Showing papers in "Journal of Microscopy in 1985"

Stereological estimation of the volume‐weighted mean volume of arbitrary particles observed on random sections

Abstract: SUMMARY A stereological estimator of the weighted mean volume of particles of arbitrary shape is described. This unbiased estimator is based on simple point-sampling of linear intercept lengths. The complete absence of shape assumptions effectively breaks the long-standing ‘convexity-barrier’: the only requirement here is that individual particles can be unambiguously identified by their profiles on random sections. Practical details of the simple estimation procedure and an example with very irregular particles are reported. Finally, an estimator of the variance of the weighted distribution of particle volume is discussed. This estimator is also valid for particles of arbitrary shape. For any mixture of ellipsoids (spheres, oblates, prolates and triaxial ellipsoids) the estimator is reduced to a simple function of measurements of diameters in the section plane.

Low temperature embedding with Lowicryl resins: two new formulations and some applications

Abstract: Lowicryl K4M and HM20 are methacrylate/acrylate based low temperature embedding resins for biological material which can be used in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. K4M and HM20 are applicable over a very extended temperature range, approximately 220 K to 340 K. With two new resins, K11M and HM23, one can reach even lower temperatures, c. 200 K. Freeze-substitution combined with low temperature embedding allows for very mild or no chemical fixation which seems to increase the sensitivity of immunocytochemical localization of antigens on sections.

Optical fluorescence microscopy in three dimensions: microtomoscopy

Abstract: SUMMARY A unique property of confocal fluorescence microscopy is utilized to obtain scanned images of a number of samples in three dimensions. This kind of microscopy for the first time enables the direct study of the 3-D arrangement of fluorescently labelled materials in living cells. The technique is based on a home build, confocal, scanning laser microscope. The entire volume of the object is scanned mechanically through the well-defined focus of a laser illuminated, high numerical aperture objective. An identical optical path, arranged confocally, is used to detect the fluorescence emission from the focal volume. A pinhole in the image plane of the detection channel prevents fluorescence light, generated outside the focal volume, to reach the detector. Consequently a true resolution is obtained along the optical axis (identified as z-axis) and three-dimensional imaging without the need for mathematical operations is possible. A high resolution along the z-axis (0.9 μm) is shown as well as in the object plane (0.25 μm) using Rhodamine as the fluorescent dye. The resolution along the z-axis is explained and experimentally demonstrated using an object which contains only a z-axis dependent structure. Pictures of sections through living cells are shown, both parallel to the object plane and parallel to the optical axis.

Unbiased estimation of particle density in the tandem scanning reflected light microscope

Abstract: SUMMARY The tandem scanning reflected light microscope has the property of being able to obtain information from ‘inside’ solid objects by taking a thin optical section at the focal plane of the objective lens. This plane can be focused up and down through the specimen. We describe an unbiased 3-D counting rule for the TSRLM, which is applied to the estimation of osteocyte lacunar density in whole bone. This is shown to be an extremely efficient way of making such an estimate. Further possibilities for the application of the microscope in the field of stereology are discussed.

Specimen preparation methods for the examination of surfaces and interfaces in the transmission electron microscope

Abstract: SUMMARY Various techniques for the preparation of cross-sectional and plan view TEM specimens of surfaces and interfaces are described. Particular emphasis is given to preparative methods which are both generally applicable and which minimize differential thinning of the materials present on either side of the interface of interest, thereby improving the reliability of the approach.

Orthogonal triplet probes: an efficient method for unbiased estimation of length and surface of objects with unknown orientation in space.

