Freeze-substitution of scleractinian coral for confocal scanning laser microscopy and X-ray microanalysis

Freeze-substitution in combination with confocal scanning laser microscopy provides a unique means by which the microstructure of undecalcified, and chemically untreated, polyps of scleractinian corals can be examined free of preparation artefacts. Mucocytes and intercellular spaces are particularly well preserved and the relationships of cell layers to each other and to the skeleton are undisturbed. Freeze-substitution also permits X-ray microanalysis of bulk samples and thin sections, a procedure which has hitherto been impossible to carry out on corals except on fixed tissue samples. Analyses indicated a high retention of Na⁺ and Cl^? in spaces thought to be filled with fluid similar in composition to sea water. This increases confidence in freeze-substitution as a means of retaining diffusible ions. Zooxanthellae contained metal/Ca ratios within the range of those previously reported for extracted zooxanthellae. It is shown that the mucocytes contain very high concentrations of S, K, Ca and Sr which are specifically localized in mucous granules. The concentrations differ between, and are characteristic of, different epithelial cell layers. Remarkably good correspondence was obtained between the two entirely different X-ray analytical methods. This is the first time such a comparision between methods has been made. It is suggested that the uptake of transepithelially transported Ca²⁺ and Sr²⁺ by mucocytes may be a means of regulating the deposition of these ions in the skeleton.     

Localization of metals in cells of saprophagous soil arthropods (Isopoda, Diplopoda, Collembola)

This review summarizes results on the intracellular distribution of metals in cells of woodlice (Isopoda), millipedes (Diplopoda), and springtails (Collembola), which are three major groups of saprophagous arthropods contributing to the turnover of soil organic matter. Although the impact of metals and also metal pollution has inevitably been shown at levels of higher biological organization than subcellular mechanisms in these animal groups, the aim of this review is to focus exclusively on storage sites and aspects of intracellular metal metabolism. Thus, methodologically, results obtained by microscopical techniques such as histochemistry, X-ray microanalysis, energy filter transmission electron microscopy, or laser microprobe mass spectrometry were given preference. Results from atomic absorption spectrophotometry of cellular fractions were kept to a minimum. In all three taxa, the main intracellular metal storage sites are various types of "granules" which are widely distributed throughout cell types associated with the digestive system.     

Subcellular imaging of isotopically labeled carbon compounds in a biological sample by ion microprobe (NanoSIMS)

Here we demonstrate the technique of nanoscale secondary ion mass spectrometry, utilizing the Cameca NanoSIMS50 ion microprobe, to detect and image the metabolism of an isotopically labeled compound (NaH(13)CO(3)) in a biological sample. In particular, we have designed and verified protocols for imaging the subcellular distribution and determining the relative abundance of labeled (13)C, within the coral Galaxea fascicularis. Analyses were conducted on 1-mum thick sections of resin-embedded material, using both scanned (mapping) and static (spot analysis) Cs(+) primary ion beam of approximately 100 nm diameter. Using these samples we establish that NanoSIMS has adequate mass resolution to reliably distinguish (13)C from potential isobaric interference by (12)C(1)H and that data extracted from ion maps are comparable to those acquired by spot analyses. Independent of the method of acquisition, ratioing of (13)C to the naturally abundant (12)C is essential if meaningful data, which can be statistically compared to standard and control samples, are to be obtained. These results highlight the potential of NanoSIMS for intracellular tracking of a variety of organic and inorganic compounds labeled with stable isotopes of C, N, O, S, P, and halogens.     

Quantitative 3D imaging of yeast by hard X-ray tomography

Full-field hard X-ray tomography could be used to obtain three-dimensional (3D) nanoscale structures of biological samples. The image of the fission yeast, Schizosaccharomyces pombe, was clearly visualized based on Zernike phase contrast imaging technique and heavy metal staining method at a spatial resolution better than 50 nm at the energy of 8 keV. The distributions and shapes of the organelles during the cell cycle were clearly visualized and two types of organelle were distinguished. The results for cells during various phases were compared and the ratios of organelle volume to cell volume can be analyzed quantitatively. It showed that the ratios remained constant between growth and division phase and increased strongly in stationary phase, following the shape and size of two types of organelles changes. Our results demonstrated that hard X-ray microscopy was a complementary method for imaging and revealing structural information for biological samples.     

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.     

