Structural and antigenic preservation of plant samples by microwave-enhanced fixation, using dedicated hardware, minimizing heat-related effects

We explored the use of microwave technology in fixation with the objective of achieving quicker fixation regimes, lower concentrations of toxic and volatile reagents, and enhanced antigen detection. We used a modified domestic microwave oven (900 W) and a low-power (5 W) microwave bench. The work was done on plant materials. The oven was supplemented with a cooling device, a stirring system, and a record of the sample temperature and the time of effective irradiation. The sample, immersed in a fixative solution of 1% paraformaldehyde (PFA) in PBS, was irradiated for only 10 minutes. The sample temperature did not exceed 37 degrees C. In these mild conditions, the quality of the (ultra)structural preservation of the samples, morphometrically assessed, was at the same level as obtained with the same fixative, using conventional methods. On the contrary, samples fixed in the same conditions without irradiation showed a poor structural preservation. The antigenic preservation of the irradiated samples was excellent, since the labeling levels of two nucleolar proteins, detected by immunogold, were three times higher than in conventionally fixed samples. In the so-called microwave bench, the pathway of microwaves is guided, so that low-power microwaves directly hit the sample and there is no dispersion of energy. Temperature of fixative did not increase after microwave irradiation. Fixation in the bench with either 4% PFA, or 1% PFA, for 20 minutes resulted in structural preservation of samples similar in quality as obtained with conventional fixation and in a similar or better level of antigen preservation. Therefore, controlling temperature and effective irradiation is crucial in order to obtain optimal structural and antigen preservation with microwave-enhanced fixation. The dramatic differences observed between microwave-irradiated samples and samples fixed in the same conditions without irradiation, strongly support the existence of specific effects of microwaves on fixation, independent from the mere heating of the samples.     

Comparison of conventional and microwave-assisted processing of mouse retinas for transmission electron microscopy

Conventional fixation and processing of mammalian retinal tissues for transmission electron microscopic (TEM) examination is slow and produces ultrastructural artefacts in the photoreceptor cell layer. Among these artefacts are gaps between photoreceptor outer segment disc membranes and between photoreceptor cells in the region of the retina where the cell nuclei are located. A study was undertaken to determine whether a much more rapid microwave‐assisted fixation and processing protocol would have an effect on the quality of ultrastructural preservation of the retina, particularly on the photoreceptor cell artefacts. The overall ultrastructural preservation of the retina was similar for the conventional and microwave‐assisted techniques. However, the magnitudes of the photoreceptor artefacts were significantly reduced when microwave irradiation was used during primary fixation and processing. It is clear that, at least for the retina, employing microwave irradiation during specimen preparation for TEM results in superior ultrastructural preservation with a substantial reduction in the time required for sample preparation.     

Microwave energy fixation of plant tissue: An alternative approach that provides excellent preservation of ultrastructure and antigenicity

Immunocytochemical techniques are confronted with the problem of obtaining adequate tissue preservation together with retention of protein antigenicity. Various methods, including freeze-drying and freeze-substitution, have been devised to circumvent this problem. In the present study, we report that microwave energy used in combination with low concentrations of glutaraldehyde (0.1%) and paraformaldehyde (2%) preserves the structural integrity of plant tissue and antigenicity of proteins. Tobacco leaf samples fixed in a time as brief as 15–20 s exhibited excellent preservation of fine structures. By contrast, specimens irradiated for shorter (5–10 s) or longer (30–40 s) periods showed poor morphological preservation. Microwave irradiation for 15–20 s was found useful for immobilizing large amounts of soluble antigens. The fast microwave fixation method was successfully used to preserve pathogenesis-related (PR) proteins, which were subsequently localized by a postembedding immunogold procedure. In addition to soluble antigens, cellulose subunits and pectic substances, two major plant cell wall components, were found to be highly preserved in microwave-irradiated tobacco plant tissue. The present study demonstrates that microwave fixation of plant tissue is a simple and inexpensive method that is easy to perform with commercially available microwave ovens. The incubation time for fixation is reduced from 2 h to 15–20 s without loss of fine structural details. This method will undoubtedly acquire increasing applicability and relevance in plant biology.     

