Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy

Cryofixation is widely held to be superior to chemical fixation for preserving cell structure; however, the use of cryofixation has been limited chiefly to electron microscopy. To see if cryofixation would improve sample structure or antigenicity as observed through the light microscope, we cryofixed Nicotiana alata and Lilium longiflorum pollen tubes and Tradescantia virginina stamen hairs by plunge freezing. After freeze-substitution, and embedding in butylmethylmethacrylate, we found using the light microscope that the superiority of cryofixation over chemical fixation was obvious. Cryofixation, unlike chemical fixation, did not distort cell morphology and preserved microtubule and actin arrays in a form closely resembling that of living cells.
Additionally, to test further the usefulness of cryofixation for light microscopy, we studied the appearance of cells and the retention of antigenicity in a plunge-frozen multicellular organ. Roots of Arabidopsis thaliana were either chemically fixed or plunge frozen, and then embedded in the removable methacrylate resin used above. We found that plunge freezing preserved cell morphology far better than did chemical fixation, and likewise improved the appearance of both actin and microtubule arrays. Plunge-frozen roots also had cells with more life-like cytoplasm than those of chemically fixed roots, as assessed with toluidine-blue staining or high-resolution Nomarski optics. Damage from ice crystal formation could not be resolved through the light microscope, even in the interior of the root, 40–75 μm from the surface. We suggest that plunge freezing would enhance many investigations at the light microscope level, including those of multicellular organs, where damage from ice crystals may be less severe than artefacts from chemical fixation.     

Routine cryofixation of plant tissue by propane jet freezing for freeze substitution

Cryofixation and freeze substitution methods were developed for ultrastructural studies of cells in complex plant tissues. Leaf tissues and root tips of tobacco (Nicotiana tabacum L. var. Maryland Mammoth) were frozen with a RMC MF7200 propane jet freezer and freeze substituted sequentially with tannic acid and osmium tetroxide/uranyl acetate in acetone. High quality preservation was consistently obtained for epidermal and phloem cells of the leaf, and epidermal, cortical, meristematic, and cap cells of the root tip. Leaf mesophyll cells were also often well frozen. Organelles, including nuclei, endoplasmic reticulum, mitochondria, Golgi bodies, and plastids, showed excellent structural integrity and contrast. Most notable is the superior preservation of the cytoskeleton. Our results demonstrate that the propane jet freezer can be used routinely for high quality cryofixation of higher plant cells in certain complex tissues. This could have important implications for the use of cryofixation approach in a wide range of research in plant biology.     

Morphometric analysis of cryofixed muscular tissue for intraoperative consultation

For diagnostic purposes, cryofixation of tissues is a daily routine technique to investigate rapidly about the presence of tumours during a surgical procedure in patients. We performed morphometric analysis of cryofixed muscular tissues according to different techniques. About 1,000 muscle fibers and 1,493 nuclei, were automatically examined. After freezing, ice tissue interfaces shrinkage of the cells were present. Liquid isopentane or liquid nitrogen produced a statistical increase of fractal dimension, D, of the ice‐tissue interfaces, P < 0.001 respect to the formalin‐fixed samples, cryofixation performed inside the cryostat chamber at t = ?20°C produced a D value close to the formalin‐fixed samples. Shrinkage of the muscle fibers was higher in the samples cryofixed inside the cryostat chamber (P < 0.001). Cryofixation inside cryostat or by liquid nitrogen caused decreases of the nuclei dimensions and altered nuclear morphology (P < 0.01), liquid isopentane appeared not affecting the nuclei of the fibers. Cryofixation inside the cryostat chamber produced the highest shrinkage but it was reduced performing cryofixation in liquid nitrogen or isopentane. Freezing damage inside the muscle cells was absent in the samples cryofixed inside the cryostat, it was present after cryofixation by liquid nitrogen or isopentane. Subcellular components like the nuclei were preserved by isopentane. This paper present, for the first time, an objective method able to quantify and characterize the damages produced by cryofixation in biological sample for intraoperative consultation. Microsc. Res. Tech. 79:155–161, 2016. © 2016 Wiley Periodicals, Inc.     

