Application of scanning electron microscopy to x-ray analysis of frozen- hydrated sections. III. Elemental content of cells in the rat renal papillary tip

The electrolyte and water content of cellular and interstitial compartments in the renal papilla of the rat was determined by x-ray microanalysis of frozen-hydrated tissue sections. Papillae from rats on ad libitum water were rapidly frozen in a slush of Freon 12, and sectioned in a cryomicrotome at -30 to -40 degrees C. Frozen 0.5- micrometer sections were mounted on carbon-coated nylon film over a Be grid, transferred cold to the scanning microscope, and maintained at - 175 degrees C during analysis. The scanning transmission mode was used for imaging. Structural preservation was of good quality and allowed identification of tissue compartments. Tissue mass (solutes + water) was determined by continuum radiation from regions of interest. After drying in the SEM, elemental composition of morphologically defined compartments (solutes) was determined by analysis of specific x-rays, and total dry mass by continuum. Na, K, Cl, and H2O contents in collecting-duct cells (CDC), papillary epithelial cells (PEC), and interstitial cells (IC) and space were measured. Cells had lower water content (mean 58.7%) than interstitium (77.5%). Intracellular K concentrations (millimoles per kilogram wet weight) were unremarkable (79-156 mm/kg wet weight); P was markedly higher in cells than in interstitium. S was the same in all compartments. Intracellular Na levels were extremely high (CDC, 344 +/- 127 SD mm/kg wet weight; PEC, 287 +/- 105; IC, 898 +/- 194). Mean interstitial Na was 590 +/- 119 mm/kg wet weight. CI values paralleled those for Na. If this Na is unbound, then these data suggest that renal papillary interstitial cells adapt to their hyperosmotic environment by a Na-uptake process.     

Elemental Composition and Water Content of Neuron and Glial Cells in the Central Nervous System of the North American Medicinal Leech (Macrobdella decora)

Elemental (Na, P, S, Cl, K, Ca, Mg) composition and water content of neurons and glial cells of the leech (Macrobdella decora) were determined by x-ray microanalysis of frozen hydrated and dried section techniques. Results are reported as elemental mass fractions (mass/mass) and water content as percent mass. Specific cell compartments and cell types had distinct elemental patterns and water content which suggests that chemical composition of specific cell types is unique and may represent an expression of cell differentiation analogous to morphological specialization. Water content of cells was also cell specific and ranged from 55% (neurons) to 90% (vacuolated zone of glial cells). K and Na were present in concentrations greater than predicted by ion-selective microelectrode measurements, indicating that not all the K and Na were simultaneously accessible to such electrodes.     

The structure of organelles of the endocytic pathway in hydrated cryosections of cultured cells

The structure of cellular organelles, in particular those involved in endocytosis, was studied by electron microscopy with hydrated cryosections. In this technique no chemical treatment is used, and the native structure of organelles can be observed in sections viewed at temperatures below -140 degrees C, using a cold stage accessory on the electron microscope. The compartments of the endocytic pathway were prelabeled with gold markers in the living cell, facilitating the identification of different structures in the cryosections. The structure of most identifiable cellular organelles, including those involved in endocytosis, appeared very similar in the hydrated cryosections to that seen after conventional plastic and cryosections of chemically fixed cells. In particular, the internal membranes of the structure we refer to as the prelysosomal compartment (Griffiths et al., Cell 52, 329-341 (1988] could be clearly visualized in these sections indicating that the organization of these membranes is not a consequence of the chemical fixation process.     

Optical sectioning of microbial biofilms.

Scanning confocal laser microscopy (SCLM) was used to visualize fully hydrated microbial biofilms. The improved rejection of out-of-focus haze and the increased resolution of SCLM made it preferable to conventional phase microscopy for the analysis of living biofilms. The extent of image improvement was dependent on the characteristics of individual biofilms and was most apparent when films were dispersed in three dimensions, when they were thick, and when they contained a high number of cells. SCLM optical sections were amenable to quantitative computer-enhanced microscopy analyses, with minimal interference originating from overlying or underlying cell material. By using SCLM in conjunction with viable negative fluorescence staining techniques, horizontal (xy) and sagittal (xz) sections of intact biofilms of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Vibrio parahaemolyticus were obtained. These optical sections were then analyzed by image-processing techniques to assess the distribution of cellular and noncellular areas within the biofilm matrices. The Pseudomonas biofilms were most cell dense at their attachment surfaces and became increasingly diffuse near their outer regions, whereas the Vibrio biofilms exhibited the opposite trend. Biofilms consisting of different species exhibited distinctive arrangements of the major biofilm structural components (cellular and extracellular materials and space). In general, biofilms were found to be highly hydrated, open structures composed of 73 to 98% extracellular materials and space. The use of xz sectioning revealed more detail of biofilm structure, including the presence of large void spaces within the Vibrio biofilms. In addition, three-dimensional reconstructions of biofilms were constructed and were displayed as stereo pairs. Application of the concepts of architectural analysis to mixed- or pure-species biofilms will allow detailed examination of the relationships among biofilm structure, adaptation, and response to stress.     

