Vitrification of aqueous suspensions from a controlled environment for electron microscopy: An improved plunge-cooling device

In the process of vitrifying aqueous suspensions for cryotransmission electron microscopy, water is solidified without crystallization. Vitrification can be achieved by rapidly plunging an aqueous thin film into a liquid cryogen. The preparation of aqueous thin films prior to vitrification must be performed in an environmental cabinet at controlled temperature and humidity in order to prevent evaporation and temperature-induced phase changes in the thin film. The device described here incorporates several important features which make the apparatus simpler and more convenient to use than similar devices described in the literature. One of these features includes the use of a totally enclosed environmental cabinet in which the grid, sample, micropipette and absorbent paper are equilibrated before thin-film preparation. Other features include a cryogen dewar on a swing arm for easy refilling, a guillotine shutter which is used to trigger the plunger electrically and a semiautomatic system which facilitates rapid transfer of the vitrified specimen from liquid propane to liquid nitrogen for storage and reduces handling of the specimen. To demonstrate the utility of the device, results showing the influence of temperature on the morphology of phospholipid vesicles are presented. A commercial cryotransfer apparatus (which is used for transportation of the vitrified specimen to the electron microscope cold-stage) has been modified to reduce the possibility of reversion of the vitreous phase to the crystalline ice phases.     

X-ray microanalysis of freeze-dried and frozen-hydrated cryosections

The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze-drying of samples. Freeze-dried cryosections 100-nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively. The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze-drying. By combining the X-ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined. In comparison to freeze-dried cryosections frozen-hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X-ray spectra made reproducible microanalysis of ultrathin (about 100-nm thick) frozen-hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X-ray microanalysis. Therefore, at present only bulk specimens or at least 1-μm thick sections can be used for X-ray microanalysis of frozen-hydrated biological samples.     

Ice crystal damage in frozen thin sections: freezing effects and their restoration

Thin sections of unfixed kidney, fast frozen without cryoprotectants, were fixed in osmium tetroxide vapour directly after freeze drying or after 30 min in a moist atmosphere. Dry sections fixed in vapour showed ice crystal damage characteristic for the freezing procedure. This was demonstrated with freeze fracture replicas from the same preparation. Ice crystal holes were obscured in serial sections which were freeze dried and allowed to rehydrate in a moist atmosphere. The same ultrastructural appearance was observed in frozen sections brought to room temperature immediately after cutting. Frozen thin sections from unfixed tissue, if freeze dried, are very sensitive to atmospheric conditions and need some form of stabilization (e.g. osmium vapour fixation, sealing with an evaporated carbon film) before electron microscope images can be interpreted as representative for the frozen state. Restoration of ice crystal damage can occur by melting frozen sections or by rehydration of freeze dried frozen sections. Restoration phenomena will impair studies aimed at the localization of diffusible substances by autoradiography or X-ray microanalysis.     

A technique for studying one and the same section of a cell in sequence with the light and electron microscope

Methods for mounting and staining relatively thin sections on electron microscope grids, in order that one and the same section of a cell can be photographed in sequence with the light and electron microscope are described. Toluidine blue is used as a stain and hexachlorabuta-1,3 diene as a medium which enables the grid carrying the stained sections to be temporarily mounted under a coverslip and examined with an oil-immersion lens. Results obtained with pollen mother cells of Fritillaria lanceolata at zygotene are illustrated.     

Cold-stage microscopy system for fast-frozen liquids

The least artifact-laden fixation technique for examining colloidal suspensions, microemulsions, and other microstructured liquids in the electron microscope appears to be thermal fixation, i.e., ultrafast freezing of the liquid specimen. For rapid-enough cooling and for observation in TEM/STEM a thin sample is needed. The need is met by trapping a thin layer ( approximately 100 nm) of liquid between two polyimide films ( approximately 40 nm thickness) mounted on copper grids and immersing the resulting sandwich in liquid nitrogen at its melting point. For liquids containing water, polyimides films are used since this polymer is far less susceptible to the electron beam damage observed for the commonly used polymer films such as Formvar and collodion in contact with ice. Transfer of the frozen sample into the microscope column without deleterious frost deposition and warming is accomplished with a new transfer module for the cooling stage of the JEOL JEM-100CX microscope, which makes a true cold stage out of a device originally intended for cooling specimens inside the column. Sample results obtained with the new fast-freeze, cold-stage microscopy system are given.     