The observation of intact hepatic endothelial cells by cryo-electron microscopy

Liver sinusoidal endothelial cells (LSECs) can optimally be imaged by whole mount transmission electron microscopy (TEM). However, TEM allows only investigation of vacuum‐resistant specimens and this usually implies the study of chemically fixed and dried specimens. Cryo‐electron microscopy (cryo‐EM) can be used as a good alternative for imaging samples as whole mounts. Cryo‐EM offers the opportunity to study intact, living cells while avoiding fixation, dehydration and drying, at the same time preserving all solubles and water as vitrified ice. Therefore, we compared the different results obtained when LSECs were vitrified using different vitrification conditions. We collected evidence that manual blotting at ambient conditions and vitrification by the guided drop method results in the production of artefacts in LSECs, such as the loss of fenestrae, formation of gaps and lack of structural details in the cytoplasm. We attribute these artefacts to temperature and osmotic effects during sample preparation just prior to vitrification. By contrast, by using an environmentally controlled glove box and a vitrification robot (37 °C and 100% relative humidity), these specific structural artefacts were nearly absent, illustrating the importance of controlled sample preparation. Moreover, data on glutaraldehyde‐fixed cells and obtained by using different vitrification methods suggested that chemical prefixation is not essential when vitrification is performed under controlled conditions. Conditioned vitrification therefore equals chemical fixation in preserving and imaging cellular fine structure. Unfixed, vitrified LSECs show fenestrae and fenestrae‐associated cytoskeleton rings, indicating that these structures are not artefacts resulting from chemical fixation.     

Effects of microwave irradiation and chemical fixation on the localization of perisinusoidal cells in rat liver by gold impregnation

Modified gold impregnation is one of the methods that are used in light microscopical demonstration of hepatic perisinusoidal cells. This method has some disadvantages, such as restriction of fixation time to 16 h, which allows limited time for processing the tissues, especially when dealing with a large amount of material, and a long impregnation time (16–24 h). We investigated the effect of prolonged fixation on the staining of sections, to shorten the time needed for gold impregnation by using microwave irradiation. Liver specimens were fixed in Baker's calcium–formalin for different periods of time. After fixation, frozen sections were impregnated in gold chloride solution either at room temperature or in a microwave oven. The staining quality of the sections which had been impregnated in the microwave oven for a much shorter time were equal to or even superior to the ones impregnated at room temperature. Prolonging the fixation time up to 7 days did not affect the staining results by microwave irradiation, whereas satisfactory results were not obtained from sections stained at room temperature and fixed for more than 3 days. We conclude that microwave irradiation can be used to shorten the impregnation time in gold chloride solution and the duration of fixation can be prolonged up to 3 days in the original method and up to 7 days when microwave irradiation is used during impregnation.     

Microwave-assisted rapid plant sample preparation for transmission electron microscopy

The preparation of plant leaf material for transmission electron microscopical investigations can be a very time- and labour-consuming task as the reagents infiltrate the samples quite slowly and as usually most steps have to be performed manually. Fixation, buffer washes, dehydration, resin infiltration and polymerization of the resin-infiltrated leaf samples can take several days before the specimen can be cut ultrathin and used for ultrastructural investigations. In this study, we present a microwave-assisted automated sample preparation procedure that reduces preparation time from at least 3 days to about 5 h – with only a few steps that have to be performed manually – until the plant sample can be ultrathin sectioned and observed with the transmission electron microscope. For studying the efficiency of this method we have compared the ultrastructure of different leaf material ( Arabidopsis thaliana , Nicotiana tabacum and Picea abies ) which was prepared with a conventional, well-established chemical fixation and embedding protocol and a commercially available automated microwave tissue processor. Despite the massive reduction in sample preparation time no negative effects on cutting properties of the blocks, stability of the sections in the electron beam, contrast and ultrastructure of the cells were observed under the transmission electron microscope when samples were prepared with the microwave-assisted protocol. Additionally, no negative effects were detected on the dimensions of fine structures of grana stacks (including membranes, inter- and intrathylakoidal spaces), the nuclear envelope and the plasma membrane as the diameter of these structural components did not differ between leaf samples (of the same species) that were processed with the automated microwave tissue processor or by conventional fixation and embedding at room temperature.     

Transpiration-assisted perfusion fixation provides in situ preservation of developing ray parenchyma cells in Eucalyptus nitens

The technique of animal vascular perfusion fixation was adapted for in situ fixation of the fragile and difficult to access cells of the ray parenchyma system in stems of 10-year-old Eucalyptus nitens trees. In situ fixation enabled tissue to be safely dissected for histological processing without risk of damage to microstructure or initiation of wound response. Fixative was perfused through the active vascular system under hydrostatic and transpiration pressure directly to vessel-associated ray cells. Diffusion from vessels allowed fixative to access nonvessel-associated ray cells. Acrolein was included to aid diffusion fixation and safranin dye was included to define fixed regions within the xylem. Sections prepared for light and electron microscopy from samples cut from regions showing intense safranin staining showed good microstructural preservation and were free of artefacts caused by mechanical injury or wound response.     

