Here we describe refinements in the processing of high-pressure frozen samples of delicate plant tissues for immuno-electron microscopy. These involve: shortened freeze-substitution schedules, lower temperatures during processing and polymerisation, the avoidance of temperature fluctuations and the optimisation of heat transfer from the specimens using small disposable aluminium containers. The application of these modifications leads to very good structural preservation and selective membrane contrast. As a result, the versatility of the method is increased since not only immuno-electron microscopical studies can be performed but often the quality is also quite suitable for structural investigations.

The higher plant Golgi apparatus consists of hundreds of individual Golgi stacks which move along the cortical ER, propelled by the actomysin system. Anterograde and retrograde transport between the endoplasmic reticulum (ER) and the plant Golgi occurs over a narrow interface (around 500 nm) and is generally considered to be mediated by COP-coated vesicles. Previously, ER exit sites (ERES) have been identified on the basis of to localization of transiently expressed COPII-coat proteins. As a consequence it has been held that ERES in higher plants are intimately associated with Golgi stacks, and that both move together as an integrated structure: the “secretory unit”. Using a new COPII marker, as well as YFP-SEC24 (a bona fide COPII coat protein), we have made observations on tobacco leaf epidermis at high resolution in the CLSM. Our data clearly shows that COPII fluorescence is associated with the Golgi stacks rather than the surface of the ER and probably represents the temporary accumulation of COPII vesicles in the Golgi matrix prior to fusion with the cis-Golgi cisternae. We have calculated the numbers of COPII vesicles which would be required to provide a typical Golgi-associated COPII-fluorescent signal as being much less than 20. We have discussed the consequences of this and question the continued usage of the term “secretory unit”.

Confocal microscopy is a suitable method for measurements and visualization of skeletal muscle fibres and the neighbouring capillaries. When using 3D images of thick sections the tissue deformation effects should be avoided. We studied the deformation in thick sections of the rat skeletal muscle from complete stacks of images captured with confocal microscope. We measured the apparent thickness of the stacks and compared it to the slice thickness deduced from calibrated microtome settings. The ratio of both values yielded the axial scaling factor for every image stack. Careful sample preparation and treatment of the tissue cryosections with cold Ringer solution minimize the tissue deformation. We conclude that rescaling by the inverse of the axial scaling factor of the stack of optical slices in the direction of the microscope optical axis satisfactorily corrects the axial deformation of skeletal muscle samples.

We implement a filterless illumination scheme on a hyperspectral fluorescence microscope to achieve full-range spectral imaging. The microscope employs polarisation filtering, spatial filtering and spectral unmixing filtering to replace the role of traditional filters. Quantitative comparisons between full-spectrum and filter-based microscopy are provided in the context of signal dynamic range and accuracy of measured fluorophores’ emission spectra. To show potential applications, a five-colour cell immunofluorescence imaging experiment is theoretically simulated. Simulation results indicate that the use of proposed full-spectrum imaging technique may result in three times improvement in signal dynamic range compared to that can be achieved in the filter-based imaging.

Combined light microscopic (LM) and field emission scanning electron microscopic (FESEM) techniques with FluoroNanogold labelling allowed quantification and high resolution analysis of 3D distribution of the centromere-specific histone H3 variant CENH3 in barley mitotic chromosomes. Chromosomes were investigated with fluorescence LM, conventional FESEM, low-voltage FESEM and combined FIB/FESEM techniques for unprecedented comprehensive analysis to determine chromatin distribution patterns in the centromere. Using data from FIB/FESEM sectioning of centromeric regions of chromosomes, it was possible to render 3D reconstruction of the CENH3 distribution with highest resolution achieved to date. Complementary data derived from each approach show that CENH3 localizes not only to the primary constriction, but also in the pericentric regions and is distributed exclusively in the interior, rather than on the surface, of the centromere. This is relevant for understanding kinetochore assembly and digresses from current models of centromere structure. We emphasize here this broad microscopic approach, focusing on technical aspects of combined FESEM techniques, for which advantages and limitations are discussed, providing a relevant example—in the field of centromeric research—for application to investigations of other subcellular biological structures.