Abstract: SUMMARY If the axis of anisotropy of oriented, tubular or lamellar objects is unknown, the unbiased stereological estimation of length density and surface density (Lv and Sv) requires counts on sections with isotropic uniform random (IUR) orientation. It is shown theoretically that in homogeneous, anisotropic specimens the precision of Lv and Sv estimation is considerably augmented if IUR-oriented sets of three mutually perpendicular sections (orthogonal triplet probes=ortrips) are used instead of three directionally independent IUR sections. The mechanism of variance reduction results from a negative covariance between sections within ‘ortrips’ and corresponds to the antithetic variate principle of Monte Carlo work. Heterogeneity decreases the efficiency of the ortrip method, but this effect can often be counteracted by systematic sampling of ortrips within specimens. Practical estimation of length and surface area of the highly anisotropic, tubular myocardial capillaries per tissue volume in the left ventricles of eight normal, adult, male perfusion-fixed Wistar rats provided estimates of excellent precision with CEs of 3.3% (Lv) and 2.1% (Sv) of the group mean. The method will hopefully allow stereologists dealing with arbitrary anisotropic structures to apply the same simple, efficient, and unbiased sampling designs that have long been used in the study of liver, lung, and kidney tissue.

A microscope diffusion chamber for the determination of the equilibrium and non-equilibrium osmotic response of individual cells.

Abstract: SUMMARY A microscope diffusion chamber has been developed which allows direct observation of the dynamic osmotic response of individual cells in micro-volume suspensions. Continuous observation of stationary cells is possible including short experimental times while the extracellular chemical composition is changed. Multiple changes of solute type or concentration are easily imposed upon a single sample volume. Response times are a function of chamber configuration but response times as low as 1–10 s are possible with negligible solute concentration gradients within the sample region. The chamber is simple and economical to construct and use. It is the size of an ordinary glass microslide and it can be adapted easily to any common laboratory microscope. All standard optical techniques may be used with the chamber. Construction details and operating characteristics including important limitations are discussed. Example photomicrographs and osmotic data are included.

The structure of the stalk surface layer of a brine pond microorganism: correlation averaging applied to a double layered lattice structure.

Abstract: SUMMARY The surface layer of the stalk of a prosthecate halophilic microorganism is a periodic array (space group p3ml) comprised of electron dense trimers with a centre to centre spacing of 9.0nm. The structure is reminiscent of E. coli porin. We have demonstrated that the method of correlation averaging can be effectively used to separate overlapping lattices, and that this results in a higher fidelity reconstruction, when compared to the established method of Fourier filtration. Two statistical methods are used to determine the resolution of the correlation average as a function of the number of ‘windows’ averaged, (i) The phase residual of spatial frequencies in Fourier space is computed between independently obtained subaverages, and (ii) a new method of Q factor analysis examines the cumulative vector sum in Fourier space as a function of the number of windows averaged. Both methods give a resolution of 1/2.1−nm.

Absorption microanalysis with a scanning soft X-ray microscope: mapping the distribution of calcium in bone.

Abstract: This article describes the scanning transmission X-ray microscope operated at the National Synchroton Light Source. The application of the instrument to elemental analysis is detailed. In particular, qualitative results on the calcium distribution in human skull tissue are presented.

The relative merits of various cooling methods

Abstract: SUMMARY Three different methods of rapidly cooling specimens prior to microscopical analysis are currently in use, namely, slamming, plunging and spraying. The heat transfer processes and specimen cooling rates which are theoretically achievable by each method are discussed and compared with some of the published experimental data. For the slamming method it is concluded that very little improvement in specimen freezing will be achieved by pre-cooling the block below about 20 K and where pure silver is the most efficient block material. The highest cooling rates and best microscopy by either the plunging or spraying method will be achieved using nitrogen at supercritical pressures. At subcritical pressures propane will produce the best results for metal samples whereas ethane is superior to propane for exposed biological specimens. Future experimental work which relates ice crystal size to cooling rate must be based on the cooling rate at the instant of freezing or nucleation and not on some average cooling rate.

Vitrification of pure liquid water

Abstract: SUMMARY The methods for vitrifying pure liquid water are reviewed: jet-freezing of water/oil emulsions, vitrification of thin water layers on an electron-microscope specimen grid, high-pressure jet-freezing and rapid cooling of aerosol droplets on a cryoplate. The first three need a liquid cryomedium for heat transfer in contrast to the last one. Small sample size in at least one dimension, with a diameter in the μm range or even less, is essential for vitrification. Vitrified liquid water has been characterized by electron and X-ray diffraction, infrared spectroscopy and by differential thermal analysis warm-up curves. These properties are compared with those of amorphous solid water prepared by deposition of the vapour.

