Glycol methacrylate embedding of bone and cartilage for light microscopic staining

A method is described for embedding bone and cartilage in glycol methacrylate (GMA) for light microscopy. Dehydration-infiltration of the hard tissue is with aqueous GMA solutions minimizing solvent and dehydration artefact, and polymerization is by UV light in the cold to minimize thermal damage. Over fifty stains, enzyme localizations and related histochemical methods for 0·5–3·0 μm thick sections of GMA embedded tissue are listed. The increased resolution plus the localization of cellular and extracellular chemical moieties is now easier and more accurate providing an improved method for the study of the musculo-skeletal system by light microscopic histochemistry.     

A grinding method for producing sections of large areas of plastic embedded tissues

We have developed a method utilizing relatively thick ground sections of plastic embedded tissue which affords the resolution obtained with 0·5 μm cut sections. The sections, which are permanently affixed to plastic microscope slides, are much larger in area than ultramicrotome sections. Additional advantages are: sections can be destained and restained and selected areas can be examined with various forms of electron microscopy. Autoradiographic studies are also possible. Although the method has a broader application, it is particularly useful in examining the interface between hard and soft tissues.     

A rapid methylene blue-basic fuchsin stain for semi-thin sections of peripheral nerve and other tissues

Differential histological staining of various tissues is achieved in epon, araldite or glycol methacrylate sections 0·25–0·5 μ thick, from human, guinea-pig and rat origin. The staining procedure is as follows: 1% methylene blue in 1% borax 15–30 sec at 70°C; wash in hot water; 2% basic fuchsin in distilled water 1–2 min at room temperature; wash and mount in DPX. Results: with all tissues: nuclei pale violet, cytoplasm blue. Special histological components: collagen red, myelin deep blue, elastin deep violet, axoplasm pale blue. Although of general utility the method is especially useful for peripheral nerve and blood vessels.     

Dimensions of active cytochrome c oxidase in reconstituted liposomes using a gold ball shadow width standard: a freeze-etch electron microscopy study

The preparation and characterization of a distribution of gold balls on a thin, flat carbon film is described. The relation of the platinum carbon shadow width distribution means to a gold ball size is reported. Freeze-etched cytochrome oxidase vesicles and gold ball calibration grids were simultaneously shadowed with platinum/carbon. The shadow width distribution of the cytochrome oxidase located in and spanning the membrane was measured. The membrane fracture face edge and cross-fractured bilayer membrane edge were also measured. Dimensions of the cytochrome oxidase were found to be 5·8 ± 0·3 nm in diameter parallel to the membrane and 8·2 ± 0·3 nm long across the membrane. The bilayer membrane dimensions were 3·0 ± 0·3 nm for the half bilayer and 5·8 ± 0·3 nm for the cross-fractured bilayer membrane edge thickness. The length of the cytochrome oxidase was observed to span the bilayer membrane. Previous X-ray diffraction measurements on similar hydrated liquid crystalline artificial membranes were found to be in good agreement with the freeze-etched results. Membrane widths from thin-sectioned cytochrome oxidase vesicles were measured and found to be 5·8–5·9 nm in non-post-stained sections. Post-staining with uranyl acetate and/or lead citrate was shown to increase this average thickness. The technique of freeze-etching electron microscopy in conjunction with the gold ball shadow width calibration experiment has been shown to provide accurate and precise measurements of membranes and a functional intramembrane protein in a hydrated non-crystalline sample.     

An improved analysis of experimental data from 125 I hot-line autoradiographs: allowing for the effects of background grains

In this paper we suggest a new statistical method of correcting the results of hot-line experiments for the effects of background sources and we use the new method to reassess the adequacy of three probability distributions proposed in the literature for image spread from line sources. The data are from sources labelled with ¹²⁵I in semi-thin resin sections 0·4-0·8 μm in thickness. The new method reveals that two of the models describe the empirical distributions more closely than earlier analysis had suggested, and it confirms an increasing relationship between half distance of image spread and the thickness of the source. However, it also confirms that considerable ‘inter hot-line’ experimental variation remains, even after background correction. This suggests that multiple experiments are needed to produce reliable estimates of half distance.     

