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Summary: By being at the interface of the exocytic and endocytic pathways, the plant trans‐Golgi network (TGN) is a multitasking and highly diversified organelle. Despite governing vital cellular processes, the TGN remains one of the most uncharacterized organelle of plant cells. In this review, we highlight recent studies that have contributed new insights and to the generation of markers needed to answer several important questions on the plant TGN. Several drugs specifically affecting proteins critical for the TGN functions have been extremely useful for the identification of mutants of the TGN in the pursuit to understand how the morphology and the function of this organelle are controlled. In addition to these chemical tools, we review emerging microscopy techniques that help visualize the TGN at an unpreceded resolution and appreciate the heterogeneity and dynamics of this organelle in plant cells. [ABSTRACT FROM AUTHOR]

Summary: Herein we present a study on the formation of irreversibly adsorbed layer of polystyrene molecules on silicon oxide surfaces. Various scanning probe microscopy techniques have been employed to study both the morphology and the mechanical properties of these self‐assembled thin polymeric layers. More in detail, standard contact mode, force versus distance spectroscopy and ultrasonic force microscopy have been employed to obtain spatially‐resolved maps and, thus, observe the physisorption of polystyrene on native silicon oxide substrate in function of time. Thick films, spin coated from a toluene solution, have been annealed at a temperature above the glass transition for increasing time intervals, and finally thoroughly rinsed in toluene. We have found that isolated islands of adsorbed chains are already present after an annealing time of half an hour. Prolonged annealing determines a progressive increase of the covered areas, whereas the formation of a complete flat layer requires 24 h. The pattern observed is in line with expected evolution of an unstable system, corresponding to the phenomenon of spinodal dewetting. Adhesion measurements show that the films present a reduced snap‐off and the formation of a meniscus between tip and surface for annealing time up to 8 h. On the other hand, elastic measurements allow us to observe a progressive increase of the elastic modulus, with a complete transition for annealing time above 20 h. This is indication that a dense packing of the polystyrene molecules occurs, in line with the predictions of current models on the kinetics of irreversible adsorption. Lay Description: Herein we present a study on the formation of irreversibly adsorbed layer of polystyrene molecules on silicon oxide surfaces. Various scanning probe microscopy techniques have been employed to study both the morphology and the mechanical properties of these self‐assembled thin polymeric layers. Thick polystyrene films, spin coated from a toluene solution, have been thermally annealed at a temperature above the glass transition for increasing time intervals, and finally thoroughly rinsed in toluene. We have found that isolated islands of adsorbed chains are already present after an annealing time of half an hour. Prolonged annealing determines a progressive increase of the covered areas, whereas the formation of a complete flat layer requires twenty‐four hours. The adsorption pattern observed is in line with expected evolution of an unstable system, corresponding to the phenomenon of spinodal dewetting. Adhesion and elastic measurements have allowed us to observe a progressive increase of the packing density of the polystyrene molecules, in agreement with the predictions of current models on the kinetics of irreversible adsorption. [ABSTRACT FROM AUTHOR]

Summary: Bird feather shafts are light, stiff and strong, but the fine details of how their structure, mechanics and function relate to one another remains poorly understood. The missing piece in our understanding may be the various fibrous layers that make up the shaft's cortex. Detailed imaging techniques are needed to enable us to capture, analyse and quantify these layers before we can begin to unravel the relationship between their structure, mechanics and function. We show that Serial‐Block‐Face scanning electron microscopy, scanning confocal polarised microscopy and synchrotron‐based computed tomography are three suitable techniques to investigate layer thickness and fibre orientation in the feather cortex. These techniques and other are discussed in terms of their ability to resolve the fibrous laminar structure of the feather cortex, on sample preparation, and on throughput. Annotated images are presented for each and less suitable techniques are presented in the Supplementary Material. Lay Description: Bird feathers have a light, stiff and strong central shaft. However, the fine details of how their structure, mechanics and function relate to one another remains poorly understood. The missing piece in our understanding may have to do with how fibrous layers within the shaft vary in thickness and alignment. Detailed imaging techniques are needed so that we can quantify some of this variation before we can revisit some long‐unanswered questions about the feather shaft's structure, mechanics and function. We investigate a number of microscopy techniques and show that three techniques are suitable for the sort of investigation that is required. These techniques and others are discussed in terms of their ability to resolve the layers' thickness and alignment, on sample preparation, and on the sample sizes they are able to process. Annotated images are presented and discussed for each of the three techniques and unsuitable techniques receive the same examination in the Supplementary Material. [ABSTRACT FROM AUTHOR]

