Freeze Substitution Research Articles

Ultrastructure of Ehrlichia mineirensis, a new member of the Ehrlichia genus

Recently, we reported the in vitro isolation and the molecular characterization of a new species of Ehrlichia (Ehrlichia mineirensis) from haemolymph of Brazilian Rhipicephalus (Boophilus) microplus ticks. This organism shows an ortholog of Ehrlichia canis major immunogenic protein gp36 with a new structure of tandem repeats. In the present study, we used electron microscopy (high pressure freezing and freeze substitution preparative techniques) to characterize morphologically this new agent growing in IDE8 tick cells. The results showed that E. mineirensis shares ultrastructural features with other members of the genus Ehrlichia (Ehrlichia muris, E. canis and Ehrlichia chaffeensis); typical parasitophorous vacuoles (morulae) contain electron-dense and reticulated Ehrlichiae embedded inside a fibrillar matrix. We observed the characteristic Gram-negative-type cell wall composed of both cytoplasmic and rippled outer membrane. We found organisms undergoing binary fission and rarely altered cells with unusual invagination of the cytoplasmic membrane.

Feasibility of high pressure freezing with freeze substitution after long‐term storage in chemical fixatives

Fixation of biological samples is an important process especially related to histological and ultrastructural studies. Chemical fixation was the primary method of fixing tissue for transmission electron microscopy for many years, as it provides adequate preservation of the morphology of cells and organelles. High pressure freezing (HPF) and freeze substitution (FS) is a newer alternative method that rapidly freezes non-cryoprotected samples that are then slowly heated in the FS medium, allowing penetration of the tissue to insure adequate fixation. This study addresses several issues related to tissue preservation for electron microscopy. Using mice liver tissue as model the difference between samples fixed chemically or with HPF immediately after excision, or stored before chemical or HPF fixation were tested with specific focus on the nuclear membrane. Findings are that immediate HPF is the method of choice compared to chemical fixation. Of the chemical fixatives, immediate fixation with 2.5% glutaraldehyde (GA)/formaldehyde (FA) is the best in preserving membrane morphology, 2.5% GA can be used as alternative for stored and then chemically processed samples, with 10% formalin being suitable as a storage medium only if followed by HPF fixation. Overall, storage leads to lower ultrastructural preservation, but HPF with FS can minimize these artifacts relative to other processing protocols.

Novel features of Brassica napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning

Induction of embryogenesis from isolated microspore cultures is a complex experimental system where microspores undergo dramatic changes in developmental fate. After ~40 years of application of electron microscopy to the study of the ultrastructural changes undergone by the induced microspore, there is still room for new discoveries. In this work, high pressure freezing and freeze substitution (HPF/FS), the best procedures known to date for ultrastructural preservation, were used to process Brassica napus microspore cultures covering all the stages of microspore embryogenesis. Analysis of these cultures by electron microscopy revealed massive processes of autophagy exclusively in embryogenic microspores, but not in other microspore-derived structures also present in cultures. However, a significant part of the autophagosomal cargo was not recycled. Instead, it was transported out of the cell, producing numerous deposits of extracytoplasmic fibrillar and membranous material. It was shown that commitment of microspores to embryogenesis is associated with both massive autophagy and excretion of the removed material. It is hypothesized that autophagy would be related to the need for a profound cytoplasmic cleaning, and excretion would be a mechanism to avoid excessive growth of the vacuolar system. Together, the results also demonstrate that the application of HPF/FS to the study of the androgenic switch is the best option currently available to identify the complex and dramatic ultrastructural changes undergone by the induced microspore. In addition, they provide significant insights to understand the cellular basis of induction of microspore embryogenesis, and open a new door for the investigation of this intriguing developmental pathway.

