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.     

Nanometre scale mechanical properties of extremely thin diamond-like carbon films

Abstract

Extremely thin diamond-like carbon (DLC) films are deposited by the filtered cathodic vacuum arc (FCVA) and plasma chemical vapour deposition (p-CVD) methods. The target thicknesses of the extremely thin protective DLC films deposited on a Si (100) surface by FCVA and p-CVD are 0·1, 0·4, 0·8, 1·0, 2·0, 5·0 and 100·0 nm. Nanoindentation hardness and nanowear resistance are evaluated by atomic force microscopy (AFM). The nanoindentation hardnesses of 100 nm thick DLC films deposited by FCVA and p-CVD are 57 and 25 GPa respectively. The nanowear test by AFM clarifies the mechanical properties of extremely thin DLC films. The wear depths of 1 and 2 nm thick FCVA-DLC films are extremely shallow. The wear depths of the 1·0 and 2·0 nm thick p-CVD-DLC films exceed the film thicknesses after five sliding cycles. These results reveal differences in the wear resistance of extremely thin DLC films and the superior mechanical properties of FCVA-DLC thin films.     

Corrosion and wear behaviour of ZrN thin films

Abstract

ZrN coating is an alternative candidate to replace the conventional TiN coating especially for high temperature oxidation resistance applications. ZrN coatings of varying thickness (1·5, 2·0, 2·5, 3·0 and 4·0 μm) were deposited on 316 stainless steel substrates by cathodic arc evaporation in a reactive nitrogen atmosphere. The influences of lamellae thickness on the microstructure, tribological and corrosive properties of the films were investigated. The coefficient of steady state friction of the films ranged from 0·213 to 0·659. The corrosion resistance of the coatings was tested in 1 N H₂SO₄ solution. The results indicate that the microstructure, wear and corrosion properties of the films were dependent on lamellae thicknesses and film structure.     

Effect of polar groups in lubricants on sliding speed dependent friction coefficients of DLC coatings

The speed dependent friction coefficients of two types of DLC coatings, a-C:H and ta-C, were evaluated when lubricated with 1-hexadecene, which did not contain any functional group, and with oleic acid and oleyl alcohol that did. The friction coefficient measured for ta-C at a low sliding speed of 0·01 mm s^?1 with the 1-hexadecene lubricant that did not contain any functional group was 0·22, which was higher than the value of 0·11 seen for a-C:H. The friction coefficients measured for a-C:H and ta-C at a high sliding speed of 50 mm s^?1 with 1-hexadecene were 0·10 and 0·06 respectively. The friction coefficients measured with oleic acid and oleyl alcohol were 0·02 for a-C:H and less than 0·001 for ta-C. The results showed that the friction coefficient of ta-C was more strongly influenced by the functional group in the lubricants than that of a-C:H. It is assumed that this difference between the two coatings can be attributed to a difference in the capability to form a tribochemical reacted film under boundary lubrication. Under mixed lubrication, differences in lubricity also affected this friction coefficient difference, in addition to the properties of the tribochemical reacted film.     

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.     

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.     

Preparation of P-47 scintillators for STEM

Uniform P-47 powder scintillators can be prepared with reproducible mass thicknesses by sedimenting the powder in an 0·01% solution of formvar in chloroform. A reflecting coating can be glued by floating a 60 nm aluminium film on the scintillation layer in an 0·1% solution of gelatine in water. The optimum thickness of P-47 for 20–100 keV electrons increases slower than the electron range due to the absorption and scattering of light with increasing thickness. An aluminium coating increases the light output by a factor 1·85 for 20 keV and 1·4 for 100 keV electrons. The noise of the emitted light quanta and the photomultiplier detection system shows a minimum for the optimum thickness of largest light output and is only a factor 1·3–1·6 larger than the calculated shot noise of the incident electron beam.     