A comparison of subcellular element concentrations in frozen-dried, plastic-embedded, dry-cut sections and frozen-dried cryosections

Biological X-ray microanalysis of diffusible elements within cellular and subcellular compartments requires preparation methods to retain electrolytes in the compartments they occupied in vivo. X-ray microanalysis of frozen-dried, plastic-embedded samples has been used to quantitate electrolytes at the cellular level. We have compared the subcellular elemental distribution in dry cut sections from such samples with that in ultrathin frozen-dried cryosections. Rat pancreases were quench-frozen onto a helium-vapor-cooled copper block. Cryosections were cut at 130-150 K, transferred using a Gatan cold stage, frozen-dried in the column and analysed at 190 K. Tissue fragments were frozen-dried at 190 K, and cut on a dry knife at 293 K. Both samples provided images permitting unambiguous identification of all major compartments except the Golgi complex. Intracellular potassium-to-sodium ratios obtained on frozen-dried plastic-embedded sections were lower than for cryosections (e.g. 1.77 in basal cytoplasm in plastic sections as compared to 4.34 for cryosections) and varied with the pre-embedding procedure (e.g. 1.77 in formaldehyde-fixed as compared to 2.87 in osmium-fixed plastic sections). Potassium gradients between adjacent organelles were large in cryosections and insignificant in plastic-embedded material. Higher cytoplasmic phosphorus, potassium and sulfur concentrations were observed in cryosections. Therefore, a redistribution of electrolytes and covalently bound elements occurred subcellularly in the plastic sections. Calcium was quantifiable in most organelles in cryosections but the plastic lowered sensitivity too much to permit routine calcium quantification. We conclude that in our hands frozen-dried, plastic-embedded samples were compromised and provided lower sensitivity than cryosections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Imaging Coral II: Using Ultrasound to Image Coral Skeleton

We report on imaging of coral skeletal densities using pulse-echo ultrasound. Focused ultrasound transducers with frequencies ranging from 1 to 5 MHz were used to image coral skeletal samples. Each transducer was scanned over the surface of a coral and the reflected signal from the coral was recorded. Post-processing of the ultrasound signal was used to generate images of the acoustic impedance of the coral—a quantity that is related to the density of the coral. The ultrasound images were qualitatively consistent with X-ray images of the coral samples—X-ray absorption is also related to coral density. Both images showed a banding structure related to the annual growth cycle of the coral. The results indicate that ultrasound can be used for nondestructive imaging of coral skeletal structure. The ultimate goal would be an in situ system for quantitatively measuring skeletal densities of live corals underwater. The skeletal density of several massive reef coral species is strongly correlated with ocean temperature. A time series reconstructions of the skeletal density of long lived specimens would provide a history of surface ocean temperatures over time periods extending over several centuries, far beyond the available instrumental record.     

Cryopreparation of mammalian tissue for X-ray microanalysis in STEM

A cryopreparation technique for studies of ultrastructure and distribution of diffusible elements in biological tissue is described. Electron microscopical contrast and characteristic X-ray spectra are found to be poor in completely frozen-hydrated ultrathin cryosections of fresh chemically untreated tissue. Both STEM contrast and detection of characteristic X-rays are enhanced by careful freeze-drying in the microscope. Although the ultrastructure is affected by ice crystals, intracellular compartments can be identified by STEM without staining and studied by X-ray microanalysis.     

Calibration for quantitative X-ray microanalysis of skeletal muscle cells in culture.

Skeletal muscle in culture is used to demonstrate that the intracellular concentration of elements in tissue culture cells, especially of diffusible ions such as sodium, chlorine, potassium, and calcium can be measured by X-ray microanalysis in the scanning electron microscope. Quantitative analysis is possible by comparison with X-ray spectra of cells incubated in electrolyte solutions of known concentration. The minimum detectable concentration is approximately 2 mM.     

钙、镧和锶复合添加对AZ91D合金显微组织及力学性能的影响

利用X射线衍射仪、光学显微镜、扫描电镜和能谱仪等研究了在AZ91D合金中复合添加钙?镧?锶对其显微组织和力学性能的影响。结果表明:在AZ91D合金中同时加入钙、镧、锶后,其显微组织相比于钙、镧或钙、锶二元添加得到组织明显细化,析出相Mg17Al12由半连续分布变为断续状;其抗拉强度得到显著提高,达到了201 MPa,比AZ91D合金的提高了28.8%。     

3D visualization of subcellular structures of Schizosaccharomyces pombe by hard X-ray tomography

Cellular structures of the fission yeast, Schizosaccharomyces pombe, were examined by using hard X-ray tomography. Since cells are nearly transparent to hard X-rays, Zernike phase contrast and heavy metal staining were introduced to improve image contrast. Through using such methods, images taken at 8 keV displayed sufficient contrast for observing cellular structures. The cell wall, the intracellular organelles and the entire structural organization of the whole cells were visualized in three-dimensional at a resolution better than 100 nm. Comparison between phase contrast and absorption contrast was also made, indicating the obvious advantage of phase contrast for cellular imaging at this energy. Our results demonstrate that hard X-ray tomography with Zernike phase contrast is suitable for cellular imaging. Its unique abilities make it have potential to become a useful tool for revealing structural information from cells, especially thick eukaryotic cells.     