Freeze substitution followed by low melting point wax embedding preserves histomorphology and allows protein and mRNA localization techniques

Fixation and embedding are major steps in tissue preservation for histological analysis. However, conventional fixatives like aldehyde‐based solutions usually mask tissular epitopes preventing their immunolocalization. Alternative fixation methods used to avoid this drawback, such as cryopreservation, alcohol‐ or zinc salts‐based fixatives do not efficiently preserve tissue and cell morphology. Likewise, paraffin and resin embedding, commonly used for thin sectioning, frequently damage epitopes due to the clearing agents and high temperatures needed along the embedding procedure. Alternatives like cryosectioning avoid the embedding steps but yield sections of poorer quality and are not suitable for all kinds of samples. To overcome these handicaps, we have developed a method that preserves histoarchitecture as well as tissue antigenic properties. This method, which we have named CryoWax, involves freeze substitution of the samples in isopentane and methanol, followed by embedding in low melting point polyester wax. CryoWax has proven efficient in obtaining thin sections of embryos and adult tissues from different species, including amphioxus, zebrafish, and mouse. CryoWax sections displayed optimal preservation of tissue morphology and were successfully immunostained for fixation‐ and temperature‐sensitive antigens. Furthermore, CryoWax has been tested for in situ hybridization application, obtaining positive results. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Optimal preservation of the shark retina for ultrastructural analysis: An assessment of chemical, microwave, and high-pressure freezing fixation techniques

Recent advances in microwave chemical fixation (MCF) and/or high pressure freezing (HPF) combined with transmission electron microscopy have resulted in superior ultrastructural detail in a variety of tissue types. To date, selachian tissue has been fixed and processed using only standard chemical fixation (CF) methods, and the resulting ultrastructure has been less than ideal. In this study, we compared the ultrastructure of the fragile retinal tissue from the brown banded bamboo shark, Chiloscyllium punctatum, obtained using CF, MCF, and HPF methods. For all fixation protocols, ultrastructural preservation was improved by keeping the tissue in oxygenated Ringer solution until the time of fixation. Both MCF and HPF produced superior retinal ultrastructure compared to conventional CF. Although HPF occasionally resulted in very high quality ultrastructure, microwave fixation was almost comparable, quicker and far more consistent. Microsc. Res. Tech. 75:1218–1228, 2012. © 2012 Wiley Periodicals, Inc.     

Preparation of resin embedded unicellular organisms without the use of fixatives and dehydration media.

A method is presented for processing single cells for conventional ultrathin sectioning without the use of fixatives and dehydration media. The cells were fixed by a physical method--spray freezing--which provides extremely high cooling rates, needs no pretreatment with cryoprotective agents and is therefore assumed to maintain the in vivo morphology of the cell. Hitherto cells prepared in this way have been investigated exclusively by freeze etching. To combine the advantages of this method with those of conventional ultrathin sectioning we have processed spray frozen cells with widely varying water contents (spermatozoa and lymphocytes) by freeze drying at 188 K and vacuum embedding. When compared to conventional chemical fixation the differences found in ultrastructural preservation of spermatozoa using this kind of preparation were confined to the arrangement of spermhead membranes and middlepiece structures. Lymphocyte structure was much closer to that known from chemical preparation, the only differences being a denser cytoplasm, denser mitochondrial matrices and thicker plasma membranes. These differences are probably due to the absence of eluating and dissolving effects present in conventional chemical preparations. The ultrastructural preservation of spray frozen cells is not different after freeze etching or after freeze-drying and vacuum embedding. This indicates clearly that drying and resin embedding does not produce artefacts and that structural preservation is therefore limited by the quality of cryofixation. Therefore this method is considered a contribution to the problem of preservation of the in vivo assembly of cellular substructure. Furthermore it seems to be a potential basis for preparation of soluble or diffusible substances or cellular compounds which would be influenced by fixatives and dehydrating agents.     