Automated Microscope-Absorption-Spectrophotometry Of Rock-Forming Minerals In The Range 40,000–5000 Cm−1 (250–2000 Nm)

To measure polarized absorption spectra of microcrystals of 3dⁿ-ion bearing silicate minerals, computer processed, microscope-spectrophotometric methods have been developed. Absorbance, log (I₀/I), can be measured with high relative accuracy (near u.v. and vis: ±0·002 to 0·001; n.i.r.: ±0·004 to 0·002), and relatively small spectral band widths are available. Hence, weak spin-forbidden dd-bands of 3dⁿ-ions can be recognized alongside spin-allowed dd-transitions without artificial broadening of absorption bands due to finite resolution. The smallest area from which absorption spectra can be taken is 8 μm in diameter. As one example of the many applications in mineralogy and material sciences, absorption spectra of a natural spessartine garnet, Spess_69·7Alm_30·0Gross_0·05, containing Mn²⁺, Fe²⁺, and traces of Fe³⁺ as 3dⁿ-ions, and of a pure Mn²⁺-garnet, Spess₆₇Gross₃₃, are presented. From these it is evident that bands in natural spessartines at ～ 26,900, 23,200 cm^?1 which were assigned to dd-transitions in Fe³⁺⁽⁶⁾, have to be reassigned to Mn²⁺⁽⁸⁾. Comparison of spectra obtained with the microscopic equipment described with those obtained by means of conventional macroscopic equipment prove that the methods described produce true spectra.     

A new confocal scanning beam laser MACROscope using a telecentric,f-theta laser scan lens

A new confocal scanning beam system (MACROscope) that images very large-area specimens is described. The MACROscope uses a telecentric, f-theta laser scan lens as an objective lens to image specimens as large as 7·5 cm × 7·5 cm in 5 s. The lateral resolution of the MACROscope is 5 μm and the axial resolution is 200 μm. When combined with a confocal microscope, a new hybrid imaging system is produced that uses the advantages of small-area, high-speed, high-resolution microscopy (0·2 μm lateral and 0·4 μm axial resolution) with the large-area, high-speed, good-resolution imaging of the MACROscope. The advantages of the microscope/MACROscope are illustrated in applications which include reflected-light confocal images of biological specimens, DNA sequencing gels, latent fingerprints and photoluminescence imaging of porous silicon.     

The point-spread function of a confocal microscope: its measurement and use in deconvolution of 3-D data

We have measured the point-spread function (PSF) for an MRC-500 confocal scanning laser microscope using subresolution fluorescent beads. PSFs were measured for two lenses of high numerical aperture—the Zeiss plan-neofluar 63 × water immersion and Leitz plan-apo 63 × oil immersion—at three different sizes of the confocal detector aperture. The measured PSFs are fairly symmetrical, both radially and axially. In particular there is considerably less axial asymmetry than has been demonstrated in measurements of conventional (non-confocal) PSFs. Measurements of the peak width at half-maximum peak height for the minimum detector aperture gave approximately 0·23 and 0·8 μm for the radial and axial resolution respectively (4·6 and 15·9 in dimensionless optical units). This increased to 0·38 and 1·5 μm (7·5 and 29·8 in dimensionless units) for the largest detector aperture examined. The resulting optical transfer functions (OTFs) were used in an iterative, constrained deconvolution procedure to process three-dimensional confocal data sets from a biological specimen—pea root cells labelled in situ with a fluorescent probe to ribosomal genes. The deconvolution significantly improved the clarity and contrast of the data. Furthermore, the loss in resolution produced by increasing the size of the detector aperture could be restored by the deconvolution procedure. Therefore for many biological specimens which are only weakly fluorescent it may be preferable to open the detector aperture to increase the strength of the detected signal, and thus the signal-to-noise ratio, and then to restore the resolution by deconvolution.     

The microstructure of non-ohmic zinc oxide ceramics

The microstructure and the nature of some crystal phases in non-ohmic ZnO ceramics containing 0·5 Bi₂O₃, 0·5 CoO, 0·5 MnO and 0·5 TiO₂ mol% were investigated by using various analytical techniques. The ceramics consist primarily of ZnO grains, and in addition, two titanium containing phases were found, a needle-like Zn₂TiO₄ spinel phase doped with bismuth and cobalt, and a cubic-Zn₂Ti₃O₈ phase with some cobalt and manganese. The Bi₂O₃ phase among the grains was found to be TiO₂ stabilized γ* Bi₂O₃.     

A simple microscope warm stage

Microscopical examination of cells, particularly spermatozoa, often necessitates the preservation of a temperature of 37°C on the stage itself. A microscope warm stage should provide a surface at this temperature and allow microscopical examination of a series of pre-warmed sample slides. A further desirable quality would be the total absence of ancillary apparatus. An apparatus has been designed and built which effectively provides these features. It measures 10·5 × 6·5 × 1·3 cm, weighs 320 g with wiring and plug and provides a thermostatically controlled temperature of 37 ± 0·5°C 5 min after connection with the electrical supply (240 V A.C.).     