Quantitative X-ray microanalysis of biological specimens

Qualitative X-ray microanalysis of biological specimens requires an approach that is somewhat different from that used in the materials sciences. The first step is deconvolution and background subtraction on the obtained spectrum. The further treatment depends on the type of specimen: thin, thick, or semithick. For thin sections, the continuum method of quantitation is most often used, but it should be combined with an accurate correction for extraneous background. However, alternative methods to determine local mass should also be considered. In the analysis of biological bulk specimens, the ZAF-correction method appears to be less useful, primarily because of the uneven surface of biological specimens. The peak-to-local background model may be a more adequate method for thick specimens that are not mounted on a thick substrate. Quantitative X-ray microanalysis of biological specimens generally requires the use of standards that preferably should resemble the specimen in chemical and physical properties. Special problems in biological microanalysis include low count rates, specimen instability and mass loss, extraneous contributions to the spectrum, and preparative artifacts affecting quantitation. A relatively recent development in X-ray microanalysis of biological specimens is the quantitative determination of local water content.     

X-ray microanalysis of ultrathin frozen and freeze-dried sections of human sperm cells

X-ray microanalysis of air dried and ultrathin frozen and frozen dried sections of human sperm cells has indicated a large deviation of elemental composition between cells in a single sample and between samples. Analysis of air dried cells indicates a similar subcellular elemental distribution to that found in sectioned material. Sodium to potassium ratios are found to be similar in both preparations. Air drying is considered a valid method for the preparation of sperm cells for analysis.     

Correlated measurements of free and total intracellular calcium concentration in central nervous system neurons.

Transient changes in the intracellular concentration of free calcium ([Ca2+])i) act as a trigger or modulator for a large number of important neuronal processes. Such transients can originate from voltage- or ligand-gated fluxes of Ca2+ into the cytoplasm from the extracellular space, or by ligand- or Ca2+(-)gated release from intracellular stores. Characterizing the sources and spatio-temporal patterns of [Ca2+]i transients is critical for understanding the role of different neuronal compartments in dendritic integration and synaptic plasticity. Optical imaging of fluorescent indicators sensitive to free Ca2+ is especially suited to studying such phenomena because this approach offers simultaneous monitoring of large regions of the dendritic tree in individual living central nervous system neurons. In contrast, energy-dispersive X-ray (EDX) microanalysis provides quantitative information on the amount and location of intracellular total, i.e., free plus bound, calcium (Ca) within specific subcellular dendritic compartments as a function of the activity state of the neuron. When optical measurements of [Ca2+]i transients and parallel EDX measurements of Ca content are used in tandem, and correlated simultaneously with electrophysiological measurements of neuronal activity, the combined information provides a relatively general picture of spatio-temporal neuronal total Ca fluctuations. To illustrate the kinds of information available with this approach, we review here results from our ongoing work aimed at evaluating the role of various Ca uptake, release, sequestration, and extrusion mechanisms in the generation and termination of [Ca2+]i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. Our observations support the long-standing speculation that the dendritic endoplasmic reticulum acts not only as an intracellular Ca2+ source that can be mobilized by a signal cascade originating at activated synapses, but also as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca2+ influx.     

A sample preparation for quantitative determination of magnesium in individual lymphocytes by electron probe X-ray microanalysis

We present a sample preparation method for measuring magnesium in individual whole lymphocytes by electron probe X-ray microanalysis. We use Burkitt's lymphoma cells in culture as the test sample and compare X-ray microanalysis of individual cells with atomic absorption analysis of pooled cell populations. We determine the magnesium peak-to-local continuum X-ray intensity ratio by electron probe X-ray microanalysis and calculate a mean cell magnesium concentration of 39± 19 mmol/kg dry weight from analysis of 100 cells. We determine a mean cell magnesium concentration of 34 ±4 mmol/kg dry weight by atomic absorption analysis of pooled cells in three cell cultures. The mean cell magnesium concentrations determined by the two methods are not significantly different. We find a 10% coefficient of variation for both methods of analysis and a 30% coefficient of variation in magnesium concentration among individual cells by electron probe X-ray microanalysis. We wash cells in ammonium nitrate for microanalysis or in buffered saline glucose for atomic absorption analysis. We find cells washed in either solution have the same cell viability (85%), recovery (75%), cell volume (555 μm³) and cytology. We air dry cells on thin film supports and show by magnesium X-ray mapping that magnesium is within the cells. We conclude that: (a) our microanalysis cell preparation method preserves whole intact lymphocytes; (b) there is no systematic difference in results from the two methods of analysis; (c) electron probe X-ray microanalysis can determine the variation in magnesium concentration among individual cells.     