Improved transfer of two-dimensional crystals from the air/water interface to specimen support grids for high-resolution analysis by electron microscopy

Electron crystallographic analysis of two-dimensional crystals grown on lipid layers at the air/water interface has been limited by loss or damage during transfer of the crystals to an electron microscope support grid. Two methods of transfer are described which are applicable on a small scale (10 microliters of protein solution) and which give greatly improved results for streptavidin crystals on biotinylated lipid layers. In the first method, a hydrophobic grid surface was produced by coating a carbon support film with a thin layer of SiO2, followed by alkylation with dimethyloctadecylchlorosilane. The transfer efficiency of protein crystals approached 50% coverage of the alkylated grid surface. The degree of order of crystals transferred to the alkylated grid surface and preserved in negative stain was significantly improved over that of crystals transferred directly to a carbon support film. In the second method, crystals at the air/water interface were transferred to a holey carbon support film. The efficiency of transfer across the holes was virtually 100% as nearly every hole was completely covered with crystals. After preservation of the crystals in 1% glucose and cooling to liquid nitrogen temperature, electron diffraction was obtained that extended to 1/2.8 A-1 resolution. This demonstrates that two-dimensional crystals grown on lipid layers at the air/water interface can be sufficiently well-ordered, even after transfer to a support grid, to yield high-resolution structural information.     

The preparation and X-ray microanalysis of bulk frozen hydrated vacuolate plant tissue

A series of modifications have been devised which allow the peak to background ratio X-ray analytical method to be used more effectively to measure elemental concentrations in large vacuolate plant cells. Planar, frozen-hydrated fracture faces of bulk plant tissue are coated with a thin film of evaporated chromium, which prevents surface charging. Provided the film is sufficiently thin, c. 5–10 nm, there is no attenuation of the electron beam and only a small absorption of soft X-rays. The chromium makes a small but measurable contribution to the spectral background and suitable corrections may be made to the quantitative results. An improved back-scattered imaging system is described, which helps to overcome the problem of spurious X-ray signals from rough surfaces. The microscope column has been modified to permit a continuous readout of beam current, sensu stricta, during X-ray microanalysis and to allow rapid exchange of the electron gun assembly during low temperature operation. Calculations are given relating the size of the X-ray interactive volume to electron penetration and X-ray emission in both frozen hydrated and frozen dried cells. The problems of X-ray microanalysis are discussed in relation to the highly vacuolate cells found in most mature plant tissues and an example given of the distribution of four major cations in tobacco leaves.     

Specimen thickness dependence of hydrogen evolution during cryo‐transmission electron microscopy of hydrated soft materials

The evolution of hydrogen from many hydrated cryo‐preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm^?2 and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy‐loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge‐shaped specimens of frozen‐hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen‐hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy‐loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation‐sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen‐based artefacts.     

Observations on the production of frozen-dried thin sections for electron microscopy using unfixed fresh liver, fast-frozen without cryoprotectants.

Thin sections of unfixed liver, fast-frozen without cryoprotectants, have been cut using conditions under which momentary thawing of the sections is unlikely to occur. A transfer stage which facilitates this procedure is described. Sections show hole damage probably due to ice-crystal formation during the freezing process and have well defined edges, but despite hole damage, some morphological features of the cell are discernible. Presumptive mitochondria appear smaller in frozen sections than in conventional Araldite sections. Sections devoid of hole damage have indistinct edges and are presumed to have undergone transient thawing. Carbon coating of freeze-dried sections to exclude atmospheric moisture during transference of sections to the electron microscope (EM) appear unnecessary as regards preservation of morphological structure. The results are discussed in relation to the limitations of the method and the potential value of the technique.     

Histological and ultrastructural features of the rectum in Poecilimon cervus Karabağ, 1950 (Orthoptera: Tettigoniidae)

The morphology and ultrastructure of the rectum in Poecilimon cervus Karaba?, 1950 (Orthoptera, Tettigoniidae) were analyzed by light microscope, scanning (SEM) and transmission electron microscopes (TEM). The rectum is the final part of the digestive tract that plays an important role in water reabsorption in insects and so provides osmoregulation. In the transverse sections, six rectal pads and columnar epithelium can be distinguished. The cuticular intima lines the lumen at the apical side of the epithelium. In the cytoplasm, there are numerous mitochondria, some endocytic vesicles, secreting vesicles whose sizes differ according to the area in the cell, and a nucleus with globular in shape. With this study, we aimed to demonstrate the ultrastructure of the rectum of P. cervus and differences or similarities of with other species.     