Cryo preparation of isolated rabbit pancreatic islets

A Flotronic silver membrane has been used as a vehicle to process, via freeze substitution, collagenase-derived rabbit pancreatic islets. The procedure provides: (1) a simple, inexpensive method for handling larger numbers of tightly clustered islet aggregates; (2) a metal surface for rapid heat transfer from specimen to cryogen resulting in an increased circumferential zone of fine structural preservation; (3) the elimination of possible artifacts associated with impact or rotation of biological specimens against a cooled, highly polished metal block; (4) superior preservation of structural components not usually observed by conventional modes of fixation; (5) retention of metabolic components which may subsequently be available for immunocytochemical or X-ray energy dispersive procedures.     

Electron spectroscopic study (ESI,EELS) of Nanoplast-embedded mammalian lung

The potential of Nanoplast melamine resin embedding for the study of mammalian lung parenchyma was examined by means of electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). Samples were either fixed with glutaralde-hyde-paraformaldehyde or glutaraldehyde-tannic acid, or were directly transferred to the embedding medium without prior fixation. Organic dehydrants, as well as fixatives containing heavy metals and stains, were omitted. A very high level of ultrastructural detail of chromatin, ribosomes, mitochondria and plasma membranes was achieved by ESI from the Nanoplast-embedded samples. The most prominent gain in ultrastructural detail was achieved when moving from an energy loss just below the L_2,3 edge of phosphorus at 132 eV to an energy loss just beyond this edge. This reflects the prominent P L_2,3 edge observed by EELS of Nanoplast-embedded samples in comparison with conventionally processed samples. Thus, taking into account possible sectioning artefacts, excellent heterochromatin images which rely on the phosphorus distribution can be obtained from Nanoplast-embedded samples by computer-assisted analysis of electron spectroscopic images. In this respect glutaraldehyde-paraformaldehyde fixation is preferable to glutaraldehyde-tannic acid fixation because the presence of silicon, revealed by EELS, in tannic-acid-fixed samples may introduce artefacts in phosphorus distribution images obtained by the three-window method because of the close proximity of the L_2,3 edges of silicon and phosphorus.     

Scanning force images through the 'Milliscope'– a probe microscope with very wide scan range

The effectiveness and adequacy of a home-built scanning force microscope (SFM) able to cover a volume of ∼1.2 × 1.2 × 0.13 mm³ (X × Y × Z) were tested on calibrating objects, as well as on cytological and histological samples. The instrument was designed for matching the magnification range of an optical microscope (∼ 20–1200×) but its dynamics were one or two orders of magnitude higher, thanks to a lateral resolution of about 10 nm. Images ranging in size from 1.2 × 1.2 mm² to 1 × 1 µm² showed a quality comparable to that given by other SFMs on similar materials. The 'Milliscope' is a curious but effective imaging tool whose operating range overlaps at one extreme with a goldsmith's eyepiece, and at the other with an electron microscope. The intrinsic limits of scanning probe techniques and of the available SFM cantilevers prevented us taking complete advantage of the wide height range of our scanner. However, our results show that an instrument having a very wide scan area, obtained through simple, inexpensive and intrinsically linear techniques, can give a good performance even at small scan sizes. This encourages us to develop wide scan instruments, which could further increase the already extensive use of scanning force microscopy in biology.     

Improved preservation of the ram spermatozoan plasma membrane using betaine in the primary fixative

Improved preservation of ram spermatozoa was obtained by filtration onto a Millipore filter followed by fixation in a fixative containing 23 m m betaine, which raised fixative osmolality only slightly. In samples fixed in control preparations with betaine omitted the plasma membrane had a ruffled appearance, while the betaine-containing fixative gave the plasma membrane a smooth contour which closely followed underlying structures.
Betaine is known to function as an osmoprotectant in other situations, and may protect the cells from osmotic damage during the initial stages of fixation.     

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. Microsc. Res. Tech. 76:942–946, 2013. © 2013 Wiley Periodicals, Inc.     