Autophagy is a principal degradation pathway for the turnover of intracellular proteins or cytoplasmic organelles in response to starvation. During autophagic activation, autophagosomes fuse with lysosomes to form autolysosomes where incorporated materials are degraded. However, the dynamics of autolysosomes in neurites of live cells was still poorly known. In this study, various subsets of microscope were applied to analyse the autophagy induction and highly dynamic transport of autolysosomes in rat PC12 neurites. Beading formation was found in degenerating PC12 neurites under phase contrast light microscope after serum deprivation. The monomeric red fluorescence protein-green fluorescence protein-light chain 3-labelled autolysosomes accumulated throughout PC12 neurites after 18 h of serum deprivation as revealed by fluorescence microscope analysis. The single-membrane autolysosomes were also visualized in PC12 cells under transmission electron microscope. Moreover, fluorescence recovery after photobleaching experiment, which was conducted by confocal laser scanning microscope, demonstrated that autolysosomes were motile vesicles and moved along PC12 neurites during starvation. The directional transport of monomeric red fluorescence protein -labelled autolysosomes in neurites was further monitored by a motorized video microscope. Both anterograde and retrograde transport of autolysosomes were observed. In addition, the autolysosomes were precisely mapped by using 2D Gaussian fitting and then their highly dynamic movement was robustly tracked by using multidimensional assignment. Collectively, by using different microscopical techniques, our results confirmed the dynamic transport of autolysosomes in starved PC12 neurites and may provide valuable insight into understanding the biophysical characteristics of autolysosomes in neurites under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

In order to observe the fine details of biomedical specimens, various kinds of high-magnification microscopes are used. However, they suffer from a limited field of view when visualizing highly magnified specimens. Image mosaicing techniques are necessary to integrate two or more partially overlapping images into one and make the whole specimen visible. In this study, we propose a new system that automatically creates panoramic images by mosaicing all the microscopic images acquired from a specimen. Not only does it effectively compensate for the congenital narrowness in microscopic views, but it also results in the mosaiced image containing as little distortion with respect to the originals as possible. The system consists of four main steps: (1) feature point extraction using multiscale wavelet analysis, (2) image matching based on feature points or by projection profile alignment, (3) colour difference adjustment and optical degradation compensation with a Gaussian-like model and (4) wavelet-based image blending. In addition to providing a precise alignment, the proposed system also takes into account the colour deviations and degradation in image mosaicing. The visible seam lines are eliminated after image blending. The experimental results show that the system performs well on differently stained image sequences and is effective on acquired images with large colour variations and degradation. It is expected to be a practical tool for microscopic image mosaicing. [ABSTRACT FROM AUTHOR]

Intravital microscopy imposes the particular problem of the combined control of the body temperature of the animal and the local temperature of the observed organ or tissues. We constructed and tested, in the rat ileum microcirculation preparation, a new organ-support platform. The platform consisted of an organ bath filled with physiological solution, and contained a suction tube, a superfusion tube, an intestine-support hand that was attached to a micromanipulator and a thermometer probe. To cover the intestine we used a cover glass plate with a plastic ring glued on its upper surface. After a routine procedure (anaesthesia, monitoring and surgery), the intestine segment (2–3 cm long) was gently exteriorized and placed on the ‘hand’ of the organ support. A small part of the intestine formed a small ‘island’ in the bath that was filled with physiological salt solution. The cover glass was secured in place. The physiological salt solution from the superfusion tube, which was pointed to the lower surface of the cover glass, formed a ‘hanging drop’. The objective of the microscope was then immersed into distilled water that was formed by the cover glass plastic ring. The ‘hanging drop’ technique prevented any tissue quenching, ensured undisturbed microcirculation, provided for stable temperature and humidity, and permitted a clear visual field. [ABSTRACT FROM AUTHOR]