Development of the gametophyte of the fern Schizaea pusilla

Schizaea pusilla is a pteridophyte with several unique developmental characteristics. In contrast to most other fern species, S. pusilla gametophytes remain filamentous throughout their development, and the gametophytes are associated with an endophytic fungus which appears to be mycorrhizal. In terms of tropistic responses, apical filament cells of young gametophytes are negatively phototropic compared with germ filaments of other ferns which exhibit positive phototropism. Cryofixation (propane jet freezing and high-pressure freezing) in conjunction with freeze substitution electron microscopy was used to study young gametophytes. The results demonstrate that apical filament cells have a distinctive structural polarity and that rhizoids also can be successfully frozen by these methods. The cytoskeleton and endomembrane system were particularly well preserved in cryofixed cells. In addition, Schizaea gametophytes were used as a test system to evaluate potential artifacts of propane jet freezing and high pressure freezing. There was little apparent difference in ultrastructure between cells cryofixed by either freezing method. These gametophytes will be useful in determining the effectiveness of cryofixation techniques and as a model system in tip growth studies.     

Candida albicans biofilms: comparative analysis of room‐temperature and cryofixation for scanning electron microscopy

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde‐based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room‐temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room‐temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze‐substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.     

Cryofixation rapidly preserves cytoskeletal arrays of leaf epidermal cells revealing microtubule co-alignments between neighbouring cells and adjacent actin and microtubule bundles in the cortex

Accurate preservation of microtubule and actin microfilament arrays is crucial for investigating their roles in plant cell development. Aldehyde fixatives such as paraformaldehyde or glutaraldehyde preserve cortical microtubule arrays but, unless actin microfilaments are stabilized with drugs such as m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS) or phalloidin, their arrays are often poorly preserved. Cryofixation, used primarily for electron microscopy, preserves actin microfilaments well but is used rarely to fix plant cells for optical microscopy. We developed a novel whole-mount cryofixation method to preserve microtubule and microfilament arrays within Tradescantia virginiana leaf epidermal cells for investigation using confocal microscopy. Cortical microtubule arrays were often oriented in different directions on the internal and external faces of the epidermal cells. A number of arrays were aligned in several directions, parallel to microtubules of neighbouring cells. Actin microfilaments were particularly well preserved possibly due to the speed with which they were immobilized. No transverse cortical microfilament arrays were observed. On occasion, we observed co-aligned microfilament and microtubule bundles lying adjacent to the plasma membrane and positioned side by side suggesting a potential direct interaction between the cytoskeletal filaments at these locations. Cryofixation is therefore a valuable tool to investigate the interactions between cytoskeletal arrays in plant cells using confocal microscopy.     

Fast cryofixation technique for X-ray microanalysis

The proposed cryofixation technique uses a tubule-shaped needle chilled in liquid propane for simultaneous excision and freezing of a tissue specimen. Due to this simultaneity, ionic shifts created by traumatic influences are avoided even in the outermost cells of the specimen. Moreover, it is shown here that stopping the blood flow for more than about 10 s results in notable ionic shifts between cells and extracellular space in rat heart and liver. Such preparative ischaemic injury is minimized by the Fast Cryofixation Technique because it can be easily performed on organs within the circulatory system, whilst the heart of the animal is still beating. Intracellular concentrations of the monovalent ions in rat heart and liver, obtained by this method, tally well with recent results from different independent techniques reported in the literature. As demonstrated by cross-sectioning and freeze-fracturing, the structural preservation of the freezing technique is sufficient for X-ray microanalytical work.     