Backscattered electron SEM imaging of cells and determination of the information depth

Backscattered electron imaging of HT29 colon carcinoma cells in a scanning electron microscope was studied. Thin cell sections were placed on indium‐tin‐oxide‐coated glass slides, which is a promising substrate material for correlative light and electron microscopy. The ultrastructure of HT29 colon carcinoma cells was imaged without poststaining by exploiting the high chemical sensitivity of backscattered electrons. Optimum primary electron energies for backscattered electron imaging were determined which depend on the section thickness. Charging effects in the vicinity of the SiO₂ nanoparticles contained in cell sections could be clarified by placing cell sections on different substrates. Moreover, a method is presented for information depth determination of backscattered electrons which is based on the imaging of subsurface nanoparticles embedded by the cells.     

Method for preparing thin sections of untreated equine hoof horn for electron microscopic examination

The preparation of hard tissues such as the equine hoof horn for electron microscopic examination is very difficult. In particular the penetration of fixatives and chemicals used during fixation and embedding is a problem. The objective of this study was to find and implement an alternative method enabling the preparation of high-quality thin sections of hoof horn and other hard tissue, which maintains the hard tissue ultrastructure and can be used for immuno-labeling. Compared to commonly used fixation and embedding techniques, the preparation of thin sections from untreated material method saves time and material and provides equivalent ultrastructural information. Furthermore, thin sections from untreated material are significantly larger and more homogeneous, more resistant to the electron ray, as well as more suitable for sectioning. The electron microscopical pictures obtained allow a comparison to previous test results achieved with fixed and embedded material. Using the preparation of thin sections from untreated material method, fixation and embedding artifacts are avoided, providing a clearer interpretation of the electron microscopical findings. Considerable advantages are achieved by using immunohistochemical techniques with untreated horn specimens because fixation invariably decreases antigenicity.     

Recent developments in analytical electron microscopy

Recent years have seen the way in which the analytical electron microscope has been applied to problems involving thin specimens in metallurgy, mineralogy and many branches of biology. The limits of sensitivity have been explored and its potential usefulness in these fields investigated. Problems concerning the interaction of electrons with the specimen are discussed in relation to the correct choice of operating conditions and specimen preparation. In biological work, frozen sections provide new information about subcellular elemental localization of mobile electrolytes, while analysis of tissue prepared by conventional means is used to detect physiological levels of some naturally occurring elements. Examination of mineral dispersions provides analysis of particles just 10 nm thick, visible only in transmission electron microscopy, and further work with thin metal foils confirms the value of high resolution transmission imaging as a complementary facility to micro-analysis. Work has been done to investigate the possibilities of improving sensitivities both by changing operational parameters and instrumental design, and the value of quantitation in thin specimen analysis explored.     

Freeze-substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry

Cryo-fixation followed by freeze-substitution without aldehyde or osmium fixation has been investigated as a method for preparing biological specimens with a view to minimizing antigenic alteration. Samples of both solid tissues (mouse small intestine and human kidney) and a human tumour cell line grown in vitro were rapidly frozen by impact (slammed) onto a copper block cooled with liquid nitrogen. They were freeze-substituted at ?80°C in methanol, and embedded at low temperature in Lowicryl K4M or HM20. Resin blocks were polymerized by ultraviolet light. Well-preserved ultrastructure was observed in the outer 10–15 μm of all samples. Positive immunocytochemical localization of fixation-resistant and fixation-labile antigens was obtained on sections of human kidney and the human breast tumour cell line ZR-75-1 at both light and electron microscope levels.     

The usefulness of glutaraldehyde-carbohydrazide copolymerization in biological specimen stabilization for scanning electron microscopy

Biological specimens can be prepared for scanning electron microscopy by means of copolymerizing the fixing agent glutaraldehyde with carbohydrazide prior to air drying. Such preparations are more stable in the electron microscope, show less internal cellular disruption and retain more of their native elemental composition than specimens prepared by means of dehydration and critical-point drying. Specimens observed in the scanning electron microscope can often be recovered for thin sectioning with no additional embedment, and can then be observed by means of transmission elecltron microscopy. The preparation (termed GACH) can be performed in almost any laboratory with no specialized equipment and, for the most part, may be carried out at room temperature. The technique appears to provide the promise of further research applications in scanning electron microscopy which may employ conjugated procedures of immunocytochemistry and cathodoluminescence as well as X-ray microanalysis in limited situations.     