4’-6-Diamidino-2-phenylindole is a fluorescent dye commonly used to visualize deoxyribonucleic acid or cell nuclei in fixed cell preparations, and is often used together with fluorescein or green fluorescent protein, which can be excited without exciting 4’-6-Diamidino-2-phenylindole. It is assumed that when using typical fluorescein or green fluorescent protein filter cubes, 4’-6-Diamidino-2-phenylindole will not be observed. In this paper, we show that following observation of 4’-6-Diamidino-2-phenylindole using UV or violet excitation, it may become sensitive to the blue/cyan excitation used in fluorescein/green fluorescent protein filter cubes. This has serious implications for the use of 4’-6-Diamidino-2-phenylindole together with widely used green fluorophores in double labelling experiments.

Cryogenic temperature scanning electron microscopy (cryo-SEM) is an excellent technique for imaging liquid and semi-liquid materials of high vapour pressure, which are highly viscous or contain large (>0.5 μm) aggregates, in which nanometric details are to be studied. However, so far there have been no adequate tools for controlled cryo-specimen preparation. The specimen preparation stage is critical, because most of those samples are very sensitive to concentration and temperature changes, leading to nanostructural artefacts in the specimens. We designed and built a system for easy and reliable cryo-SEM specimen preparation under controlled conditions of fixed temperature and humidity. We describe this new methodology, and demonstrate its applicability, by showing imaging data of three liquid material systems. We have studied carbon nanotubes (CNTs) dispersions in superacid. We also characterized a number of systems made of water/isooctane/nonionic and cationic surfactant that showed different microemulsion phases as function of the system composition and temperature. In all of the examples given, we demonstrate artefact- and contamination-free specimens, which have preserved their native nanostructure. Our new system paves the way for a new methodology for the newly emerging field of cryo-SEM.

The determination of grain boundary planes in multicrystalline material has only been restricted to transmission electron microscope investigations (Jang et al., 1992; Elgat et al., 1985) or to metallograpical investigations of the grain boundary (Randle et al., 1993). The first method is expensive, and both are complex and time consuming in grain boundary preparation. This paper proposes the determination of grain boundary planes in semiconductor wafer by a combined application of Electron Back Scatter Diffraction and Infrared Transmission Microscopy. In particular, the new method is demonstrated with directional solidificated multicrystalline silicon.

A focused ion beam–scanning electron microscope (FIB–SEM) technique for three-dimensional reconstruction and representation of material microstructures was applied to a silica-filled synthetic rubber for the first time. Backscattered electron imaging allowed differentiation between rubber matrix, silica filler and zinc oxide (used as an activator for the sulphur vulcanisation reaction). Subsequent image processing allowed three-dimensional isosurface model generation of the particulate structure within the rubber composite and separation of zinc oxide from the silica filler. The potential for development and application of this technique using finite element analysis modelling is also highlighted.

Many fundamental biological processes, such as the search for food, immunological responses and wound healing, depend on cell migration. Video microscopy allows the magnitude and direction of cell migration to be documented. Here, we present a simple and inexpensive method for simultaneous tracking of hundreds of migrating cells over periods of several days. Low-magnification dark-field microscopy was used to visualize individual cells whereas time-lapse video images were acquired by computer for future analysis. We employed an automated tracking algorithm to identify individual cells on each video image allowing migration paths to be tracked using a nearest neighbour algorithm. To test the method, we followed the time-course of migration of 3T3 fibroblasts, endothelial cells and individual amoeba in the absence of any chemical stimulus gradient. All cell types showed a ‘random walk’ behaviour in which mean squared displacement in position increased linearly with time. We defined a ‘migration coefficient’ (D_mig), analogous to a diffusion coefficient, which gave an estimate of cell migration rate. D_mig depended on cell type and temperature. When amoebas were made to undergo chemotaxis, the cells no longer followed a random walk but instead moved at a near constant velocity (V_av) towards the chemotactic stimulus.