Resolution in low voltage scanning electron microscopy

Abstract: SUMMARY As the energy of an electron beam is reduced, the range falls and the secondary electron yield rises. A low voltage scanning electron microscope can therefore, in principle, examine without damage or charging samples such as insulators, dielectrics or beam sensitive materials. This paper investigates the way in which the choice of beam energy affects the spatial resolution of a secondary electron image. It is shown that for samples which are thin compared to the electron range, the edge resolution and contrast in the image improve with increasing beam energy. In samples that are thicker than the electron range, the resolution can be optimized at either high or low energies, but low energy operation will produce images of higher contrast. At an energy of 2 keV or less beam interaction limited resolutions of the order of 3 nm should be possible.

Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes.

Abstract: SUMMARY Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO4 fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO4 fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO4 fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.

The stabilization of labile configurations of plant cytoplasm by freeze‐substitution

Abstract: SUMMARY The system of elongated, vacuole-like structures that are seen in phase-contrast images of living plant cytoplasm, which has been called the pleomorphic canalicular system, has been shown previously to be distorted and vesiculated by conventional fixation methods The stabilization of this labile structure in tomato hair cells by freeze-substitution is described Structural features of the freeze-substituted cytoplasm as seen in whole mounts of resin-embedded cells resemble closely those of the living cells

A microcomputer based digital imaging system for ion microanalysis

Abstract: SUMMARY A microcomputer based digital imaging system was developed for a Cameca IMS-3f ion microscope permitting real-time digital acquisition of secondary ion images. Image signal-to-noise enhancement results from random noise reduction by real-time ensemble averaging and from a reduction of pattern noise in the charge injection device (CID) array by subtraction of blank frames. Acquired images comprise 244times248 pixel arrays with 8-bit intensity resolution. Images are displayed on a colour monitor in a grey scale or pseudo-colour using one of four programmable lookup tables. Image processing software permits off-line ion image enhancement and manipulation as well as multitechnique digital image correlations. System capability is illustrated by a biological example involving digital imaging studies of Al distribution in osteomalacic bone tissue, including correlative light microscopy and ion microanalysis.

Point cathodes for use in virtual source electron optics

Abstract: SUMMARY A comparison is made for three point cathodes which are used in an electron gun forming a virtual rather than real source. The three cathodes operate in the field emission (FE), thermal-field (TF) and Schottky emission (SE) regimes. A low angular intensity is chosen for this comparison, typical of an SEM as contrasted to an electron-beam lithography system application. Energy spread, virtual source size and current fluctuations are compared.

Recrystallization and ice‐crystal growth in a biological specimen, as shown by a simple freeze substitution method

Abstract: SUMMARY The sensory hairs of the silkmoth, Bombyx mori, are suitable test objects to check for recrystallization and secondary freezing damage in a biological object, because cryofixation by immersion into propane (90 K) routinely yields well-preserved specimens without noticeable freezing damage. After rewarming the frozen specimens for 10 min to 230 K (boiling propane), the tissue preservation has not deteriorated, and even after 45 min at 230 K, ice-crystal ghosts rarely exceed 50 nm. Two minutes at 250 K (in deep freezer) produced moderate freezing damage with ice-crystal ghosts of 30–75 nm, whereas 90 min at 250 K resulted in severe damage with ice-crystal ghosts well over 100 nm. Secondary freezing damage by ice-crystal growth upon rewarming well-frozen biological specimens, therefore, is a relatively slow process, depending not only on the temperature, but also on the exposure time. Moreover, with some biological specimens, secondary ice-crystal growth starts at much higher temperatures than previously guessed, and with short exposure times rarely should become a hazard in fine structure work.

Systematic inquiry in tests of negative/positive replica combinations for SEM

Abstract: SUMMARY High resolution replica materials are routinely used in scanning electron microscopy. A systematic evaluating procedure for replica combinations is proposed which details fourteen points to be recorded. These points include quantitative and qualitative information useful when assessing a replica combination for a particular research problem. A case study employing one silicone-based impression material and one epoxy resin is performed as an example of the procedure.