Image mosaicing has found a number of applications such as panoramic imaging, digital terrain mapping, ophthalmology and virtual microscopy. In this study, we present an automated mosaicing technique for generating virtual slides from microscope images. We carried out robust image feature matching and global geometric and radiometric parameter estimation. The input images were transformed using the estimated geometric and radiometric parameters and mosaiced together, producing accurate registration of overlapping regions without visible seams. [ABSTRACT FROM AUTHOR]

Determining transmission electron microscope specimen thickness is an essential prerequisite for carrying out quantitative microscopy. The convergent beam electron diffraction method is highly accurate but provides information only on the small region being probed and is only applicable to crystalline phases. Thickness mapping with an energy filter is rapid, maps an entire field of view and can be applied to both crystalline and amorphous phases. However, the thickness map is defined in terms of the mean free path for energy loss (λ), which must be known in order to determine the thickness. Convergent beam electron diffraction and thickness mapping methods were used to determine λ for two materials, Si and P91 steel. These represent best- and worst-case scenario materials, respectively, for this type of investigation, owing to their radically different microstructures. The effects of collection angle and the importance of dynamical diffraction contrast are also examined. By minimizing diffraction contrast effects in thickness maps, reasonably accurate (±15%) values of λ were obtained for P91 and accuracies of ±5% were obtained for Si. The correlation between the convergent beam electron diffraction-derived thickness and the log intensity ratios from thickness maps also permits estimation of the thickness of amorphous layers on the upper and lower surfaces of transmission electron microscope specimens. These estimates were evaluated for both Si and P91 using cross-sectional transmission electron microscopy and were found to be quite accurate. [ABSTRACT FROM AUTHOR]

Summary For the validation of a cleaning method in conservation, the examination of the object's surface after the cleaning and the quantification of the cleaning efficiency are significant steps. In this study, several cleaning solutions were tested on a plasticized poly(vinyl chloride) object whose surface was characterized before and after cleaning by optical microscopy and noncontact profilometry. In addition, different methods to quantify the cleaning efficiency based on spectrocolorimetric and microscopic techniques were provided. The results showed that noncontact profilometry supported by optical microscopy and spectrocolorimetry was very useful for the characterization of the plasticized poly(vinyl chloride) surface before and after the cleaning and also for the determination of the cleaners' efficiency. [ABSTRACT FROM AUTHOR]

Automated segmentation of time-lapse images is a method to facilitate the understanding of the intricate biological progression, e.g. cancer cell migration. To address this problem, we introduce a shape representation enhancement over popular snake models in the context of confident scale–space such that a higher level of interpretation can hopefully be achieved. Our proposed system consists of a hierarchical analytic framework including feedback loops, self-adaptive and demand-adaptive adjustment, incorporating a steerable boundary detail term constraint based on multiscale B-spline interpolation. To minimize the noise interference inherited from microscopy acquisition, the coarse boundary derived from the initial segmentation with refined watershed line is coupled with microscopy compensation using the mean shift filtering. A progressive approximation is applied to achieve represented as a balance between a relief function of watershed algorithm and local minima concerning multiscale optimality, convergence and robust constraints. Experimental results show that the proposed method overcomes problems with spurious branches, arbitrary gaps, low contrast boundaries and low signal-to-noise ratio. The proposed system has the potential to serve as an automated data processing tool for cell migration applications. [ABSTRACT FROM AUTHOR]