Cytochemical techniques and energy-filtering transmission electron microscopy applied to the study of parasitic protozoa

The study of parasitic protozoa plays a major role in cell biology, biochemistry and molecular biology. Numerous cytochemical techniques have been developed in order to unequivocally identify the nature of subcellular compartments. Enzyme and immuno-cytochemistry allow the detection of, respectively, enzymatic activity products and antigens in particular sites within the cell. Energy-filtering transmission electron microscopy permits the detection of specific elements within such compartments. These approaches are particularly useful for studies employing antimicrobial agents where cellular compartments may be destroyed or remarkably altered and thus hardly identified by standard methods of observation. In this regard cytochemical and spectroscopic techniques provide valuable data allowing the determination of the mechanisms of action of such compounds. Published: August 4, 2001     

Post-embedding localization of glycoconjugates by means of lectins on thin sections of tissues embedded in LR white

Summary A simple post-embedding technique for the electron microscopical detection of lectin-binding sites using thin sections of tissues embedded in the resin LR White is described. With this technique, no prior etching of the sections is necessary. The cellular fine structure is well preserved and permits close correlation of the labelling to distinct cellular compartments. After mild aldehyde fixation (4% formaldehyde and 0.5% glutaraldehyde for 30 min), enterocyte brush border, vesicles and lysosomes as well as goblet cell Golgi apparatus and mucin are intensely stained after 30–60 min.The hydrophilia and penetrability of LR White is shown by the formation of oxidized diaminobenzidine reaction product arising from horseradish peroxidase-conjugated lectins. The precipitate not only covers the surface of the sections but is also formed within the resin, as is revealed on cross-sections through thin and semithin sections. The addition of 0.2m solutions of the appropriate inhibitory sugars prevented staining, which indicates a specific binding. Examples are given of the binding of gold-, ferritin- and peroxidase-conjugated lectins for the purpose of detecting glycoconjugates in various intracellular compartments.     

Quantitative Scanning Transmission Electron Microscopy of Ultrathin Cryosections: Subcellular Organelles in Rapidly Frozen Liver and Cerebellar Cortex

Freeze-dried, ultrathin cryosections of directly frozen mouse liver and brain have been prepared and characterized by low-dose dark-field scanning transmission electron microscopy (STEM). These improved cryosections gave images comparable to those from conventional plastic sections. They were thin enough (1.0 elastic mean free path) to use established dark-field techniques, modified for thickness-dependent nonlinearities, to measure the dry mass fraction of individual organelles, and hence to deduce their water content. Digital STEM imaging in combination with electron and X-ray spectroscopy has important biological applications, as illustrated by studies on calcium regulation in Purkinje neurons. Calcium concentrations per unit dry weight of dendritic compartments were determined by the peak/continuum method of energy-dispersive X-ray spectroscopy (EDXS), which necessarily overstates elemental concentrations because of beam-induced mass loss. The dry mass content of organelles at low dose and the percentage of dry mass retained after analysis at high dose were as follows: mitochondria (46.0 g dry mass/100 g hydrated mass, 67% mass retained); endoplasmic reticulum (27.9 g/100 g, 57%); and cytoplasm (16.3 g/100 g, 41%). These values were used to correct elemental concentrations for mass loss. Results indicated that the major calcium storage organelle in Purkinje cell dendrites is the endoplasmic reticulum, of which there are two types distinguished by their levels of calcium. Parallel electron energy loss spectroscopy of dendritic organelles corroborated EDXS measurements, with an improved sensitivity that indicates the feasibility of quantitative calcium mapping.     