Digital image tiles: a method for the processing of large sections.

Abstract: SUMMARY Segmentation of large areas of light microscopic slides into N by N fields, and each of these fields into M digital image tiles, allows the scanning, storage and digital processing of large images. Any of the original N2 fields or composites of M adjacent tiles can be recalled to the video display for analysis. Developed procedures for use on a microscope equipped with a precision scanning stage allow registration of the image coordinates (X-Y) for any original or composite field and the alignment of one of these fields along the depth (Z) axis by means of external, machined fiducial marks in serial sections. To facilitate work whenever unavoidable, we have incorporated methods for digital image panning and zooming (changes of magnification) and discuss their use and implications.

A comprehensive set of stereological formulae for embedded aggregates of not‐necessarily‐convex particles

Abstract: SUMMARY Theoretical stereological approaches to particle aggregates characteristically suppose that each particle is convex, so that upon sectioning, each particle gives rise to either 0 or 1 convex planar profile. Actually, the essential requirement in such approaches is that each planar section profile be connected; such particles need not be convex. In this paper, aggregates of quite general non-convex particles, whose individual planar section profiles may thus comprise a number of disjoint connected regions, are considered. For this, it is necessary to anticipate the ultimate technical development of ultra-sensitive devices (or other methods) capable of identifying in a planar section those disjoint connected regions stemming from the same particle. With this assumption, it is shown how, using the ‘4-linc’—a new line section characteristic—to estimate the aggregate volume moment ratio , the standard isotropic section theory fully extends. To complete the picture, the recent development by Jensen & Gundersen of estimators for is extended to the ‘restricted’ case considered here. Allowing the particle volume distribution to belong to a two-parameter family, these two parameters, Nv and the aggregate mean values may all be estimated.

Soft X-ray contact microscopy with nanosecond exposure times

Abstract: SUMMARY High resolution (better than 20 nm) contact micrographs have been produced with exposure times of about a nanosecond. The illuminating source was a short-lived carbon plasma produced by focusing a single short (∼1 ns) 100 J pulse from the Vulcan laser at the Rutherford Appleton Laboratory (RAL) to a 300 μm spot on a graphite target. This plasma emits strongly in the soft X-ray region, particularly at the CVI (3.37 nm) and CV (4.03 nm) lines. The specimens were behind a 100 nm thick Si3N4 window, at atmospheric pressure in an environmental cell. The images of diatoms recorded on X-ray resist showed features down to the limit of resolution of the SEM used to view the developed resist, which was about 20 nm.

Microchannel plate detector for low voltage scanning electron microscopes

Abstract: SUMMARY A microchannel plate detector has advantages over conventional Everhart-Thornley detectors for low voltage SEM applications. A microchannel plate can provide symmetric SEM signal detection at the low beam energies needed for non-destructive examination of integrated circuits. Microchannel plate detectors are effective for both secondary electron and backscattered electron imaging. Their relatively small size and ability to be mounted directly below the final pole piece give them improved performance relative to conventional detectors in applications requiring short working distances. A significant amount of laboratory and applications experience has shown that microchannel plates are reliable and sufficiently resistant to contamination for use in high volume, production environments which require low voltage SEM imaging.

Penetration rate of glutaraldehyde in various buffers into plant tissue and gelatin gels

Abstract: SUMMARY The choice of buffer vehicle during fixation with glutaraldehyde influences not only the rate of crosslinking in a bovine serum albumin model system, but also the penetration rate of the glutaraldehyde into plant tissue and gelatin gels. The rate of penetration into plant tissue and the crosslinking rate of bovine serum albumin by glutaraldehyde are independent of each other in any one buffer. Buffer penetration into gelatin gels is not as fast as glutaraldehyde penetration into the same gels. The highest rate of crosslinking of bovine serum albumin by glutaraldehyde is obtained with a buffer vehicle consisting of Na2HPO4 and NaH2PO4. Glutarldehyde concentrations of 2–3% are advised for routine fixation of plant material.