Freeze substitution sample preparation for ion microscopy of plant tissue

Tomato leaf sections were freeze substituted with either ethanol, n-propanol, 20·0% acrolein: diethyl ether or acetone and then examined with an ion microscope to determine Na, K and Ca distributions. Thin sections were also examined for ultrastructural preservation using a transmission electron microscope. Generally, the 20·0% acrolein: diethyl ether and to a lesser degree the n-propanol substituted materials demonstrated better ultrastructural preservation than the ethanol or acetone substitued materials. Ion micrographs for Na, K and Ca²⁺ of the 20·0% acrolein:diethyl ether and n-propanol substituted leaves revealed a low contrast, well-graded response with more informational content while tissue substitued with ethanol gave a high contrast response with little or no gradation. Ion losses from the leaf sections after substitution were monitored with neutron activation analysis (NAA) and initial experiments revealed significant contamination by molecular sieve desiccants. Empirically derived correction factors compensated for these contaminants; however, further experimentation utilizing silica gel as a solvent desiccant demonstrated ion retention values in excess of 80%.     

An interference technique for measuring the thickness of semi-thin and thick sections

An interference method is described for measuring section thickness in the range 0·3–45 μm. An incident illumination objective incorporating a beam splitter and adjustable reference mirror is used to generate interference fringes by reflection from the upper surfaces of sections on glass slides. Sections do not require a reflective coating. The lateral displacement of the zero-order fringe generated using white light is measured in terms of sodium light fringes and photographic enlargement of the fringes allows measurement to ± 30 nm. The method is simple in operation and allows rapid assessment of any local distortions over the entire section area.     

Sectioning and imaging hard mineral fibres in biological tissues

A technique is described which overcomes the problems associated with sectioning biological tissue containing hard mineral fibres. 0·2–0·5 μm thick sections were cut with a diamond knife, placed in a folding grid, conventionally stained with uranyl acetate and lead citrate and viewed at an accelerating voltage of 200 kV in the scanning transmission mode.     

Cutting work in thick section cryomicrotomy

The forces during cryosectioning were measured using miniature strain gauges attached to a load cell fitted to the drive arm of the Porter-Blum MT-2 cryomicrotome. Work was calculated and the data normalized to a standard (1 mm × 1 mm × 0·5 μm) section. Thermal energy generated was also calculated. Five parameters were studied: cutting angle, thickness, temperature, hardness, and block shape. Force patterns could be divided into three major groups thought to represent cutting (Type I), large fracture planes > 10 μm in length (Type II), and small fracture planes < 10 μm in length (Type III). Type I and Type II produced satisfactory sections. Work in cutting ranged from an average of 78·4 μJ to 568·8 μJ. Cutting angle and temperature had the greatest effect on sectioning. Heat generated would be sufficient to cause through-section melting for 0·5 μm thick sections assuming the worst possible case, namely that all heat went into the section without loss. Presence of a Type II pattern (large fracture pattern) is thought to be presumptive evidence against thawing.     

The influence of tissue processing on quantitative histopathology in breast cancer

Objective grading of breast cancer by morphometry has been suggested for improving the precision of the prognostic prediction. However, the tissue components evaluated might be influenced by variations in the processing, reducing the clinical value. In the present study, the impact of the period of fixation, of the acidity of the fixative and of the embedding medium was investigated by allocating tissue samples from 27 surgical breast cancer specimens systematically randomly to different modes of processing. The volume-weighted mean volume of cancer cell nuclei, v?_V(nuc), was estimated using the method of point-sampled intercepts on vertical sections. In addition, estimates of the mean nuclear profile area, ā_H(nuc), the nuclear volume fraction, V_V(nuc), the nuclear profile density, ND, and the mitotic profile frequency, MF, were obtained. The quantitative histopathological estimates were stable with respect to the investigated variables of the tissue processing. No significant differences were found when comparing the estimates obtained in samples from five tumours fixed in formalin at pH 5·0, 6·0, 7·0, 7·4 and 8·0, respectively. Similarly, no significant correlations between the estimates and the period of formalin fixation (24 h, 3 days and 3 months) were found in samples from five other tumours. However, the v?_V(nuc) was 13% larger (2p = 0·004) and the mean ND 17% smaller (2p = 0·04) in hydroxyethyl-methacrylate-embedded samples from 17 tumours as compared to paraffin-embedded samples. Thus, the shrinkage observed in paraffin seems to affect nuclei less than tissue.     