The X-ray microanalysis of frozen-hydrated sections in scanning electron microscopy: An evaluation

The present status of the technique to measure concentrations of electrolyte elements and dry mass in 1 μm thick frozen-hydrated sections of soft biological tissues with electron probe X-ray microanalysis in a scanning electron microscope is critically reviewed. The technique is to quench-freeze fresh specimens to < − 180°C, cut 1 μm thick hydrated cryosections −70°C), transfer on to a cold stage (< −170°C) of a suitable microanalytical arrangement, obtain scanning transmission images to identify the cell and tissue compartments, locate an electron probe (several μm² to 100 nm) on the areas of interest and collect X-ray quanta. The X-ray quanta are collected with suitable spectrometers (WDS and EDS) and processed with a computer using a comprehensive programme based on continuum normalization procedures (‘Hall’ programme). The cryosections are analysed first in a hydrated state and second after dehydration within the microanalyser column to obtain directly elemental concentrations in mM kg⁻¹ wet wt and mM kg⁻¹ dry wt of the compartments identified under the beam. The local water-fractions are estimated and the elemental concentrations converted into mM 1⁻¹ water. In the past 7 years the technique has been applied to obtain fully quantitative information on Na, K, Cl, P, S, Ca and H₂O in more than ten types of tissue.     

Analysis of tracer experiments: VIII. Integro-differential equation treatment of partly accessible,partly injectable multicompartment systems

An integro-differential equation treatment of multi-compartment systems is developed which permits formal analysis of the incomplete data which is available from partly accessible, partly injectable systems. New transport functions are defined which can be obtained directly from the experimental data. These functions serve to characterize the communication and topology between different accessible compartments and also the reentrant contributions from inaccessible sites. The method gives solutions consistent with those of the differential equation approach when the system is uniformly contiguous and accessible, more complete solutions than those of the integral equation approach when all measured compartments are injectable, and in addition provides complete or partial solutions for certain otherwise analytically intractable systems. Detailed numerical illustrations of the method are given.     

Distribution of Elements in Rat Peripheral Axons and Nerve Cell Bodies Determined by X-Ray Microprobe Analysis

X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, myelin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellular distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.     

Osmotic and motional properties of intracellular water as influenced by osmotic swelling and shrinkage of Xenopus oocytes

Experiments were done on fully grown Xenopus oocytes to determine the extent and the properties of cellular water of hydration. The studies involved the osmotic shrinking and swelling of the oocytes under known osmotic pressure as well as proton NMR spectral, titration, and free induction decay analyses. Studies were done both on whole oocytes and on subcellular fractions. The results show that little if any of the oocyte water in situ has the motional or osmotic properties expected of pure "bulk" water. Four distinct water of hydration compartments were found and defined on the basis of distinct hydrogen bounding mechanisms. Some of the water in yolk platelets was found not to be in fast exchange with other water compartments. Osmotic shrinkage of oocytes caused an adaptive decrease in the bound water of hydration compartments. This osmotically induced decrease is attributed to decreased surface area available for the hydrogen bounding of water molecules on cellular proteins.     

Application of scanning electron microscopy to x-ray analysis of frozen- hydrated sections. II. Analysis of standard solutions and artificial electrolyte gradients

New specimen handling and analytic techniques for the application of x- ray microanalysis to studies of cell and organ biology have been recently described (Saubermann et al., 1981, J. Cell Biol. 88:257-267). Based on these techniques, absolute quantitative standardization has been established through x-ray analysis of frozen-hydrated and then dried sections of independently measured standard solutions of elements. These experiments demonstrate that the specific techniques employed have a probable error of less than 10%. Artificial electrolyte gradients established in gelatin were subjected to analysis to determine whether there was elemental displacement under non-membrane- limited conditions at the temperatures employed for sectioning (-30 degrees to -40 degrees C). No significant difference was observed between such gradients in serial sections cut at -30 degrees and -80 degrees C. Similarly, no additional ice-crystal-damage artifact was found in sections cut at -30 degrees C when compared with sections cut at -80 degrees C. Thus, in terms of ice-crystal size, gradient maintenance, and compartmental differentiation, cryosectioning at -30 degrees to -40 degrees C was not associated with redistribution incompatible with 1- to 2-micrometers spatial resolution, and absolute measurements of elemental concentration were practical within regions of this size.     

Elemental analysis of histochemically defined cells in the earthworm Lumbricus terrestris.

A method for preparation of biological specimens for electron probe X-ray microanalysis is described, that aims at retaining the original elemental distribution within the tissue at the cellular level. The tissue is without any chemical fixation, quench-frozen, and 16-micron sections are prepared with a conventional cryomicrotome, transferred to a carbon specimen holder and freeze-dried. Adjacent serial sections, collected on glass slides and stained with various histological procedures, are used to correlate the data obtained by X-ray microanalysis with other histochemical information on the same cell or tissue. To demonstrate the possibilities of the method, sections of the earthworm Lumbricus terrestris were analyzed. In the chloragogenous cells, high concentrations of Ca, Zn and P were found. The inner and outer muscle layer show slightly different properties, both with regard to elemental composition and to myofibrillar ATPase activity.     