Comparing different preparation methods to study human fibrin fibers and platelets using TEM

For the study of cellular ultrastructure, the sample needs to be stabilized by fixation, with the ultimate aim to preserve the native tissue organization and to protect the tissue against later stages of preparation. Chemical and freezing fixation are most used, and chemical fixation employs agents that permeate tissues and cells by diffusion and covalently bind with their major biochemical constituents to fix them. Most widely used chemical fixatives are aldehydes, e.g., formaldehyde and glutaraldehyde, which are noncoagulating, crosslinking agents. Cryofixation methods for ultrastructural studies are also popular, and high-pressure freezing immobilizes all cell constituents and arrests biological activity by removing the thermal energy from the system. In the current research, we used platelet-rich plasma (PRP) to study expansive fibrin fibers and platelet ultrastructure to compare the two fixation techniques. We also used thrombin and calcium chloride as a clotting agent to determine the technique most suitable for the formation of extensive fibrin networks. Chemically fixated fibrin fibers were more compact and condensed and also showed a banding pattern on longitudinal sections. High-pressure frozen samples were more dispersed while platelets fixated showed better preserved cellular membranes and organelle structure. PRP coagulated by addition of CaCl(2) showed blood platelets that are noticeably more activated compared with PRP; however, with thrombin, a sharp ultrastructure was seen. We conclude that PRP mixed with thrombin, and freeze substituted, is the most suitable method for the study of extensive fibrin fibers as well as platelets.     

Embedding in Spurr's resin is a good choice for immunolabelling after freeze drying as shown with chemically unfixed dendritic cells

Immunocytochemical reactions on biological specimens depend on many factors, the most crucial one being the maintenance of antigenicity. Antigens are vulnerable at each stage during preparation for electron microscopy. One of the least traumatic methods of preparing biological tissues for post‐embedding immunolabelling includes the following steps: (1) physical stabilization of the native biological material by rapid freezing (cryofixation) and keeping the immobilized biological sample at low temperature, thereby avoiding any movements of water, ions and macromolecules; (2) dehydrating the frozen biological material by freeze‐drying at low temperature; (3) embedding of the dehydrated specimen. Here we show that embedding of chemically unfixed dendritic cells in Spurr's resin after cryofixation and freeze‐drying enables the conservation of fine ultrastructure without cell distortion or shrinkage. Furthermore, we demonstrate the feasibility of protein localization in ultrathin sections by immunolabelling of the major histocompatibility class II molecules.     

Time‐series investigation of fused vesicles in microvessel endothelial cells with atomic force microscopy

Vesicles or caveolae within endothelial cells, fusing together to form vacuolar organelles, are implicated in macromolecular transport and cellular element transmigration across the blood–brain barrier (BBB) during inflammation and ischemia. Vacuolar organelles have been described by transmission electron microscopy and immunofluorescence, but the details of their dynamics have not been well addressed yet. Herein, by using tapping mode atomic force microscopy (AFM), we observed the time‐series changes of fused vesicles within the serum‐free cultured rat cerebral microvessel endothelial cells. The fused vesicles were certainly proved by fluorescent staining of Fm4‐64 combining simultaneous AFM imaging, as well as the field emission scanning electron microscopy technique. And energy dispersive spectrum results additionally implied that there may be specific structure and compositions around the vesicle region. These results indicate that increased vesicles in BBB may contribute to the formation of fused vesicles and a higher probability to construct the trans‐endothelial channel across endothelium layer. Furthermore, the AFM application may open up a new approach to investigate the details of transcellular process by fused vesicles. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Cryofixation methods for ion localization in cells by electron probe microanalysis: a review

Electron probe microanalysis data on the intracellular content and distribution of electrolyte ions depends critically on the functional state of the cells at the moment of cryofixation. Whereas tissue specimens often require special in-situ freezing techniques, isolated and cultured cells can be frozen within their environmental medium under physiologically controlled conditions. Thus, they represent a feasible system to study functional ion-related intracellular parameters such as the K/Na ratio. Specifically modified freezing devices allow the study of ion shifts related to dynamic processes in cells, for example, locomotion and exocytosis. The time resolution achieved by time-controlled cryofixation is approximately 1 ms.     

