Combined autoradiography and histochemistry: the simultaneous detection of 6-3H thymidine and acid phosphatase activity in cryostate sections.

A method is described for the simultaneous demonstration of 6-3H thymidine incorporation and acid phosphatase activity in cryostae sections of mouse thymus. The method enables a comparison of mitosis and acid hydrolase activity to be made in the same tissue section. In 8-week-old mice acid phosphatase positive cells reprsent 1.23 +/- 0.06% of the total population and 8.4 +/- 0.27% of the cells incorporate tritiated thymidine. Acid phosphatase activity can be used to estimate cell autolysis and death. The implication of the method in relation to tissue dynamics is discussed.     

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.     

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).     

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.     

Autoradiographic studies of DNA synthesis in lymphoid tissues

Using long exposures of stripping film autoradiographs before processing, mixtures of weakly and strongly labelled nuclei were seen in different areas of the mouse spleen. Previous results (Harris et al., 1973) led to the conclusion that many cells, not in division cycle, were labelling with (³H) thymidine and that this process was important for the development of specific antibody-producing cells following stimulation with an antigen such as sheep red cells (SRC). The present data are an analysis of the (³H) thymidine labelling kinetics in the spleens of mice reared in conventional or germ-free conditions. The labelling seen in the 24 h following an injection of (³H) thymidine could best be interpreted on the basis of synthesis of unstable DNA. The changes in the pattern, and distribution of labelled nuclei as well as the intensity of their labelling was not compatible with cell division only, but was also the result of movement of labelled material between the lymphoid cells of the organ. Germ-free mice were followed for 24 days following a single injection of (³H) thymidine. The rate of uptake of label into the spleen was much slower than has been found previously in mice reared in conventional conditions. When SRC were injected 2 h after giving (³H) thymidine the labelling of lymphoid cells in the spleen and blood was quite different to controls given (³H) thymidine alone. Detailed analysis indicated that turnover of labelled material, presumably DNA, as well as cells was involved. This turnover of DNA could be considered to be metabolic in the sense that renewal, increase in amount, loss, and transfer to other cells were involved. These, and other studies, in vivo (Harris & Olsen, 1973) and in vitro (Harris et al. 1975) indicate that such processes, involving DNA, are highly relevant to the development of antibody-producing capacity by cells responding to antigenic challenge.     

Two-photon excited lifetime imaging of autofluorescence in cells during UV A and NIR photostress

By monitoring coenzyme autofluorescence modifications. as an indicator of cell damage. the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UV A radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser. focused through highnumerical- aperture microscope optics. provided diffractionlimited mlcrobeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (_T-mapping). Upon the onset of UV or NIR exposure. Chinese hamster ovary cells exhibited blue/green autofluorescence witq a mean lifetime of 2·2 ns. which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 m W. 300 J cm^-2 ) resulted in oxidative stress correlated with increased autofluorescence intensity. onset of nuclear fluorescence. and a fluorescence lifetime decrease. The cellular response to femtosecond NIR micro beams depended significantly on peak power. Peak powers above a threshold value of about 0·5kW (average power: 6mW). 0·55kW (7mW) and 0·8kW (lOmW) at 730nm. 760nm and 800nm. respectively. resulted in the onset of short-lived luminescence with higher intensity (100x) than the intracellular NAD(P)H fluorescence. This luminescence. accompanied by destruction of cellular morphology. was localized and occurred in the mitochondrial region. In contrast. beams at a power of less than 0·5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.     

Freeze-fracture autoradiography of the red blood cell plasma membrane

A procedure for electron microscope autoradiography of red blood cell ghosts or their outer membrane leaflets is described: sheep erythrocytes, attached to positively charged mica, were either lysed and air-dried, or freeze-fractured ‘by hand’ and freeze-dried. This was followed by shadowing and carbon replication; coating with photographic emulsion; exposure in darkness at 277 K; developing and fixation; carbon coating; floating off the mica and mounting on grids. Erythrocytes iodinated with ¹²⁵I using the lactoperoxidase reaction were used as a test object. The efficiency of the procedure was found to be between 3·4% and 5·6%.     

Quantitative water mapping of cryosectioned cells by electron energy-loss spectroscopy

A direct technique based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen-hydrated biological cryosections. Previously, methods for water determination have been indirect in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum-imaging, where EELS data are collected in parallel at each pixel. Several operations are required to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point-spread function, removal of plural inelastic scattering and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. Reference spectra have been recorded from the major biomolecules (protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as ± 3·5%. With the present instrumentation the maximum allowed dose of 2 × 10³ e/nm² limits the useful spatial resolution to around 80 nm for ± 5% precision at a single pixel. By averaging pixel intensities a value of 56·8% with a precision of ± 2·0% has been determined for the water content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.     

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.     

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.).     

Surface structures of YBa2Cu3O7-x,BiCa1·7Sr0·7Cu2Ox and TlCaBaCuO4·5±x investigated by scanning tunnelling microscopy

Surface structures of the high-T_c superconductors YBa₂Cu₃O_7-x, BiCa_1·7Sr_0·7Cu₂O_x and TlCaBaCuO_4·5±x have been investigated with scanning tunnelling microscopy. The observed features include well-organized stripe corrugations in Y-Ba-Cu-O as well as orientated flake-like structures and steps with various heights in the Bi- and Tl-compounds. These observations suggest strong local variations of the elastic and electronic properties of the investigated materials.     