Current understanding of the mechanisms involved in osseointegration following implantation of a biomaterial has led to adhesion quantification being implemented as an assay of cytocompatibility. Such measurement can be hindered by intra-sample variation owing to morphological changes associated with the cell cycle. Here we report on a new scanning electron microscopical method for the simultaneous immunogold labelling of cellular focal adhesions and S-phase nuclei identified by BrdU incorporation. Prior to labelling, cellular membranes are removed by tritonization and antigens of non-interest blocked by serum incubation. Adhesion plaque–associated vinculin and S-phase nuclei were both separately labelled with a 1.4 nm gold colloid and visualized by subsequent colloid enhancement via silver deposition. This study is specifically concerned with the effects microgroove topographies have on adhesion formation in S-phase osteoblasts. By combining backscattered electron (BSE) imaging with secondary electron (SE) imaging it was possible to visualize S-phase nuclei and the immunogold-labelled adhesion sites in one energy ‘plane’ and the underlying nanotopography in another. Osteoblast adhesion to these nanotopographies was ascertained by quantification of adhesion complex formation. [ABSTRACT FROM AUTHOR]

It is widely recognized that the accuracy of colocalization measurements is dependent upon the quality of the source images. We demonstrate that, as the image quality increases, the measured colocalization, using the Pearson and Spearman rank correlation coefficients, approaches the true colocalization asymptotically. This means that in practice it is difficult to obtain images of sufficient quality for accurate measurements. We introduce the replicate-based noise corrected correlation (RBNCC) which aligns the measured colocalization with the true colocalization: a noise measurement is made for each fluorophore from a pair of replicate images, the two noise measurements are combined to generate a correction factor which is applied to the measured colocalization between the two fluorophores. In consequence, accurate measurements can be obtained even with noisy images, making RBNCC especially attractive for live imaging. Even with images of apparently good quality we found an average discrepancy of about 20% between the measured and corrected colocalization. A case is made for using the Spearman rank coefficient instead of the Pearson coefficient to measure colocalization. [ABSTRACT FROM AUTHOR]

A new fully automated microfabric analyzer (MiFA) is described that can be used for the fast collection of high-resolution spatial c-axis orientation data from a set of digital polarized light images. At the onset of an analysis the user is presented with an axial-distribution diagram (AVA –‘Achsenverteilungsanalyse’) of a thin section. It is then a simple matter to build-up c-axis pole figures from selected areas of interest. The c-axis inclination and colatitudes at any pixel site is immediately available to create bulk fabric diagrams or to select measurements in individual areas. The system supports both the interactive selection of c-axis measurement sites and grid array selection. A verification process allows the operator to exclude dubious measurements due to impurities, grain boundaries or bubbles. We present a comparison of bulk and individual c-axis MiFA measurements to pole figures measured with an X-ray texture goniometer and to data collected from a scanning electron microscope furnished with electron backscatter diffraction (EBSD) facility. A second sample, an experimentally deformed quartzite, illustrates that crystal orientations can be precisely linked to any location within an individual grain. [ABSTRACT FROM AUTHOR]

Synchrotron-generated X-rays provide scientists with a multitude of investigative techniques well suited for the analysis of the composition and structure of all types of materials and specimens. Here, we describe the properties of synchrotron-generated X-rays and the advantages that they provide for qualitative morphological research of millimetre-sized biological organisms and biomaterials. Case studies of the anatomy of insect heads, of whole microarthropods and of the three-dimensional reconstruction of the cuticular tendons of jumping beetles, all performed at the beamline ID19 of the European Synchrotron Radiation Facility (ESRF), are presented to illustrate the techniques of phase-contrast tomography available for anatomical and structural investigations. Various sample preparation techniques are described and compared and experimental settings that we have found to be particularly successful are given. On comparing the strengths and weaknesses of the technique with traditional histological thin sectioning, we conclude that synchrotron radiation microtomography has a great potential in biological microanatomy. [ABSTRACT FROM AUTHOR]