Stereological measures of trabecular bone structure: comparison of 3D micro computed tomography with 2D histological sections in human proximal tibial bone biopsies

Stereology applied on histological sections is the ‘gold standard’ for obtaining quantitative information on cancellous bone structure. Recent advances in micro computed tomography (µCT) have made it possible to acquire three-dimensional (3D) data non-destructively. However, before the 3D methods can be used as a substitute for the current ‘gold standard’ they have to be verified against the existing standard. The aim of this study was to compare bone structural measures obtained from 3D µCT data sets with those obtained by stereology performed on conventional histological sections using human tibial bone biopsies. Furthermore, this study forms the first step in introducing the proximal tibia as a potential bone examination location by peripheral quantitative CT and CT. Twenty-nine trabecular bone biopsies were obtained from autopsy material at the medial side of the proximal tibial metaphysis. The biopsies were embedded in methylmetacrylate before µCT scanning in a Scanco µCT 40 scanner at a resolution of 20 × 20 × 20 µm³, and the 3D data sets were analysed with a computer program. After µCT scanning, 16 sections were cut from the central 2 mm of each biopsy and analysed with a computerized method. Trabecular bone volume (BV/TV) and connectivity density (CD) were estimated in both modalities, whereas trabecular bone pattern factor (TBPf) was estimated on the histological sections only. Trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp), and structure model index (SMI) were estimated with the µCT method only. Excellent correlations were found between the two techniques for BV/TV (r = 0.95) and CD (r = 0.95). Additionally, an excellent relationship (r = 0.95) was ascertained between TBPf and SMI. The study revealed high correlations between measures of bone structure obtained from conventional 2D sections and 3D µCT data. This indicates that 3D µCT data sets can be used as a substitute for conventional histological sections for bone structural evaluations.     

Coalignment of the muscle cell and nucleus,cell geometry and Vv in the tunica media of monkey cerebral arteries,by electron microscopy

We undertook to ascertain how well aligned is the rod-shaped nucleus within the spindle-shaped cell of vascular muscle in order that we might use the darkly staining nucleus in histological sections to indicate precisely the directional alignment of the cell. We fixed cerebral arteries from five monkeys under physiological pressure and embedded portions of the tissue so that mid-plane longitudinal sections of the arteries were obtained; the circumferentially arranged muscle cells were cut in cross-section. From the electron micrographs we obtained the cross-sectional profile of the cell and its nucleus, determining that the centre of the nucleus was on average 9·5 ± 5·8% (SD) away from the centre of the cell (expressed as a ratio of the cellular diameter). We calculated the alignment between the cell and nucleus to be from 0 to 3°, and obtained a volume fraction of 59% for muscle tissue in the tunica media of these arteries.     

An electron diffraction study of paramylon storage granules from Euglena gracilis

Paramylon storage granules from Euglena gracilis were characterized by electron diffraction techniques using electrons of various accelerating voltages: 2 MV for the thick granules and 100 kV for the thinner ones. Intact granules gave well resolved, characteristic (1→3)-β-d glucan fibre diffraction diagrams with the glucan molecular orientation parallel to the longer axis of the granule. Hydrated electron diffraction patterns with better resolution were obtained from thinner granules by examination at low temperatures of quench-frozen specimens. In this case, the pattern indexed on an hexagonal system with a = b= 1·55 + 0·01 nm and c (fibre axis) = 1·86 ± 0·01 nm. Sections of embedded granules provided single crystal-like electron diffraction patterns corresponding to various sections of the reciprocal lattice of (1→3)-β-d glucan (including the hko section). Finally, by scanning electron microscopy, it was shown that the granules swell on contact with water and take up a characteristic ribbed, pumpkin-like shape.     

Quantitative analysis of electrolytes in frozen dried sections

During recent years our group has employed the technique of electron microprobe analysis to determine the electrolyte concentrations in various epithelial tissues. The specimen preparation is characterized by shock-freezing of small tissue pieces in liquid propane/isopentane mixtures at 77 K, cryosectioning of 1 μm thick serial sections at 170 K and subsequent freeze-drying at 190 K and 10^?4 Pa. The analysis of the frozen dried cryosections is performed in a scanning electron microscope which is equipped with an energy dispersive X-ray detector. The measuring conditions selected are 17–20 kV acceleration voltage and 0·1–0·5 nA probe current. For quantification, the cellular X-ray spectra are compared with those of an internal albumin standard layer. The evaluation of the characteristic X-ray intensities is performed using a computer program. Some critical points of this technique will be discussed.     

An electron microscopy study of the phase Al3Fe

The phase Al₃Fe (monoclinic C2/m, a = 1·549 nm, b = 0·808 nm, c = 1·248 nm, β = 107·8°) has been studied by transmission electron microscopy (TEM) and high resolution electron microscopy (HREM). Crystals were obtained from a direct chill-cast ingot of an Al-0·25 wt% Fe-0·13 wt% Si alloy. Extracted crystals were prepared by dissolving the aluminium phase in butanol and filtering off the particles. The extracted Al₃Fe crystals were mainly (100) platelets. The monclinic lattice was confirmed by tilt experiments and the mirror plane was confirmed by convergent beam electron diffraction. Experimental HREM images from the [100] and [110] projection agreed with images calculated by the multislice method. The interpretation of images in terms of a projected crystal structure is discussed. Common defects in Al₃Fe crystals were: twins on (100) and faults on (001). The (001) faults could be described by a displacement 1/2·[100] on a fault plane at z = 0·5 in the unit cell. A model for (001) faults, based on multiple twinning, is proposed.     