Effect of specimen preparation and section transfer techniques on the preservation of ultrastructure,lipids and elements in cryosections

Cryofixation, cryoultramicrotomy, and proper transfer of the cryosections into the electron microscope are important for the preservation of good ultrastructure and the measurement of subcellular elemental distributions. These techniques are applicable to tissue systems which can be rapidly frozen so that minimal to no ice damage occurs during the cryofixation step. For the transfer step we have compared the cryotransfer of hydrated sections and subsequent freeze-drying in the electron microscope with the transfer of sections into an external freeze-dryer, followed by exposure to room temperature and humidity before introduction into the electron microscope. The use of a cryotransfer stage for section transfer from the cryoultramicrotome to the electron microscope and low temperature observation of the thin sections avoids the potential problem of rehydration damage to freeze-dried sections as well as provides protection from the possibility of melting of the lipids in the sections. Both of these problems may lead to loss of in situ elemental distribution and morphology. In this report, observations are presented which show the damaging effects of temperatures above 273 K on ultrastructure due to lipid melting in tissues with high lipid content and the redistribution of elements which can be encountered when thin sections become inadvertantly rehydrated.     

Quick-freeze,deep-etch studies of endothelial components,with special reference to cytoskeletons and vesicle structures

A three-dimensional study of the ultrastructure of endothelial cells is helpful in understanding important endothelial functions such as vascular transport and cell permeability. For this purpose, in addition to serial sectioning electron microscopy and high-voltage electron microscopy, the quick-freeze, deep-etching technique also enables us to analyze structures at the molecular level by its high resolution and is useful for three-dimensional morphological studies. Some modifications on the conventional deep-etching method were made in this study to reduce the undesirable aggregation of proteins and salts during etching. Using this technique, we examined the rat aortic endothelium, particularly the membrane structures and cytoskeletons. The luminal surface of the endothelium was covered with a fine filamentous coat, which was anchored to the plasma membrane. In the cytoplasm, actin filaments were prominent and were oriented randomly or in a parallel fashion near the plasma membrane. Of the vesicles seen in the endothelium, some had basket coats of clathrin, and others had striped coats on the cytoplasmic membrane surface. These surface structures of the vesicles suggest the transport mechanism of the vesicles in association with the fine filaments attached to the vesicles.     

Freeze-fracture electron microscopic and low temperature X-ray scattering studies of the effect of cryofixation upon serum low density lipoprotein structure

We report here a correlated X-ray diffraction and freeze-fracture electron microscope study of the effects of several cryofixation procedures upon human serum low density lipoprotein (LDL₂) structure. Only when the LDL₂ solutions contained 75%, by weight, glycerol were the room temperature and post cryofixation low temperature LDL₂ X-ray scattering curves indistinguishable from one another. Other cryofixation procedures, slow or rapid, with or without glycerol, resulted in differences between the room temperature and low temperature LDL₂ X-ray scattering curves, in part due to the effect of quenching upon the solvent. Freezeetching electron microscopy of the slowly cryofixed LDL₂ showed marked aggregation of the particles and an unusual morphological appearance. In contrast, after rapid cryofixation or cryofixation in the presence of glycerol, freeze-etch electron microscopy revealed well-isolated particles which had a knobby morphology. The results demonstrate that under certain conditions (in the presence of 75% glycerol) cryofixation results in minimal, if any, structural alteration of, at least, the LDL₂ lipid moiety. Further, this study underlines the more general conclusion that any high resolution structural study employing a cryofixation step must be interpreted with caution and the effect of cryofixation upon the sample structure need be evaluated by independent means.     

