer level. Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mitochondria, in time, then the sample is fixed and the exactly same region is investigated by electron microscopy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either transfections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluorescence labelling of a series of sections can be correlated with the ultrastructure of the individual sections to get 3D information of the distribution of the marked proteins: array tomography. More and more efforts are put in either converting a fluorescence label into an electron dense product or preserving the fluorescence throughout preparation for the electron microscopy. Here, we will review successful protocols and where possible try to extract common features to better understand the importance of the individual steps in the preparation. Further the new instruments and software, intended to ease correlative light and electron microscopy, are discussed. Last but not least we will detail the approach we have chosen for correlative microscopy.

Disruption of the mitochondrial network in a mouse model of Huntington's disease visualized by in-tissue multiscale 3D electron microscopy.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the coding sequence of huntingtin protein. Initially, it predominantly affects medium-sized spiny neurons (MSSNs) of the corpus striatum. No effective treatment is still available, thus urging the identification of potential therapeutic targets. While evidence of mitochondrial structural alterations in HD exists, previous studies mainly employed 2D approaches and were performed outside the strictly native brain context. In this study, we adopted a novel multiscale approach to conduct a comprehensive 3D in situ structural analysis of mitochondrial disturbances in a mouse model of HD. We investigated MSSNs within brain tissue under optimal structural conditions utilizing state-of-the-art 3D imaging technologies, specifically FIB/SEM for the complete imaging of neuronal somas and Electron Tomography for detailed morphological examination, and image processing-based quantitative analysis. Our findings suggest a disruption of the mitochondrial network towards fragmentation in HD. The network of interlaced, slim and long mitochondria observed in healthy conditions transforms into isolated, swollen and short entities, with internal cristae disorganization, cavities and abnormally large matrix granules.

Cellular organization in germ tube tips of Gigaspora and its phylogenetic implications.

Comparative morphology of the fine structure of fungal hyphal tips often is phylogenetically informative. In particular, morphology of the Spitzenkörper varies among higher taxa. To date no one has thoroughly characterized the hyphal tips of members of the phylum Glomeromycota to compare them with other fungi. This is partly due to difficulty growing and manipulating living hyphae of these obligate symbionts. We observed growing germ tubes of Gigaspora gigantea, G. margarita and G. rosea with a combination of light microscopy (LM) and transmission electron microscopy (TEM). For TEM, we used both traditional chemical fixation and cryo-fixation methods. Germ tubes of all species were extremely sensitive to manipulation. Healthy germ tubes often showed rapid bidirectional cytoplasmic streaming, whereas germ tubes that had been disturbed showed reduced or no cytoplasmic movement. Actively growing germ tubes contain a cluster of 10-20 spherical bodies approximately 3-8 µm behind the apex. The bodies, which we hypothesize are lipid bodies, move rapidly in healthy germ tubes. These bodies disappear immediately after any cellular perturbation. Cells prepared with cryo-techniques had superior preservation compared to those that had been processed with traditional chemical protocols. For example, cryo-prepared samples displayed two cell-wall layers, at least three vesicle types near the tip and three distinct cytoplasmic zones were noted. We did not detect a Spitzenkörper with either LM or TEM techniques and the tip organization of Gigaspora germ tubes appeared to be similar to hyphae in zygomycetous fungi. This observation was supported by a phylogenetic analysis of microscopic characters of hyphal tips from members of five fungal phyla. Our work emphasizes the sensitive nature of cellular organization, and the need for as little manipulation as possible to observe germ tube structure accurately.

Characterization of Complex Drug Formulations Using Cryogenic Scanning Electron Microscopy (Cryo-SEM).

The physicochemical properties of complex drug formulations, including liposomes, suspensions, and emulsions, are important for understanding drug release mechanisms, quality control, and regulatory assessment. It is ideal to characterize these complex drug formulations in their native hydrated state. This article describes the characterization of complex drug formulations in a frozen-hydrated state using cryogenic scanning electron microscopy (cryo-SEM). In comparison to other techniques, such as optical microscopy or room-temperature scanning electron microscopy, cryo-SEM combines the advantage of studying hydrated samples with high-resolution imaging capability. Detailed information regarding cryo-fixation, cryo-fracture, freeze-etching, sputter-coating, and cryo-SEM imaging is included in this article. A multivesicular liposomal complex drug formulation is used to illustrate the impact of different cryogenic sample preparation conditions. In addition to drug formulations, this approach can also be applied to biological samples (e.g., cells, bacteria) and soft-matter samples (e.g., hydrogels). © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Cryo-fixation to preserve the native structure of samples using planchettes Alternate Protocol: Cryo-fixation to preserve the native structure of biological samples on sapphire disks Basic Protocol 2: Sample preparation for cross-sectional cryo-SEM imaging Basic Protocol 3: Cryo-SEM imaging and microanalysis.