An improved method for combined autoradiography and histochemistry: the simultaneous detection of 6-3H thymidine and acid phosphatase activity in cryostat sections of mouse thymus and duodenum

A new method is described that enables the simultaneous detection of 6-³H thymidine incorporation and acid phosphatase activity in the same tissue section. Histochemically, naphthol AS B1 released by tissue based acid phosphatase activity from the substrate naphthyl AS B1 phosphoric acid is coupled with a range of diazonium salts to produce insoluble azo dyes. The azo dye tests result in a particulate localization of lysosomal acid phosphatase and also label diffuse sources associated with cell death. The tests selected permit the application of photographic emulsion without the necessity of an inert barrier layer to separate the emulsion from the histochemically treated cryosections. The localization of 6-³H thymidine incorporation and cell death in mouse thymus and duodenum is demonstrated and comparative counts estimating the distribution of 6-³H thymidine incorporation and hydrolase labelled cell death in the thymus are presented. Young mouse thymus (5 weeks) was found to contain 1·36 ± 0·12% dying cells and 6·78 ± 0·03% thymidine incorporating cells, whilst old mouse thymus (53 weeks) was found to contain 2·34 ± 0·6% dying cells and 5·29 ± 0·37% thymidine incorporating cells.     

The use of high-voltage electron microscopy and semi-thick sections for examination of cyanobacterial thylakoid membrane arrangements

High-voltage electron microscopy (HVEM) of semi-thick sections was evaluated as a technique for studying thylakoid membrane arrangements in cyanobacterial cells. Semi-thick sections (0·25 μm) provided important information that was relatively difficult or impractical to obtain by viewing either randomly or serially cut thin sections. Specifically, the semi-thick sections were better suited for visualizing (i) overall thylakoid arrangements and (ii) interconnections between the thylakoids and the cytoplasmic membrane. By comparison, randomly cut thin sections frequently yielded deceptively incomplete or inconsistent data in regard to these specific features. Tilting of thick sections about two perpendicular axes served to improve the clarity of complex membranous intersections and other cell features.     

A new method to correlate acoustic spectroscopic microscopy (30 MHz) and light microscopy

A powerful new method is used to investigate the correlation between light microscopic and acoustic properties of biological tissues. Specimens of liver were sectioned into successive slices, 250 μm and 10 μm thick. The thick sections were investigated acoustically, the thin sections by means of light microscopy. Markers that could be detected and located, both optically and acoustically, were used to find and reconstruct corresponding regions in the acoustic and optical sections (2·5 × 2·5 mm). Parameter images were reconstructed from the sections investigated acoustically. The acoustic parameters were attenuation at 30 MHz, the slope of the attenuation spectrum (between 10 and 50 MHz), backscattering at 30 MHz, the slope of the backscattering spectrum (between 10 and 50 MHz) and the local ultrasound velocity. Acoustic images were obtained in the frequency range from 10 to 50 MHz, yielding a lateral resolution of about 50 μm. The sections for light microscopy were stained according to the Goldner trichrome staining technique. The histological composition was determined quantitatively, using digital image segmentation techniques. The percentage of collagen-rich fibrous tissue, luminal structure and interstitial spaces, and the number of nuclei were calculated for regions of 250 × 250 μm. These histological features were correlated with the acoustic parameters obtained from the corresponding regions in adjacent sections. It was thus possible to find the histological components responsible for acoustic parameters.     

X-ray microtomography

A microscope system based on the principles of computerized axial tomography is described for determining the distribution of the X-ray absorption coefficient in a slice from a solid object without cutting sections. An application is given to determining the distribution at a resolution of about 15 μm through a shell of about 0·5 mm diameter.     

Ponceau 2R staining of proteins and periodic acid bleaching of osmicated subcellular structures on semi-thin sections of tissues processed for electron microscopy: a simplified procedure

An aqueous solution (pH 1·5) of 0·5% Ponceau 2R (C.I. 16150) and 2% periodic acid was used at 318 K to stain proteins (brilliant red) and simultaneously to bleach the osmicated non-proteinaceous cell structures on 1–2 μm thick sections of tissues fixed in glutaraldehyde-osmium tetroxide and embedded in epoxy resins. This H₅IO₆ bleaching-Ponceau 2R staining procedure only stains the proteins so that black-and-white films can be used.