X-ray microscopy using computerized axial tomography

Abstract: Computerized axial tomography with MoK alpha X-radiation has been used to study a 08 X 08 mm column of human femoral bone at a resolution of 15 micron This non-destructive X-ray microscopic technique allowed 'sections' to be made with 25 micron separation and the distribution of linear absorption coefficient in each section to be determined

Cryo-electron microscopy of helical particles TMV and T4 polyheads.

Abstract: Vitrified Tobacco Mosaic Virus (TMV) and T4 polyhead suspensions have been studied by cryo-electron microscopy. The order of TMV particles is preserved to distances better than 0.3 nm when vitrified. In spite of the high beam sensitivity of unstained biological material, the resolution of images extends to 1.15 nm. Radial density distributions calculated from images of TMV and the helical aggregate of TMV protein (TMVP) show that the RNA is likely visualized in the virus. However, due to the imaging conditions of unstained vitrified specimens, the position of the RNA is erroneous. While TMV and TMVP do not show any departure from cylindrical symmetry, T4 polyheads are sensitive to the thickness of the ice layer in which they are embedded. To avoid flattening, T4 polyheads have to be embedded in an ice layer thicker than their diameter.

A simple device for low temperature polymerization of Lowicryl K4M resin

Abstract: SUMMARY Difficulties concerning low temperature polymerization of Lowicryl K4M have been overcome by constructing and using a simplified polymerization chamber with plastic syringe. Five Beem capsules without cover containing the resin together with the specimen can be inserted into this syringe. For polymerization it is filled with nitrogen gas and closed tightly. Then the device is exposed to long wave UV-light at 253 K. Using this polymerization chamber Lowicryl K4M resin blocks hardened completely and homogeneously.

An X-ray diffraction analysis of rat tail tendons treated with Cupromeronic Blue.

Abstract: Cupromeronic Blue was used to stain selectively the proteoglycans in rat tail tendons under 'critical electrolyte' conditions. Earlier electron microscopical observations indicated that at least one type of proteoglycan filament is associated with tendon collagen fibrils at the positive staining band 'd'. To ensure that this was not an artefact caused by specimen preparation or the subsequent positive staining of the collagen fibrils, we have analysed low angle meridional diffraction patterns from stained but not dehydrated, embedded or counterstained tissues. Axial electron density profiles of Cupromeronic Blue-stained compared with unstained rat tail tendons revealed the axial locations and relative amounts of dye in both mature and young wet specimens. In mature tendons, the difference electron density profile contained a broad peak centred near residue 180 along the 234-residue D-period. This corresponds to the electron-optical staining band 'd'. In young tendons a similar distribution of stain was observed although in this case there was evidence of a doublet of peaks, one centred near residue 182 (band 'd') and the other near residue 165 (midway between bands d and e1). The wet proteoglycan--Cupromeronic Blue complexes distribute over about 30 nm along the collagen fibril axis. Comparison with the images of filaments seen in the electron microscope suggests that the dye complexes collapse significantly on dehydration and embedding.

Automatic analysis of the motion of microorganisms

Abstract: SUMMARY An automatic, interpretative, off-line computer-television-microscope system for studying the behaviour of microorganisms is presented. The equipment should be available even in relatively modestly equipped laboratories. The movement of microorganisms freely swimming in the chamber of a depression slide is recorded on a video tape with the aid of a microscope and an infrared TV camera. The images are acquired by means of a frame grabber in an image controller and elaborated by a PDP 11/23 computer. The procedures used for data processing are discussed in detail. Trajectories reconstructed by the computer are shown to be exactly superimposed to the real ones, and the motion parameters can be easily determined.

Weighted least squares estimation of background in EELS imaging.

Abstract: In quantitative Electron Energy Loss Spectrometry, a weighted least squares estimation should theoretically be used to estimate the background law below core edge energy, since the variances of the data vary. However, it is found that proper weighting makes the above edge signal-to-noise ratio decrease rather than increase. This result is discussed, and the influence of the bias introduced by the logarithmic transformation of the data is quantified.