Toluidine blue and gold chloride stains are both well-known for staining nervous tissue. Toluidine blue is a general method to stain neurons and glia, used because of the speed of action. Gold sublimate is a delicate procedure that requires extreme purity of reagents and materials, and it stains astrocytes and neurons. The aim of this study was to combine these two special staining methods for embryonic neural tissue and to obtain a repeatable and easily manageable new staining method with improved overall visualization of neural tissue. Fertilized Broiler hatching eggs were incubated at 36°C and on day 8 prepared for histology. Sections were cut at 7 μm thickness and slides were stained for 30 s using a 0.1% Toluidine blue solution. These slides were rinsed with dH2O followed by placing them in gold sublimate for 4 h at room temperature in the dark. Rinsing with dH2O and transferal to a 5% sodium thiosulfate solution was done, followed by another rinsing and mounting of slides. Results showed that the combination of the two methods offers a fast, reliable method with which chick embryonic neural tissue can be evaluated. [ABSTRACT FROM AUTHOR]

Sectioning vitrified cells and tissues for cryo-electron microscopy is more challenging than room-temperature sectioning of plastic-embedded samples. As the sample must be kept very cold (<−130 °C) and because there is no liquid upon which the sections can float as they are cut, transferring the sections from the knife edge to a grid is one of the more difficult steps in the process. We employed a micromanipulator to hold and control the cryo-sections as they come off the knife. This allows slower cutting speeds than are typically used in vitreous cryo-sectioning and contributes to better control during cutting, which facilitates repeatable placement of a ribbon of sections onto a grid. The ribbon is kept under tension during the entire cutting process, which may decrease folding and/or compression, features that are inherent to vitreous sections. Furthermore, the added control afforded by this technique makes it easier for multiple ribbons to be placed on a single grid, thereby increasing the number of sections that can be examined and imaged during a microscopy session. It even allows for serial cryo-electron microscopy. As such, this approach is an advance in the cryo-microtomy of vitreous sections. [ABSTRACT FROM AUTHOR]

We show a new atomic force microscopy technique for obtaining high-resolution topographic images of large bio-samples. To obtain high-resolution topographic images for the samples, we fabricated a long polymeric tip with a small protrusion using two-photon adsorbed photo-polymerization techniques. The obtained tip length was over 50 µm, and the tip was used directly to visualize COS-1 and 293 cells. Compared with commercial tips, the long tip made it easier to obtain topographic images of the large cells. In the magnified topographic images, the sub-100-nm resolution was confirmed with the long tips. This long probe tip is expected to broaden large sample-related studies and applications in the future. [ABSTRACT FROM AUTHOR]

Epifluorescence, confocal and total internal reflection microscopy are the most widely used techniques for optical single-molecule experiments. Employing these methods, we recorded the emission intensity of the same single molecule as a function of the excitation rate under otherwise identical experimental conditions. Evaluation of these data provides a quantitative comparison of the signal-to-background ratios that can be achieved for the three microscopic techniques. [ABSTRACT FROM AUTHOR]

Examines the femtosecond near-field spectroscopy. Relaxation and transport in single quantum wires; Combination of femtosecond spectroscopy with microscopy techniques; Investigation of carrier dynamics on nanometer length scales.

Analyzes the plant microtechnique and microscopy. Application of the method on various scientific studies; Effectiveness of the microtechnique and microscopy; Textual parameters on the microscopic images.

Soft X-ray microscopy is a powerful tool for investigations of, for example, polymers or soils in their natural liquid environment. This requires a wet-specimen chamber. Compact X-ray microscopy allows the horizontal mounting of such samples, thereby reducing the influence of gravitational forces. We have developed a wet-specimen chamber for such compact X-ray microscope. The chamber is vacuum compatible, which reduces the exposure time. The vacuum sealing is achieved by a combination of mechanical sealing and sealing by bio-compatible glue. With the wet-specimen chamber the specimens can be kept in an aqueous environment in a vacuum of 10−4 mbar for several hours. Imaging of lipid droplets in water demonstrates the function of the wet-specimen chamber. [ABSTRACT FROM AUTHOR]