X-ray microanalysis of ion distribution in frozen salt/dextran droplets after freeze-substitution and embedding in anhydrous conditions

A model system using dextran droplets of different salt solutions either frozen together, or sandwiched together after freezing and then freeze-substituted, embedded and dry sectioned has been investigated by X-ray microanalysis. X-ray maps and spot measur ements taken in transects through the interface of both droplets have shown that the P, K and Ca remain well localized in their original droplet. This validates freeze-substitution as a method for localization of these elements in biological samples. Results with Na were more variable and not always explainable. Success was achieved by the use of super-dry solvents and maintenance of a dry environment at all stages. We emphasize the need to avoid water contamination not only during freeze-substitution but also during sectioning, storage and section transfer to the electron microscope.     

Use of primary cell cultures and intact isolated glandular epithelia for X-ray microanalysis

Changes in the elemental composition of cells during isolation of glandular epithelia were studied by electron probe X-ray microanalysis. Fine chopping of rat submandibular gland followed by enzymatic treatment for 15 min caused marked increases in Na and Cl and a decrease in K concentrations in acinar cells. After enzymatic treatment for 50 min, Na, Cl and K concentrations returned to close to the control level. Mechanical disaggregation of the acinar clumps following enzymatic treatment resulted again in minor increases in Na and Cl and a marked decrease in K concentration. Exposure of isolated acini to cholinergic stimulation in vitro resulted in secretion of Cl and K from the acinar cells. Dissection of the sweat gland from human skin caused a decrease in the K/Na ratio. Incubation of the gland for 30–45 min with collagenase gave rise to a gradual decrease in the K/Na ratio. After mechanical separation of the gland into the secretory coil and reabsorptive duct, a further reduction of the K/Na ratio was seen. However, the duct cells had a much lower K/Na ratio and higher Ca concentration than the coil cells. In primary cultures, the K/Na ratios of the coil and duct cells returned to the in situ level. The elemental composition of sweat gland cells incubated in collagenase-containing medium was no different from that in cells incubated in collagenase-free medium. In the intact collagenase-isolated tissue, Cl^? secretion in the coil was elicited by carbachol but not by cAMP, whereas in the duct cells the reverse was the case. In primary cell cultures, Cl^? efflux in both coil and duct cells could be elicited by both carbachol and cAMP. In conclusion, although changes in elemental composition of gland cells during the isolation procedure occur, physiological responses can be detected. When primary cell cultures are used, it should be borne in mind that cultured cells may have physiological properties different from those of the intact tissue.     

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.     

Gallium replication in aquatic and nonaquatic organism scanning electron microscopy.

A technique using pure gallium metal as a replication material is reported for biological surface scanning electron microscopy (SEM). The technique first directly enables aquatic organisms in water to be replicated due to gallium's low melting point and, second, reproduces surface structures and images of the two-dimensional (2D) distribution of substances transferred from the original surface to the gallium surface due to gallium's high surface tension. An aquatic protozoan in water was directly replicated to show its typical surface structures. The technique was then used to visualize human hair surface structures and 2D transferred substance distribution using X-ray microanalysis.     

X-ray microanalysis of wet biological specimens in the environmental scanning electron microscope. 1. Reduction of specimen distance under different atmospheric conditions

X-ray microanalysis of non-biological and biological specimens was carried out in the environmental scanning electron microscope (ESEM) under different conditions of specimen distance (the distance travelled by the electron probe within the specimen chamber) and chamber atmosphere. Using both water vapour and argon atmospheres, it was shown that reduction in specimen distance had no effect on atmospheric gas X-ray signal in either case. Unlike water vapour, increased levels of argon (up to 10 torr) caused a marked depression of specimen P/B ratios, with a decrease in both characteristic and background (continuum) counts. These effects in argon were not altered by reduction in specimen distance. Specimen distance was important in relation to beam skirting and elemental analysis. With an extended assembly (short specimen distance), beam skirting in a water-vapour atmosphere was much reduced – leading to enhanced element detectability in a discrete biological specimen (Anabaena cyclindrica).