Freeze-substitution techniques for preparing nematodes for scanning electron microscopy

The effect of different substitution times, temperatures and the incorporation of fixatives on the preservation of three species of nematode for scanning electron microscopy by freeze substitution with methanol, followed by critical point drying, is investigated. Hammerschmidtiella diesingi adults and Trichostrongylus colubriformis infective juveniles were successfully preserved using methanol at 253 K as the substitution medium. Preservation deteriorated with long substitution times, suggesting the extraction of material and that substitution times should be kept as brief as possible. Panagrolaimus davidi was not successfully preserved using pure methanol, but preservation was improved by using fixatives in the substitution medium, the best results being obtained with 1% OsO₄/3% glutaraldehyde in methanol. A substitution temperature of 193 K did not give any improvement in preservation. The differences in the quality of preservation between the three species may be due to the relative ability of the cuticle to withstand collapse during critical point drying. Chemical fixation using cold fixative resulted in the retention of a natural posture but poor preservation, whereas hot fixatives resulted in good preservation but the loss of a natural posture. Freeze substitution in methanol may prove useful in the preparation of specimens possessing cuticles or cell walls which have sufficient strength to withstand the drying process (e.g. arthropods, plants, fungi, nematodes). More delicate specimens may require the incorporation of fixatives into the substitution medium or conventional fixation.     

Phase identification of individual crystalline particles by electron backscatter diffraction

Recently, an electron backscatter diffraction (EBSD) system was developed that uses a 1024 × 1024 CCD camera coupled to a thin phosphor. This camera has been shown to produce excellent EBSD patterns. In this system, crystallographic information is determined from the EBSD pattern and coupled with the elemental information from energy or wavelength dispersive X-ray spectrometry. Identification of the crystalline phase of a sample is then made through a link to a commercial diffraction database. To date, this system has been applied almost exclusively to conventional, bulk samples that have been polished to a flat surface. In this investigation, we report on the application of the EBSD system to the phase identification analysis of individual micrometre and submicrometre particles rather than flat surfaces.     

An X-ray microscopy perspective on the effect of glutaraldehyde fixation on cells

X-ray microscopy (XRM) is the only microscopy technique that can provide high-resolution (30 nm) imaging of biological specimens without the need to fix, stain or section them. We aim to determine the effect, if any, of glutaraldehyde fixation on algae cells from the XRM perspective and thus provide beneficial information for both X-ray and electron microscopists on artefacts induced by glutaraldehyde fixation. Three species of microalgae, Microcystis aeruginosa, Anabaena spiroides and Chlorella vulgaris, were used in this study. XRM images were obtained from unfixed and glutaraldehyde-fixed cells and cell diameter and percentage X-ray absorbency were measured. The mean diameter of cells from fixed preparations was smaller than from unfixed preparations; the mean diameter of M. aeruginosa cells was significantly reduced from 3.92 µm in unfixed cells to 3.43 µm in fixed cells (P < 0.05); in C. vulgaris the diameter of cells was also significantly reduced from 3.50 µm in unfixed to 2.98 µm in fixed samples (P < 0.05); whereas there was no significant reduction in the diameter of A. spiroides cells (4.04–3.90 µm). The protein crosslinking mechanism of glutaraldehyde probably generated free water molecules, which play an important role in radiation damage induced by X-rays. This was seen as mass loss and cell shrinkage, which in the present study occurred more frequently in fixed cells than in unfixed cells. In addition, we demonstrated that the uptake of glutaraldehyde by cells makes all protein constituents in the cell organize into a closely packed configuration, thus causing a rise in the percentage of X-ray absorbency. In fixed cells, this rise was approximately by a factor of two compared with unfixed samples in which protein constituents inside the cell are arranged in their native form.     

Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution

Freezing of bulk biological objects was investigated by X-ray cryodiffraction. Freezing at atmospheric pressure of most microscopic biological samples gives rise to large hexagonal crystals and leads to poor structural preservation of these specimens. High-pressure freezing induces the formation of different ices (hexagonal, cubic and a high-pressure form) consisting of crystals having sizes smaller than those formed at atmospheric pressure. With both freezing methods, a cryoprotectant has to be added to the biological object to avoid the formation of ice crystals. However, special cases can be encountered: some biological objects contain large amounts of natural cryoprotectant or have a low water content. In these cases, vitrification can be achieved, especially using high-pressure freezing. Cryo-sectioning can be performed on vitrified samples, and the sections studied by electron cryomicroscopy. Images and electron diffraction patterns having a resolution better than 2 and 0.2 nm, respectively, can be obtained with such sections. Because samples containing crystalline ices cannot be cryosectioned, their structure has to be studied using cryosubstitution and resin embedding. We show that bacteria, yeast, and ciliate and marine worm elytrum have cellular compartments with an organization that has not been described by classical techniques relying on chemical fixation of the tissues. A high-pressure artefact affecting the Paramecium trichocysts is described. Such artefacts are not general; for example, we show that 70% of high-pressure frozen yeast cells survive successive high-pressure freezing and thawing steps.     

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.