The dimensions and numbers of small vesicles in blood capillary endothelium in the hind legs of dogs, and their relation to vascular permeability

The dimensions and numbers of vesicles were determined in the blood capillary endothelium of the gastrocnemii muscle of dogs. These results permitted more accurate calculations of the number of vesicles crossing the endothelium in one direction/sec/(μm² (～6·2), and of the median vesicular attachment time (～8 sec). The probability of fusion occurring when a vesicle contacts a plasma membrane (α= 0·004) was unchanged: hence it was concluded, from the mean cellular width (0·21 μm) and the calculated cytoplasmic viscosity (～0·1 poise), that ～49% of the vesicles starting from one side reached the other one, and that their median transit time was ～1 sec.     

5-Cl-PADAB显色树脂相分光光度法测定微量Co(Ⅱ)

研究了在pH 7.60的KH_2PO_4—NaOH介质中,Co(Ⅱ)与4-(5-氯-2-吡啶偶氮)-1,3-二氨基苯(简称5-Cl-PADAB)生成有色配合物,酸化后与强酸性阳离子交换树脂交换吸附,进行树脂相分光光度法测定微量Co(Ⅱ)的实验条件。最大吸收波长为550nm,表观摩尔吸光系数为4.7×10~5L·mol~(-1)·cm~(-1),Co(Ⅱ)含量在0～0.20mg·L~(-1)范围内符合比耳定律。此法用于天然水和维生素B_(12)中的Co(Ⅱ)含量测定,结果满意。     

Anomalous voltage dependence of tunnelling microscopy in WSe2

We present scanning tunnelling microscopy (STM) investigations of the layered semiconductor WSe₂. The tunnelling experiments were performed in air and under silicone oil with markedly different results. In air, atomic resolution images of the hexagonally structured surface could be obtained for sample-to-tip voltages of both negative and positive polarities, from ?1·5 to ?0·3 V for negative sample and from +0·6 to +1·6 V for positive sample, respectively. Under silicone oil, however, good atomic images could be seen for negative sample biases down to at least ?14 V, while for positive sample biases no difference with respect to the tunnelling in air was found.     

Recent improvements to the Cambridge University 600 kV High Resolution Electron Microscope

Several recent modifications to the Cambridge University 600 kV HREM have resulted in increased levels of performance and greater operating efficiency. Changes include new Permendur pole-pieces with a reduced gap leading to improved aberration coefficients, and an attachment to the tilt/shift supplies which facilitates accurate beam alignment. Resolution figures include: (a) an axial CTF extending, without zeros, beyond 1·8 Å at 575 kV; (b) tilted beam CTFs extending beyond 1·1 Å; and (c) lattice fringes of 0·72 Å spacing.     

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.     

Direct imaging of paracrystalline phospholipid structure in the electron microscope

A long chain amphiphilic molecule—the phospholipid 1,2-dihexadecyl sn glycerophosphoethanolamine—has been crystallized epitaxially so that the interlamellar molecular periodicity is parallel to the substrate and hence normal to the electron beam in the electron microscope. This has permitted the direct resolution of the 55·6 Å lamellae in unstained crystals at room temperature. The lattice images have shown the presence of line dislocations and lenticular cracks in the crystals. Of significance to their biological properties is that the lattice is undulating with a periodicity of 0·1–0·5 μm. This would also account for the difficulties encountered by X-ray and electron diffraction techniques when examining these crystals.     

Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spread bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64°, both the dorsal and ventral membranes were illuminated, while images excited above 66° illuminated only focal contacts. Above 74° the fluorescence of focal contacts was dominated by background noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 ± 13 nm. Focal contact areas estimated by TIRFM (1·3 ± 0·7 μm²) agreed closely with areas observed by immunofluorescence staining of vinculin (1·5 ± 0·3 μm²).     

Empirically determined freezing time for quick-freezing with a liquid-nitrogen-cooled copper block

A method is presented to determine freezing time empirically. The method is based on determining the amount of stretch of a skinned muscle fibre while it is being frozen. Freezing time, as determined with this method, lies in between 0·5 and 1·5 ms.