A new sample holder that allows combined microtomy for atomic force microscopy (AFM) and transmission electron microscopy (TEM) is described. The main feature of this sample holder is a small central part holding the sample. This central part fits into the head of an atomic force microscope. AFM measurements can be performed with a sample mounted in this central part of the sample holder. This makes the alignment of a microtomed bulk sample unnecessary, and offers the opportunity of an easy and fast combined sample preparation for AFM and TEM. [ABSTRACT FROM AUTHOR]

A new microscopical technique based on the principle of confocal theta microscopy (Stelzer, E.H.K., Lindek, S. & Pick, R. (1996) Konfokales Mikroskop. German Patent Office DE 43 26 473 (filed 6.8.1993, granted 6.12.1996)) is described. It uses a single objective lens in combination with a mirror unit to achieve the theta configuration that leads to axial and volume resolution improvements. In this paper we present technical details of possible microscopical set-ups, and we discuss different versions of mirror units. [ABSTRACT FROM AUTHOR]

Summary: The existence of membrane contact sites (MCS) has been reported in different systems in the past decade, and their importance has been recognised by the cell biology community. Amongst all endomembrane structures, the endoplasmic reticulum (ER) plays vital roles in organising the organelle interaction network with the plasma membrane (PM), Golgi bodies, mitochondria, plastids, endosomes and autophagosomes. A number of methods have been used to study the establishment and functions of these interactions, among them, light microscopy appears to be one of the most effective approaches. Here, we present an overview of the discovery of ER–PM contact sites, and highlight the latest developments in light microscopical‐based techniques that can be used for their study. [ABSTRACT FROM AUTHOR]

Summary: The observation with RCM of the reflection from reaction products produced by nonisotopic in situ hybridisation and a peroxidase staining, has recently facilitated the identification of single copy genes. RCM also reveals light microscope structures in stained ultrathin (0.1 μm) epon and lowicryl sections. In such preparations no out‐of‐focus blur can be observed. Confocal optics are thus not needed to obtain high‐quality images of ultrathin sections at the highest conventional light microscope optical resolution, and with higher image‐contrast than can be obtained by classical absorption microscopy. Such RCM images provide the same type of information as confocal scanning microscope images. A sequential ultrathin section of the same microscopic specimen can be examined with electron microscopy for a simple and accurate (CLEM) procedure. For some applications RCM can replace (CLSM). Lay Description: With reflection contrast microscopy (RCM), a microscope image can be observed if micromirror‐like reflecting substances are present in the reaction product of cytochemical stains. Using an antiflex objective for RCM, the reflected incident light from the stained microscope specimen can be observed with only a small amount of unwanted stray‐light in the image background. Stray‐light is caused by reflection of incident light from the surface of the lenses in the microscope objective and in the microscope tube (using a 50% beam splitter for epi‐illumination). Several authors who compared RCM with bright‐field and fluorescence microscopy, found RCM to be a sensitive microscope method in many applications. It has been reported that RCM, using in situ hybridisation methods, can detect DNA sequences in single copy genes. In the literature is mentioned that single copy genes have an estimated mass of less than a trillionth of a gram of DNA. From stained ultrathin microscope sections with less than 0.1 μm thickness, multicoloured images with a high image contrast, optimal resolution and no out‐of‐focus blur can be observed with RCM. To achieve this, RCM uses an antiflex microscope objective, with a quarter‐wave plate mounted on the front lens in combination with a polarising filter cube inserted in the epi‐illuminator. RCM can also make use of oblique illumination, for a further increase of the image‐contrast and the resolution of the microscope image. A central stop has then to be inserted in the aperture plane of an epi‐illuminator. [ABSTRACT FROM AUTHOR]

We have developed a method based on confocal laser scanning microscopy for detection and quantification of cyanobacteria from the Ebro Delta microbial mats. Cyanobacteria play a major role as primary producers in microbial mats; it is difficult, however, to apply classical methods to estimate their biomass because they establish strong interactions with detritic particles. The protocol described here allows the localization of individual cells or filaments with micrometre precision without the need to either manipulate or stain the samples. This method is suitable for studying biomass ‘in situ’ in microbial mats. [ABSTRACT FROM AUTHOR]