Quantitative histology of the rat thyroid. Influence of histologic techniques on the morphometric data

The influence of various histologic techniques on the results obtained by morphometric analysis of the rat thyroid gland was studied. The limits of thyroid follicles were more clearly defined in both silver-impregnated paraffin-embedded sections and resin-embedded semithin sections than in routinely stained paraffin-embedded sections, thus enabling more accurate measurements of thyroid structures. Due to its simplicity, the silver impregnation method is clearly useful for histomorphometric studies when large numbers of measurements are involved. C cells were easily identified in paraffin-embedded sections by immunohistochemical staining. The measurement of interstitial tissue in sections without immunostaining of C cells led to an overestimation of the volume fraction of interstitial tissue.     

Pores of Kohn are filled in normal lungs: low-temperature scanning electron microscopy.

Interalveolar pores of Kohn, small uniform-sized epithelium-lined openings in alveolar walls of normal lung, have historically been demonstrated with electron-microscopic techniques that remove water. We show these pores to be present but almost invariably filled with material when water and surfactant are preserved in frozen hydrated lung examined with low-temperature scanning electron microscopy. In the normal mouse, 16 open empty pores per alveolus were found in instillation-fixed dried lung vs. less than 1 per alveolus in frozen hydrated lungs (P less than 0.001). In the normal rat, 13 pores were seen per alveolus in instillation-fixed dried lung vs. less than 1 per alveolus in frozen hydrated lungs (P less than 0.001). We suggest that pores of Kohn 1) function primarily as conduits for interalveolar movement of alveolar liquid, surfactant components, and macrophages, 2) provide distributed sites for tubular myelin storage without increasing gas diffusion pathway thickness in the alveolar subphase itself, and 3) do not function as pathways for collateral ventilation during normal breathing in the absence of atelectasis or obstruction.     

Staining Paraffin Sections Without Prior Removal of the Wax

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10-20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.     

Isolation of Avian Trypanosomes by Selective Centrifugation and Subsequent Processing for Electron Microscopy

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10–20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.     

Histological analysis and staining techniques modified and verified on ancient mummified tissues to study microorganism infestations.

The purpose of this work is to give a brief account of the possibility to estimate the preservation of human mummified tissues using histological analysis. This method can be useful to identify injuries and to plan qualified conservative actions on ancient human remains. Some preliminary results are presented here regarding the study on 16 ancient mummies from the Egyptian Museum of Turin. Samples of mummified tissues were taken without damaging the remains; they were hydrated again and dyed with histological techniques which were specifically modified and verified in same cases. Our research identifies some agents (biological and chemical) that appear to be responsible, among others, of mummified tissue destruction. The microscopic examination reveals features that might refer to fungal and bacterial infestation. Using special staining methods on seried sections we were able to suspect, at least in one case, the presence of biologically active forms. Microbiological assays confirm the vitality of fungi. Histological tissue analysis can then be useful to guide any conservative intervention for preservation and protection of the integrity of biological remains from museum collections.     

A Stereological Approach for Estimation of Cellular Immunogold Labeling and Its Spatial Distribution in Oriented Sections Using the Rotator

Particulate gold labeling applied to ultrathin sections is a powerful approach for locating cellular proteins and lipids on thin sections of cellular structures and compartments. Effective quantitative methods now allow estimation of both density and distribution of gold labeling across aggregate organelles or compartment profiles. However, current methods generally use random sections of cells and tissues, and these do not readily present the information needed for spatial mapping of cellular quantities of gold label. Yet spatial mapping of gold particle labeling becomes important when cells are polarized or show internal organization or spatial shifts in protein/lipid localization. Here we have applied a stereological approach called the rotator to estimate cellular gold label and proportions of labeling over cellular compartments at specific locations related to a chosen cell axis or chosen cellular structures. This method could be used in cell biology for mapping cell components in studies of protein translocation, cell polarity, cell cycle stages, or component cell types in tissues. (J Histochem Cytochem 57:709–719, 2009)     

Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation

We report a new cellular mechanism of rod photoreceptor adaptation in vivo, which is triggered by daylight levels of illumination. The mechanism involves a massive light-dependent translocation of the photoreceptor-specific G protein, transducin, between the functional compartments of rods. To characterize the mechanism, we developed a novel technique that combines serial tangential cryodissection of the rat retina with Western blot analysis of protein distribution in the sections. Up to 90% of transducin translocates from rod outer segments to other cellular compartments on the time scale of tens of minutes. The reduction in the transducin content of the rod outer segments is accompanied by a corresponding reduction in the amplification of the rod photoresponse, allowing rods to operate in illumination up to 10-fold higher than would otherwise be possible.