Rapid cryofixation of rabbit muscle fibres after a temperature jump

We describe a procedure whereby structural changes that occur in muscle fibres after a rapid temperature jump can be captured by cryofixation. In the thick filament from rabbit and other mammalian skeletal muscles there is a rapid transition from a non‐helical to a helical structure as the temperature is raised from 273 K towards physiological levels. This transition is accompanied by characteristic intensity changes in the X‐ray diffraction pattern of the muscle. In our experiments to capture these changes, single fibres of glycerinated psoas muscle were subjected to a Joule temperature jump of 15–30 K from ~278 K in air. We have developed a freezing method using a modified Gatan cryosnapper in which a pair of liquid nitrogen‐cooled copper jaws were projected under pressure and closed on the fibre between 50 and 100 ms after the temperature jump. The frozen fibres were freeze‐substituted and embedded for electron microscopy. Transverse and longitudinal sections of relaxed ‘cold’ (~278 K) and temperature‐jumped fibres as well as rigor fibres were obtained. Fourier transforms of the images from the three preparations showed differences in the relative intensities of the reflections from the hexagonal filament lattice and in those of the helix‐based layer lines, similar to the differences seen by X‐ray diffraction. We conclude that we have preserved the ‘hot’ structure and that cryofixation is sufficiently fast to prevent the transition back to the ‘cold’ state.     

Cryofixation of monolayer cell cultures for freeze-fracturing without chemical pre-treatments

A cryofixation method is presented which gives excellent ultrastructural preservation of monolayer cell cultures without any chemical pretreatments. Rat hepatocytes in primary culture were used in this study. The equipment needed is inexpensive and easy to manufacture. Cells are grown on a usual tissue culture support material (Thermanox plastic sheets). For cryofixation, samples are prepared essentially by a combined sandwich-cryogen-jet technique. 3 mm large discs are punched out and sandwiched with Cu- or Au-object holders of little mass; a 15 μm spacer is put in between. The viability of the cells is not impaired by the manipulations before freezing. The sandwich sample is quickly frozen by shooting a propane jet from a simple pressure chamber on to the metal object holder. The relevant parameters were optimized by parallel freeze-fracture analyses of 5% glycerol as a model system and by thermocouple measurements. Sandwich samples are then mounted in an appropriate double replication specimen table for further analysis by freeze-fracturing. It is possible to obtain a certain selectivity of the fracture plane with regard to apical, lateral or basal aspects of the cell layer. Alternatively, disc samples can be processed by chemical fixation methods (including freeze substitution to determine the freeze-fracture plane), since the support material Thermanox is insensitive to organic solvents and easy to cut. In each case the cells remain attached to their substratum throughout the whole procedure. Thus, the ultrastructural data can be directly correlated with parallel functional analyses obtained from the same cell cultures.     

Solutions to difficulties encountered in low-temperature SEM of some frozen hydrated specimens: Examination of Penicillium nalgiovense cultures

Penicillium nalgiovense cultures, which are used in the food industry, were found to be collapsed when prepared by standard procedures for scanning electron microscopy. Neither freeze-drying nor critical point-drying preserved the structure of cultures grown on agar media. Cryofixation and preparation of frozen hydrated samples using the Hexland Cryotrans CT 1000 attachment in conjunction with an AMR 1000A scanning electron microscope yielded micrographs of uncollapsed structures which could be used for morphological characterization. Several additional steps had to be used in sample preparation to achieve satisfactory results. Samples were held in a humid chamber prior to freezing; growth substrate was trimmed as thinly as possible (less than 1 mm above the support); the sides of samples were painted with a conductive cement to their upper edge; and frozen samples were coated intermittently with gold sputtered in several 2-min bursts.     

The dimensions and numbers of small vesicles in blood capillary endothelium in the hind legs of dogs, and their relation to vascular permeability

The dimensions and numbers of vesicles were determined in the blood capillary endothelium of the gastrocnemii muscle of dogs. These results permitted more accurate calculations of the number of vesicles crossing the endothelium in one direction/sec/(μm² (～6·2), and of the median vesicular attachment time (～8 sec). The probability of fusion occurring when a vesicle contacts a plasma membrane (α= 0·004) was unchanged: hence it was concluded, from the mean cellular width (0·21 μm) and the calculated cytoplasmic viscosity (～0·1 poise), that ～49% of the vesicles starting from one side reached the other one, and that their median transit time was ～1 sec.     