A method is described that allows rapid and reliable serial sectioning down to thicknesses of 1 μm. The tissue is first embedded in celloidin and then in wax and trimmed so that the block is sandwiched between two layers of wax. This combines the virtues of both media. The celloidin gives greater support to tissue than wax and enables the cutting of semithin sections. The wax allows ribbons of serial sections to be produced as in conventional wax microtomy. This makes it easy to produce serial semithin sections as a matter of routine. [ABSTRACT FROM AUTHOR]

Ceramic composites based either on a particulate, fibre or a lamellar architecture are potentially useful as damage-tolerant high-temperature engineering materials. The ability of the interfaces in such systems to deflect cracks is vital to the damage tolerance of these materials. Transmission electron microscopy techniques enable the chemical and physical characterization of these interfaces, providing information on interlayer thicknesses, chemical species, local bonding and the microstructural features which give rise to the interfacial properties, thereby enabling a full understanding not only of composites after processing, but also after exposure to aggressive environments such as air at high temperature. Examples of the application of transmission electron microscopy to all three composite architectures are described. [ABSTRACT FROM AUTHOR]

The ultrastructure of primary cultured rat Kupffer cells was studied using transmission X-ray microscopy as well as transmission electron microscopy. X-ray microscopical images of intact, hydrated Kupffer cells demonstrated structures such as cell nucleus separated by a nuclear membrane and filaments concentrated in the perinuclear area. Within the cytoplasm, a number of vacuoles were visible; some of these were crescent-shaped vacuoles that were half X-ray lucent, half X-ray dense; others were uniformly dense. The number of crescent-shaped vacuoles was predominant. After phagocytosis of haematite particles, enlarged vacuoles containing the ingested material were visible within the cytoplasm of Kupffer cells while crescent-shaped vacuoles were no longer detectable. Densitometric analysis of the two types of vacuole revealed that the X-ray absorption of the uniform vacuole was approximately half that of the dense part of the crescent-shaped vacuoles. This observation led to speculation on the existence of only one type of vacuole in the cytoplasm of Kupffer cells. The different morphological aspects — crescent-shaped versus uniform vacuoles — might be due to different three-dimensional orientation with respect to the image plane. Using transmission electron microscopy, the morphology of vacuoles differed more widely in diameter, density and shape. Two main types of vacuole were identified: electron-lucent and electron-dense. Based on the observation of only one type of vacuole by transmission X-ray microscopy, the different morphological aspects of vacuoles obtained by transmission electron microscopy could be explained by imaging several different sections of a crescent-shaped vacuole. From the present data it can be concluded that transmission X-ray microscopy is a versatile technique that reveals the ultrastructure of intact, unsectioned biological specimens in their aqueous environment, thereby allowing a more comprehensive interpretation of... [ABSTRACT FROM AUTHOR]

Determines the significance of the microscopial technique in the study of nanotechnology and bioscience. Characterization of polysaccharides at morphological and quantitative levels; Interaction of atomic forces; Measurement of molecular volumes. [ABSTRACT FROM AUTHOR]

We have examined the feasibility of incident light microscopy for visualizing the poorly reflecting sensory epithelium in the macula of the fish lateral line in vivo. This investigation demonstrates that the apical cell surface and the hair bundles of the sensory hair cells in the epithelium can be visualized with incident light polarization microscopy (ILPM). Under the most favourable conditions of illumination, individual sensory hairs with a diameter of about 0.3 micron can be distinguished. Also, in the superficial layers of the macula up to a depth of about 200 microns branching nerve fibres and blood capillaries can be readily discerned. From measurements of the polarized light flux as a function of focal depth it followed that the image of the sensory epithelium is formed by the light reflected from the underlying nerves. Thus although the preparation is illuminated with incident light, the image of the object is actually formed under transmitted light conditions. We demonstrate further that the present microscopical technique can be successfully applied to several other poorly reflecting biological objects, which are unsuited for transmitted light microscopy because of their thickness.