THE MINERAL RELATIONS OF THE LEMMA OF PHALARIS CANARIENSIS L., WITH PARTICULAR REFERENCE TO ITS SILICIFIED MACROHAIRS

ABSTRACT The mineral relations of the lemma of Phalaris canariensis L. were investigated using standard chemical analysis and, at the ultrastructural level, using freeze substitution followed by energy dispersive X-ray analysis. Potassium, calcium, magnesium and chloride concentrations in the whole lemma declined following emergence of the panicle, as did tissue water content. Lemma dry weight doubled between emergence and inflorescence maturity. Using energy dispersive analysis silicon, potassium, chlorine and phosphorus were detectable in the lemma tissues. In a more detailed study of macrohairs originating from the outer epidermis of the lemma, silicon was shown to enter the hair walls soon after emergence. The cell walls appeared to be the major route for soluble silicon movement in the hairs. Phosphorus was mainly located in the hair cytoplasm, while potassium and chlorine were present in the vacuole, cytoplasm and cell wall. The mechanism of silica deposition in the hairs is discussed.

Cryo-electron tomography reveals four-membrane architecture of the Plasmodium apicoplast

BackgroundThe apicoplast is a plastid organelle derived from a secondary endosymbiosis, containing biosynthetic pathways essential for the survival of apicomplexan parasites. The Toxoplasma apicoplast clearly possesses four membranes but in related Plasmodium spp. the apicoplast has variably been reported to have either three or four membranes.MethodsCryo-electron tomography was employed to image merozoites of Plasmodium falciparum and Plasmodium berghei frozen in their near-native state. Three-dimensional reconstructions revealed the number of apicoplast membranes and the association of the apicoplast with other organelles. Routine transmission electron microscopy of parasites preserved by high-pressure freezing followed by freeze substitution techniques was also used to analyse apicoplast morphology.ResultsCryo-preserved parasites showed clearly four membranes surrounding the apicoplast. A wider gap between the second and third apicoplast membranes was frequently observed. The apicoplast was found in close proximity to the nucleus and to the rhoptries. The apicoplast matrix showed ribosome-sized particles and membranous whorls.ConclusionsThe Plasmodium apicoplast possesses four membranes, as do the apicoplasts of other apicomplexan parasites. This is consistent with a four-membraned secondary endosymbiotic plastid ancestor.

Homogenization ofPseudomonas aeruginosaPAO1 biofilms visualized by freeze-substitution electron microscopy

A knowledge of the mechanical properties of bacterial biofilms is required to more fully understand the processes of biofilm formation such as initial adhesion or detachment. The main contribution of this article is to demonstrate the use of homogenization techniques to compute mechanical parameters of Pseudomonas aeruginosa PAO1 biofilms. For this purpose, homogenization techniques are used to analyze freeze substitution electron micrographs of the biofilm cross-sections. The concept of a representative volume element and the study about his representativeness allows us to determine the optimal size in order to analyze these biofilm images. Results demonstrate significant heterogeneities with respect to stiffness and these can be explained by varying cell density distribution throughout the bacterial biofilms. These stiffness variations lead to different mechanical properties along the height of the biofilm. Moreover, a numerical shear stress test shows the impact of these heterogeneities on the detachment process. Several modes of detachment are highlighted according to the local strain energy in the different parts of the biofilm. Knowing where, and how, a biofilm may detach will allow better prediction of accumulation and biomass detachment.

Analysis of human cytomegalovirus secondary envelopment by advanced electron microscopy

Electron microscopy (EM) allows visualization of viruses in fixed cells with high resolution. High-pressure freezing for sample fixation in combination with freeze substitution and embedding in resin improves significantly the preservation of cellular structures and specifically of membranes. This advancement allows better visualization of human cytomegalovirus (HCMV) morphogenesis occurring at membranes. To obtain comprehensive information on viral phenotypes from ultrastructural images it is important to also quantify morphological phenotypes. This again can be much refined by three-dimensional visualization after serial sectioning. For elucidation of dynamic processes three-dimensional tomography is extremely helpful. We analysed interaction of HCMV particles with host cell membranes during final envelopment. Both wild-type virus and a viral mutant with impaired envelopment were analysed in fibroblasts, but also using in vivo relevant human endothelial cells and macrophages. The quantification of the EM data showed similar ultrastructural phenotypes regarding the envelopment efficiency in the different cell types indicating similar mechanisms in late stages of virus morphogenesis. Furthermore, thorough analysis of the viral assembly complex (AC) - a virus-induced cytosolic structure - by using three-dimensional visualization techniques combined with a quantitative analysis revealed that the events of final envelopment are equally distributed within the AC irrespective of different local membrane composition.

Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves

The anatomical localization of caffeine within young Camellia sinensis leaves was investigated using immunohistochemical methods and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in resin was deemed the best protocol as it retained most of the caffeine and allowed for the samples to be sectioned with ease. Immunohistochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localized and accumulated within vascular bundles, mainly the precursor phloem. With the use of a pressure bomb, xylem sap was collected using a micro syringe. The xylem sap was analyzed by thin-layer chromatography and the presence of caffeine was determined. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defense against various pathogens and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine's localization.

Insight into the ultrastructural organisation of sporulated oocysts of Eimeria nieschulzi (Coccidia, Apicomplexa)

Sporulated oocysts of Eimeria contain four sporocysts with two sporozoites each and a sporocyst residuum. The developing sporozoites are protected by the sporocyst wall and the robust double-layered oocyst wall. Because of problems with conventional fixatives, high-pressure freezing, followed by freeze substitution was used to achieve optimal ultrastructural preservation of oocysts, sporocysts and sporozoites. After embedding in Epon®, ultrathin sections were examined by electron microscopy to select specific oocyst regions for further investigation by electron tomography (ET). ET allows high-resolution three-dimensional views of subcellular structures within the oocysts and sporocysts. Analysis of several 300 nm sections by ET revealed a network of small tubular structures with a diameter of 70-120 nm inside the sporocysts which is decribed here for the first time. This network connects the residual body in a sporocyst with the endoplasmic reticulum (ER) of the surrounding sporozoites. The network consists of membrane-bound tubules that contain vesicles but no larger organelles like mitochondria. These tubules, named "sporocord", may have a function similar to an "umbilical cord" providing the sporozoites with metabolites for long-term survival. Small vesicular structures inside the ER of the sporozoites, multivesicular structures inside the residual bodies and vesicles in the tubules support this hypothesis.

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.

Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes

Despite the remarkable advances in electron microscopy, the difficulty in preserving the ultrastructural details of many plant cells is the major limitation to exploiting the full potential of this technology. The very nature of plant cells, including their hydrophobic surfaces, rigid cell walls and large vacuoles, make them recalcitrant to the efficient exchange of reagents that are crucial to preserving their fine structure. Achieving ultrastructural preservation while protecting the antigenicity of molecular epitopes has proven difficult. Here we describe two methods that provide good ultrastructural detail in plant cells while preserving the binding capacity of carbohydrate and protein epitopes. The first is a traditional, chemical-based protocol used to prepare developing grass (cereal) grain for electron microscopy and to locate carbohydrates as they are deposited using immunogold labeling. The second uses cryofixation techniques, including high-pressure freezing and freeze substitution, to prepare delicate, tip-growing pollen tubes and to locate the intracellular site of a polysaccharide synthase. Both procedures can take as long as 2 weeks to achieve results, but there is scope to fast-track some steps depending on the physical characteristics of the material being processed.

On the ultrastructural organization of Trypanosoma cruzi using cryopreparation methods and electron tomography

The structural organization of Trypanosoma cruzi has been intensely investigated by different microscopy techniques. At the electron microscopy level, bi-dimensional analysis of thin sections of chemically fixed cells has been one of the most commonly used techniques, despite the known potential of generating artifacts during chemical fixation and the subsequent steps of sample preparation. In contrast, more sophisticated and elaborate techniques, such as cryofixation followed by freeze substitution that are known to preserve the samples in a more close-to-native state, have not been widely applied to T. cruzi. In addition, the 3D characterization of such cells has been carried out mostly using 3D reconstruction from serial sections, currently considered a low resolution technique when compared to electron tomography (ET). In this work, we re-visited the 3D ultrastructure of T. cruzi using a combination of two approaches: (1) analysis of both conventionally processed and cryofixed and freeze substituted cells and (2) 3D reconstruction of large volumes by serial electron tomography. The analysis of high-pressure frozen and freeze substituted parasites showed novel characteristics in a number of intracellular structures, both in their structure and content. Organelles generally showed a smooth and regular morphology in some cases presenting a characteristic electron dense content. Ribosomes and new microtubule sets showed an unexpected localization in the cell body. The improved preservation and imaging in 3D of T. cruzi cells using cryopreparation techniques has revealed some novel aspects of the ultrastructural organization of this parasite.