The dimensions and numbers of small vesicles in cells, endothelial and mesothelial and the significance of these for endothelial permeability

Measurements were made of the diameters, numbers and other parameters of small, smooth-surfaced vesicles in the endothelium of blood capillaries and lymphatics, and in the mesothelial cells of the diaphragms of mice. Some measurements were also made on the aortic endothelium. With a few exceptions, there were no morphological differences between the various sites. It was found that between 25% and 35% of the non-nuclear cell volume was composed of vesicles, whose membranes accounted for about 55% of their volumes. Their internal volumes were ～ 70,000 nm³, totalling ～ 0·04 μm³/μm² of luminal surface area. For each 1 μm² there were ～ 135 vesicles attached to each surface membrane of the cell, and between ～ 200 and ～ 350 vesicles lying free in the cytoplasm. There was probably a slight amount of shrinkage during the preparation of the material, and the true linear dimensions were probably ～ 105% of those actually observed. Thus the values for the internal volumes were probably ～ 85,000 nm³ and ～005 μm³ respectively; the vesicular numbers were probably ～ 125 attached to each surface and between ～ 175 and ～ 300 free. The vesicles attached to the plasma membranes often had quite long stalks; these were estimated to be ～ 30 nm at the moment of rupture. Thus the vesicles must be released an appreciable distance away from the membrane. This modifies the conclusions of Shea & Karnovsky (1966), since it can now be shown that Brownian movement alone is capable of accounting for the release of the vesicles, their movements within the cells and their transportation of material. By combining these results with others estimating the endothelial permeability coefficients, it can be calculated that the average free lifetime of a vesicle is ～1½ sec, from union with one plasma membrane to the next. It can also be shown that the average time of such an attachment is ～ 2½ sec. There are many possible sources of error relating to these measurements; they must only be regarded as tentative. It appears likely, however, that they are of about the correct order of magnitude as they accord well with other data.     

The passage of cytoplasmic vesicles across endothelial and mesothelial cells

The proportions of labelled cytoplasmic vesicles, at increasing distances across mouse heart endothelium and diaphragmatic mesothelium, were studied using the following electron-microscopical tracers: ferritin, horseradish peroxidase and sodium ferrocyanide. The cells were incubated in Hank's solution containing the tracer for periods of 2 sec up to 30 min. Peroxidase labelled the vesicles well, but ferrocyanide may have escaped from them into the cytoplasm. Both passed down the intercellular junctions. Ferritin showed evidence of molecular sieving when entering the vesicles, and probably also suffered from this on leaving them. It was possible to allow for these effects. The vesicles containing tracers were found to have traversed the cells after only a few seconds; by approximately 10 sec a steady-state was observed, after which there were constant proportions of labelled vesicles. These proportions decreased slightly, but not sharply, across the cells. It was found that the observed distributions across the cells were very similar to those predicted by diffusion theory, assuming the following postulates: (1) the vesicles are moved solely by Brownian motion; (2) a low (i.e. α = 0.05) probability that a collision with a plasma membrane results in fusion. If however it was assumed that the fusion probability (α) were unity, the observed distributions of labelled vesicles differed very significantly from those predicted. A possible explanation of the observed low value of α is suggested, based on mutually-repelling charges on the vesicles and membranes. If α is given a value of around 0.05, the observations agree with calculated predictions that the median transit times for vesicles through cells 0.3–0.5 μm wide are approximately 3–5 sec and that the cytoplasmic viscosity is approximately 0.2–0.3 poise. The predictions of vesicular median free lives of- sec and median attachment times of 3–5 sec also received confirmation, as did the prediction that some 40% of the released vesicles regain the membrane on the opposite side of the cell.