Ultrastructural and electron spectroscopic analyses of cyanobacteria and bacteria

The extracellular sheath material and some intracellular cell components of cyanobacteria and phosphate-accumulating sewage bacteria were analysed by electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). The specimens were embedded in water-soluble Nanoplast resin without any previous fixation and ultrathin sections were examined in a Zeiss CEM 902 microscope. A high sulphur content was detected in the inner sheath of the cyanobacterium Gloeothece. The elemental composition of some cell components and inclusion bodies, such as carboxysomes and cyanophycin, was determined by ESI and EELS. In addition, the phosphate content in specific granules of phosphate-accumulating sewage bacteria was estimated by EELS and nuclear magnetic resonance spectroscopy.     

A resin slide technique to select fixed embedded cells for transmission electron microscopy

A simple technique is described to resection, for electron microscopy, 2–6 μm sections that have been observed under the light microscope. The technique can be used to select cells on the basis of light microscope ultrastructure, histochemistry and autoradiography prior to electron microscopy.     

Determination of experimental and theoretical kASi factors for a 200-kV analytical electron microscope

The relative sensitivity of an analytical electron microscope and energy-dispersive x-ray detector to x-rays of various elements is investigated through an extensive k_ASi factor study. Elemental standards, primarily National Bureau of Standards multielement research glasses, were dry-ground into submicrometer-sized particles and analyzed at 200 kV accelerating potential. The effect of self-absorption of x-rays by the particle has been corrected for, allowing the experimental k_ASi factors from this study to approximate those that could be obtained from “infinitely thin” specimens. Whenever possible, elemental k-factors were determined by the analysis of many (up to a maximum of nine) different standard materials. Experimental k_ASi factors were calculated for a wide range of K_α, L_α, and M_α x-ray lines. For comparison, theoretical k_ASi factors, employing a variety of ionization cross sections, were computed. Good agreement is obtained between several of the theoretical k-factor models and the experimental results. Mass volatilization of Na and K from the small glass particles during analysis is discussed, as are observations that the grinding and/or dispersing of standard materials in a liquid (such as ethanol) may promote leaching of certain elements from the particle matrix.     

A cryostat approach to ultrathin "dry" frozen sections for electron microscopy: a morphological and x-ray analytical study

Conventional preparative procedures for the examination of tissues in the electron microscope involve the use of fixatives, dehydration in alcohol or acetone, embedding in plastics and staining. Such procedures remove soluble components and are therefore often unsuitable for chemical analysis of naturally occurring electrolytes. Ultrathin frozen sections of unfixed, unembedded biological tissue can be cut onto dry glass knives, freeze-dried and viewed in the electron microscope without staining. Morphological detail is sufficient to identify cell types and ultrastructure. X-ray microanalysis in the analytical electron microscope (EMMA-4) has shown that highly soluble electrolytes can be detected and that intracellular compartments are retained.     

Why low temperature embedding for X-ray microanalytical investigations?

Freeze-drying followed by infiltration with resin and polymerization by UV light at low temperatures and under constant vacuum conditions is an alternative tissue preparation technique for microprobe analysis. Embedding is carried out with the nonpolar low-temperature embedding resin (Lowicryl HM20) which allows infiltration and polymerization at temperatures down to ?50°C. Sections of low temperature embedded material can be cut dry at ?60°C or at room temperature. Sectioning at low temperatures is an alternative for preparations that are difficult to cut at room temperature. The morphological preservation is adequate for the identification of structures such as mitochondria, lysosomes and different types of endoplasmic reticulum in liver cells. Some physical properties of Lowicryl resins, such as mass loss under the electron beam and high contrast, are positive characteristics for the analysis of semi-thick sections. No significant differences in the elemental composition could be detected between tissue which was freeze-dried or freeze-substituted prior to embedding. Freeze-drying is less time consuming. By avoiding contact with organic solvents the risks of ion loss and redistribution are diminished. In contrast to freeze-dried thin cryosections, low temperature embedded material can be sectioned for light microscopy and areas of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogeneous cell populations. The initial preparative step—the cryofixation— determines to a high degree the morphological preservation of freeze-dried and embedded tissue.