Utilization of cellulose microcapillary tubes as a model system for culturing and viral infection of mammalian cells

Cryofixation by high-pressure freezing (HPF) and freeze substitution (FS) gives excellent preservation of intracellular membranous structures, ideal for ultrastructural investigations of virus infected cells. Conventional sample preparation methods of tissue cultured cells can however disrupt the association between neighboring cells or of viruses with the plasma membrane, which impacts upon the effectiveness whereby virus release from cells can be studied. We established a system for virus infection and transmission electron microscopy preparation of mammalian cells that allowed optimal visualization of membrane release events. African horse sickness virus (AHSV) is a nonenveloped virus that employs two different release mechanisms from mammalian cells, i.e., lytic release through a disrupted plasma membrane and a nonlytic budding-type release. Cellulose microcapillary tubes were used as support layer for culturing Vero cells. The cells grew to a confluent monolayer along the inside of the tubes and could readily be infected with AHSV. Sections of the microcapillary tubes proved easy to manipulate during the HPF procedure, showed no distortion or compression, and yielded well preserved cells in their native state. There was ample cell surface area available for visualization, which allowed detection of both types of virus release at the plasma membrane at a significantly higher frequency than when utilizing other methods. The consecutive culturing, virus infection and processing of cells within microcapillary tubes therefore represent a novel model system for monitoring intracellular virus life cycle and membrane release events, specifically suited to viruses that do not grow to high titers in tissue culture.

The ability of the Antarctic nematode Panagrolaimus davidi to survive intracellular freezing is dependent upon nutritional status

The Antarctic nematode Panagrolaimus davidi is the best documented example of an animal surviving intracellular freezing and the only animal so far shown to survive such freezing throughout its tissues. However, a recent study found that after exposure to a freezing stress that produced intracellular freezing in a proportion of nematodes, the resulting survival levels could be explained if those nematodes that froze intracellularly had died. We have thus re-examined the survival of intracellular freezing in this nematode. The ability to survive a freezing exposure that is likely to produce intracellular freezing (freezing at -10 °C) declines with culture age. In cultures that are fed regularly, the ability to survive freezing at -10 °C increases, but in starved cultures freezing survival declines. Survival of intracellular freezing in fed cultures was confirmed using cryomicroscopy, staining of cells with vital dyes and by freeze substitution and transmission electron microscopy. We have thus confirmed that P. davidi can survive intracellular freezing and shown that this ability is dependent upon them being well fed. The effect of culture conditions on the nutrient status of the nematodes should thus be an important factor in the design of experiments.

Preserving Fluorescent Protein Signal after High Pressure Freezing and Freeze Substitution of Arabidopsis Leaf Tissue

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Effects of High-Pressure Low-Temperature Freezing&Thawing Process on Potato Qualities

The research about the high pressure technology to preserve foodstuff has been studied for a longer time, but there were few of papers about the research of the combination effects of high-pressure-freezing&thawing process on food qualities. To examine the combination effects of high-pressure-freezing&thawing process on food qualities, potato was chosen as model object. The experiments were conducted with pressure-shift-freezing processes at 0.1, 100~200MPa, and pressure-assisted-thawing processes at 0.1, 200MPa. Texture analysis was as the key index to evaluate the combination effects of high-pressure-freezing&thawing process on food qualities by Texture Analyzer. At the same time the frozen samples treated by pressure-shift-freezing process were histologically analyzed using the isothermal freezing substitution technique to contrast the pressure effects on the size and shape of ice crystal. The sizes and locations of ice crystals in samples as a result of pressure-shift-freezing were compared to those obtained by atmospheric freezing. The results showed that the combination of pressure-shift-freezing and pressure-assisted-thawing process made less change on the cell wall.

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae).