Study of solid particle erosion on AISI D2 using angular silicon carbide and steel round grit particles

Abstract

In this study, the performance of AISI D2 steel subjected to solid particle erosion tests was analysed. This material has applications for tools and dies for blanking, wood milling cutters, cold-extruding and other operations requiring high compressive strength and excellent wear resistance. The erosion tests performed by using a rig developed according to some parameters of the ASTM G76-95 standard. Two abrasive were used, angular silicon carbide (SiC) and steel round grit, both, with a particle size of 400–420 μm. This allowed comparing the erosion severity of each abrasive particle. The tests were conducted using four different incident angles 30, 45, 60 and 90° with a particle velocity of 24±2 m s^?1 and a flow rate of 21±2·5 g min^?1 for silicon carbide and 48·5±3·5 g min^?1 for the steel round grit. The exposure testing time was 10 min. Subsequently, the surface damage was analysed with a scanning electron microscope (SEM) to identify the wear mechanisms. Additionally, atomic force microscopy (AFM) was conducted in order to obtain roughness of the surface damage at 60°. The results indicated that higher amount of mass loss was obtained by angular silicon carbide particles.     

Use of differential interference contrast microscopy to determine cell renewal times in mouse intestine

Pieces of mouse mid jejunum have been cleared in glycerin and examined by differential interference contrast microscopy at low powers of magnification. The position and number of crypts and villi can be determined in the same specimen using this technique. The calculated value for crypt/villus ratio 4·53 ± 0·99 (mean ± SD) is less than a previously published value obtained using indirect techniques. A revised estimate of cell renewal time, based on this newly determined value for crypt/villus ratio, is 45 h. This agrees with earlier estimates derived from entirely different methods of analysis. The general usefulness of this form of light microscopy in helping one appreciate some three-dimensional problems in mucosal architecture is emphasized.     

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.     

Investigation of dislocation geometries in the diamond cubic structure

The weak-beam technique of electron microscopy (Cockayne, Ray & Whelan, 1969) has been applied to the examination of dislocations in germanium. These are shown to be dissociated into partial dislocations with a separation in the edge orientation of 5.5 ± 1·0 nm. A value for the stacking-fault energy of γ = 60 ± 8 mJ m^?2 (erg cm^?2) is deduced from the measured dissociation width as a function of orientation, using anisotropic elasticity theory.     

Spectrophotometric Determination of Silicon in Rice and Alloy Steel Samples

Abstract

In the medium of 0.128 mol/L nitric acid and 0.450 mol/L sulfuric acid, silicon(IV) and ammonium molybdate form molybdosilicate blue blue complex in the presence of ascorbic acid. The maximum absorption wavelength of the complex locates at 810 nm. The apparent molar absorptivity is ε_810 nm = 3.18 × 10⁴ L · mol^?1 · cm^?1. Beer's law is followed over the range of 0 ～ 1.0 µg/mLof silicon(IV). The present method has been successfully applied to the determination of silicon in rice and alloy steel samples.     

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.     

Tem Of Dislocations Under High Stress In Germanium And Doped Silicon

The stacking fault energies y of silicon (58 ± 6 mJ m^?2) and germanium (75 ± 10 mJ m^?2) were determined. Within the limits of accuracy γ was not found to change on doping with (13·8 mol m^?3 (8 × 10¹⁸ cm^?3) boron, and 1·17 mol m^?3 (7 × 10¹⁷ cm^?3) phosphorus). Freezing in dislocations under high shear stress reveals a different behaviour of screw dislocations: whereas these dislocations become wider in pure and p-silicon, they become narrower in n-silicon. From this we conclude the ratio of mobilities of the two 30° partials to be different in n- and p-silicon. Other observations on frozen dislocations are mentioned.     

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.     

The occurrence of autophagic cell death in the tegument of rabbits pre‐infested with Rhipicephalus sanguineus and exposed to selamectin (active principle of acaricide pfizer revolution®)

Ticks of Rhipicephalus sanguineus species have great medical and veterinary importance for being a vector of various diseases. In an attempt to minimize their action on the host, people have resorted to chemical control by using various acaricides, such as selamectin. Although previous studies have demonstrated its toxic action in domestic animals, no studies focused on the detection of cell death when exposed to selamectin. For this reason, the technique for detecting autophagic cell death was used in order to demonstrate the responses of rabbits' skin tissues pre‐infested with R. sanguineus and exposed to different concentrations of selamectin. The obtained results when exposed to 100 and 80% concentrations of selamectin showed a strong mark of acid phosphatase on the cells of the connective tissue of the dermis and hair follicles, whereas the ones exposed to the 50% concentration had a weak mark on the cells of the connective tissue of the dermis and moderate staining in hair follicles. It became clear that, when used at high concentrations (100 and 80%), selamectin is capable to induce a large scale occurrence of the autophagic cell death process. On the other hand, the concentration of 50% causes minor morphophysiological changes in the skin of rabbit hosts when evaluated the cell death process. Therefore, the data confirms that selamectin is a powerful dose‐dependent toxic agent causes increased activity of the enzyme acid phosphatase. Microsc. Res. Tech. 76:1171–1176, 2013. © 2013 Wiley Periodicals, Inc.