Cryo-Scanning Electron Microscopy to Study the Freezing Behavior of Plant Tissues.

A cryo-scanning electron microscope (cryo-SEM) is a valuable tool for observing bulk frozen samples to monitor freezing responses of plant tissues and cells. Here, the essential processes of a cryo-SEM to observe freezing behaviors of plant tissue cells are described.

Ultrastructural comparison of dendritic spine morphology preserved with cryo and chemical fixation.

Previously, we showed that cryo fixation of adult mouse brain tissue gave a truer representation of brain ultrastructure in comparison with a standard chemical fixation method (Korogod et al., 2015). Extracellular space matched physiological measurements, there were larger numbers of docked vesicles and less glial coverage of synapses and blood capillaries. Here, using the same preservation approaches, we compared the morphology of dendritic spines. We show that the length of the spine and the volume of its head is unchanged; however, the spine neck width is thinner by more than 30% after cryo fixation. In addition, the weak correlation between spine neck width and head volume seen after chemical fixation was not present in cryo-fixed spines. Our data suggest that spine neck geometry is independent of the spine head volume, with cryo fixation showing enhanced spine head compartmentalization and a higher predicted electrical resistance between spine head and parent dendrite.

Characterization of unusual MgCa particles involved in the formation of foraminifera shells using a novel quantitative cryo SEM/EDS protocol.

Quantifying ion concentrations and mapping their intracellular distributions at high resolution can provide much insight into the formation of biomaterials. The key to achieving this goal is cryo-fixation, where the biological materials, tissues and associated solutions are rapidly frozen and preserved in a vitreous state. We developed a correlative cryo-Scanning Electron Microscopy (SEM)/Energy Dispersive Spectroscopy (EDS) protocol that provides quantitative elemental analysis correlated with spatial imaging of cryo-immobilized specimens. We report the accuracy and sensitivity of the cryo-EDS method, as well as insights we derive on biomineralization pathways in a foraminifer. Foraminifera are marine protozoans that produce Mg-containing calcitic shells and are major calcifying organisms in the oceans. We use the cryo-SEM/EDS correlative method to characterize unusual Mg and Ca-rich particles in the cytoplasm of a benthic foraminifer. The Mg/Ca ratio of these particles is consistently lower than that of seawater, the source solution for these ions. We infer that these particles are involved in Ca ion supply to the shell. We document the internal structure of the MgCa particles, which in some cases include a separate Si rich core phase. This approach to mapping ion distribution in cryo-preserved specimens may have broad applications to other mineralized biomaterials. STATEMENT OF SIGNIFICANCE: Ions are an integral part of life, and some ions play fundamental roles in cell metabolism. Determining the concentrations of ions in cells and between cells, as well as their distributions at high resolution can provide valuable insights into ion uptake, storage, functions and the formation of biomaterials. Here we present a new cryo-SEM/EDS protocol that allows the mapping of different ion distributions in solutions and biological samples that have been cryo-preserved. We demonstrate the value of this novel approach by characterizing a novel biogenic mineral phase rich in Mg found in foraminifera, single celled marine organisms. This method has wide applicability in biology, and especially in understanding the formation and function of mineral-containing hard tissues.

Self-Pressurized Rapid Freezing as Cryo-Fixation Method for Electron Microscopy and Cryopreservation of Living Cells.

Reduction or complete prevention of ice crystal formation during freezing of biological specimens is mandatory for two important biological applications: (1) cryopreservation of living cells or tissues for long-term storage, and (2) cryo-fixation for ultrastructural investigations by electron microscopy. Here, a protocol that is fast, easy-to-use, and suitable for both cryo-fixation and cryopreservation is described. Samples are rapidly cooled in tightly sealed metal tubes of high thermal diffusivity and then plunged into a liquid cryogen. Due to the fast cooling speed and high-pressure buildup internally in the confined volume of the metal tubes, ice crystal formation is reduced or completely prevented, resulting in vitrification of the sample. For cryopreservation, however, a similar principle applies to prevent ice crystal formation during re-warming. A detailed description of procedures for cooling (and re-warming) of biological samples using this technique is provided. © 2018 by John Wiley & Sons, Inc.

Mycolicibacterium smegmatis, Basonym Mycobacterium smegmatis, Expresses Morphological Phenotypes Much More Similar to Escherichia coli Than Mycobacterium tuberculosis in Quantitative Structome Analysis and CryoTEM Examination.