Plasmodesmata are intercellular bridges that directly connect the cytoplasm of neighboring cells and play a crucial role in cell-to-cell communication and cell development in multicellular plants. Although brown algae (Phaeophyceae, Heterokontophyta) are phylogenetically distant to land plants, they nevertheless possess a complex multicellular organization that includes plasmodesmata. In this study, the ultrastructure and formation of plasmodesmata in the brown alga Dictyota dichotoma were studied using transmission electron microscopy and electron tomography with rapid freezing and freeze substitution. D. dichotoma possesses plasma membrane-lined, simple plasmodesmata without internal endoplasmic reticulum (desmotubule). This structure differs from those in land plants. Plasmodesmata were clustered in regions with thin cell walls and formed pit fields. Fine proteinaceous "internal bridges" were observed in the cavity. Ultrastructural observations of cytokinesis in D. dichotoma showed that plasmodesmata formation began at an early stage of cell division with the formation of tubular pre-plasmodesmata within membranous sacs of the cytokinetic diaphragm. Clusters of pre-plasmodesmata formed the future pit field. As cytokinesis proceeded, electron-dense material extended from the outer surface of the mid region of the pre-plasmodesmata and finally formed the nascent cell wall. From these results, we suggest that pre-plasmodesmata are associated with cell wall development during cytokinesis in D. dichotoma.

The Dual Organization of P-bodies Revealed by Immunoelectron Microscopy and Electron Tomography

Processing bodies (P-bodies) are cytoplasmic non-membranous domains involved in the regulation of eukaryotic gene expression. Since their discovery, several studies using fluorescence-based strategies have uncovered their pivotal role in mRNA metabolism, particularly during translation repression and/or mRNA degradation. Yet, P-bodies still remain a “black box” in which numerous proteins accumulate next to RNAs to regulate their fate by unknown mechanisms. In this study, we investigated the ultrastructural organization of P-bodies in human cells. Using a wide range of original electron microscopy strategies, including high-pressure freezing and freeze substitution, we found that P-bodies are huge ribonucleoprotein complexes located in the close proximity of mitochondria and ribosomes, in which regulatory factors exhibit differential localization depending on their activity on mRNAs. We describe the first experiment pairing immunogold labeling with electron tomography (immunoelectron tomography) of a human P-body. Overall, the results depict a P-body organization that comprises at least two distinct compartments: a dense core on which peripheral protrusions are anchored.

Transmission Electron Microscopy of Thin Sections of Drosophila: High-Pressure Freezing and Freeze-Substitution

The state of the art in fine-structure preservation for thin sectioning can be achieved by using fast-freezing technology followed by freeze substitution and embedding in resin. Samples prepared by high-pressure freezing are estimated to be "fixed" in 20-50 msec. Fast freezing also freezes every cell component regardless of its chemistry. Once frozen, tissues can be processed in a variety of ways before viewing in the electron microscope; here we describe only freeze substitution. In freeze substitution, cells are dehydrated at very low temperatures and cell water is replaced with organic solvent at -80°C to -90°C. At this temperature, large molecules such as proteins are immobilized, yet smaller molecules such as water (ice) can be dissolved and replaced with organic solvents, e.g., acetone. The ideal way to do freeze substitution is with a dedicated freeze-substitution device such as the Leica AFS2 system. These devices allow programming of the times and temperatures needed. Alternatively, if this equipment is not available, freeze substitution can still be performed using items commonly found around the laboratory, as is described here. This protocol is useful for preparing thin sections of Drosophila when the best possible preservation of ultrastructure and antigenicity is required.

High-Pressure Freezing/Freeze Substitution and Transmission Electron Microscopy for Characterization of Metal Oxide Nanoparticles Within Sunscreens

To date, the description of a single, suitable method to observe in detail metal oxide nanoparticles in situ within sunscreens is currently lacking, despite growing concern as to how they interact with humans. This study explores the usefulness of transmission electron microscopy to characterize the nanoparticles in sunscreens. High-pressure freezing then freeze substitution was used to prepare resin-embedded commercial sunscreen samples, and ultrathin sections of these were observed with transmission electron microscopy. Conventional room temperature processing for resin embedding was also trialed. High-pressure frozen/freeze substituted samples provided clear visualization of the size and shape of the nanoparticles and agglomerates and allowed further characterization of the composition and crystal form of the metal oxides, while conventionally processed chemically fixed samples were subject to distribution/agglomeration artifacts. Transmission electron microscopy of high-pressure frozen/freeze substituted samples is an ideal method to completely observe metal oxide nanoparticles in situ in sunscreens.