A series of structome analyses, that is, quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level, have already been achieved individually in Exophiala dermatitidis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Myojin spiral bacteria, and Escherichia coli. In these analyses, sample cells were processed through cryo-fixation and rapid freeze-substitution, resulting in the exquisite preservation of ultrastructures on the serial ultrathin sections examined by transmission electron microscopy. In this paper, structome analysis of non pathogenic Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, was performed. As M. smegmatis has often been used in molecular biological experiments and experimental tuberculosis as a substitute of highly pathogenic M. tuberculosis, it has been a task to compare two species in the same genus, Mycobacterium, by structome analysis. Seven M. smegmatis cells cut into serial ultrathin sections, and, totally, 220 serial ultrathin sections were examined by transmission electron microscopy. Cell profiles were measured, including cell length, diameter of cell and cytoplasm, surface area of outer membrane and plasma membrane, volume of whole cell, periplasm, and cytoplasm, and total ribosome number and density per 0.1 fl cytoplasm. These data are based on direct measurement and enumeration of exquisitely preserved single cell structures in the transmission electron microscopy images, and are not based on the calculation or assumptions from biochemical or molecular biological indirect data. All measurements in M. smegmatis, except cell length, are significantly higher than those of M. tuberculosis. In addition, these data may explain the more rapid growth of M. smegmatis than M. tuberculosis and contribute to the understanding of their structural properties, which are substantially different from M. tuberculosis, relating to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance in relation to the ratio of the targets to the corresponding drugs. In addition, data obtained from cryo-transmission electron microscopy examination were used to support the validity of structome analysis. Finally, our data strongly support the most recent establishment of the novel genus Mycolicibacterium, into which basonym Mycobacterium smegmatis has been classified.

The development and validation of micro-CT of large deep frozen specimens.

Repetitive freeze/thaw cycles lead to a progressive loss of structural and molecular integrity in deep frozen specimens. The aim of this study was to evaluate a micro-CT stage, which maintains the cryoconservation of large specimens throughout micro-CT imaging. Deep frozen ovine vertebral segments (-20 °C) were fixed in a micro-CT stage made of expanded polystyrene and cooled with dry ice (0 g, 60 g and 120 g). The temperature inside the stage was measured half-hourly over a time span of three hours with subsequent measurement of surface temperature. The method was validated in a series of 30 deep frozen vertebral specimens and in liver tissue after repetitive micro-CT scanning. Isolation without cooling resulted in defrosting. Cooling with 60 g of dry ice led to a temperature rise inside the stage (max. 5.1 °C) and on the specimen surfaces (max. -3 °C). Cooling with 120 g of dry ice resulted in a significant (p < 0.001) and sufficient lowering of the temperature inside the stage (max. -14 °C) and on the surface of the specimens (max. -13.9 °C). The surface temperature during the subsequent micro-CT validation study did not exceed -16 °C (processing time 1 h 45 min). The resolution was 33 µm isotropic voxel side length, enabling a binarization of bone microstructures. Temperature can reliably be maintained below -10 °C during a micro-CT scan by applying the described technique. The resulting spatial resolution and image quality permits a binarization of bone microstructure.

High-pressure freezing and freeze-substitution fixation reveal the ultrastructure of immature and mature spermatozoa of the plant-parasitic nematode Trichodorus similis (Nematoda; Triplonchida; Trichodoridae).

The spermatozoa from testis and spermatheca of the plant-parasitic nematode Trichodorus similis Seinhorst, 1963 (Nematoda; Triplonchida; Trichodoridae) were studied with transmission electron microscopy (TEM), being the first study on spermatogenesis of a representative of the order Triplonchida and important to unravel nematode sperm evolution. Comprehensive results could only be obtained using high-pressure freezing (HPF) and freeze-substitution instead of chemical fixation, demonstrating the importance of cryo-fixation for nematode ultrastructural research. The spermatozoa from the testis (immature spermatozoa) are unpolarized cells covered by numerous filopodia. They contain a centrally-located nucleus without a nuclear envelope, surrounded by mitochondria. Specific fibrous bodies (FB) as long parallel bundles of filaments occupy the peripheral cytoplasm. No structures resembling membranous organelles (MO), as found in the sperm of many other nematodes, were observed in immature spermatozoa of T. similis. The spermatozoa from the uterus (mature or activated spermatozoa) are bipolar cells with an anterior pseudopod and posterior main cell body (MCB), which include a nucleus, mitochondria and MO appearing as large vesicles with finger-like invaginations of the outer cell membrane, or as large vesicles connected to the inner cell membrane. The peripheral MO open to the exterior via pores. In the mature sperm, neither FBs nor filopodia were observed. An important feature of T. similis spermatozoa is the late formation of MO; they first appear in mature spermatozoa. This pattern of MO formation is known for several other orders of the nematode class Enoplea: Enoplida, Mermithida, Dioctophymatida, Trichinellida but has never been observed in the class Chromadorea.