The ever-increasing interest in nanostructured materials and shrinking dimensions of state-of-the-art devices pose new challenges both in synthesis and metrology. Although an extensive range of nanotubular materials of different compositions and for various applications are reported in the literature, often detailed structural characterisation of these materials is limited. This is due to the fact that techniques and characterisation protocols for structural analysis of ‘buried’ nano-scale features, defects or inhomogenities that are difficult to obtain by conventional imaging methods, are still not fully developed. In the case of 1D nanoporous structures, the continuity of the nano-tubular channels, their uniformity and orientation is of particular interest. Herein, we employ a serial sectioning technique on a dual beam FIB followed by 3D volume reconstruction for comprehensive analysis of tubular metal nanostructures encapsulated within porous anodic alumina. Using this technique, we demonstrate a nano-tomography characterisation protocol that can be used for analysis of nanoporous structures with emphasis on their channel uniformity and orientation. We demonstrate that high-resolution nano-tomography can be performed to visualise pores as small as 60 nm in diameter, with conical or globular shapes, and to quantitatively estimate their localisation and distribution along one-dimensional metal structures. We specifically chose to examine Cu-nanotubes, deposited electrochemically within anodic alumina template, because there is a great deal of debate regarding the deposition process. Hence, the comprehensive analysis shown here is not only demonstrating the applicability of the developed characterisation methodology but it is also, in conjunction with other advanced electron microscopy methods such as elemental nano-scale STEM/EDX mapping, providing conclusive evidence of the key factors at play during the deposition process.

The purpose of this study is to investigate how to scale pixel intensity acquired from one exposure time to another. This is required when comparing grayscale images acquired at different exposure times and other image processing such as autofluorescence removal. Pixel intensity is linear to exposure time as long as images are acquired at the linear range of a camera, but importantly there exists an intercept, which is set by the camera. We termed this intercept as dark pixel intensity, as it is the pixel intensity under conditions of no light and zero exposure time. Dark pixel intensity is determined by camera's readout noise (electron/pixel), gain, and DC offset. Knowing dark pixel intensity, image acquired from one exposure time can be linearly scaled to an image at a different exposure time. Dark pixel intensity can be directly measured by obtaining an image at no light and zero (or minimum) exposure time. It can be also indirectly calculated by capturing images at a series of exposure times. Finally, the prestained and poststained images were acquired at their optimal exposures and autofluorescence was completely removed by normalizing images with the exposure time ratio and dark pixel intensity followed by subtraction.

In orthodontic treatment, the frictional force between the archwire and bracket reduces the effectiveness of orthodontic treatment. The frictional force is affected not only by the geometry of the self-ligating brackets but also by physical changes between the bracket slots and archwire surfaces during sliding movement. This study examined quantitatively the effect of self-ligating treatments on the surfaces of stainless steel (SS) archwires during tooth movement in vivo by atomic force microscopy. Orthodontic 0.019″ × 0.025″ SS archwires after clinical use with the first bicuspid-extraction treatment were employed using the Damon 3MX^® SS self-ligating brackets, Clippy-C^® ceramic self-ligating brackets, and Kosaka^® SS brackets. Intact SS archwires were used as the control group. All SS archwires after clinical use showed severe scratches and significantly higher roughness caused by frictional interactions between the brackets and archwires (p < 0.0001 vs. control). The descending order of surface roughness was the SS archwires treated, with ceramic self-ligating brackets, with conventional SS brackets, and with SS self-ligating brackets (p < 0.001). These findings suggest that an orthodontic treatment with SS self-ligating brackets may require smaller orthodontic forces than that with ceramic self-ligating brackets or conventional SS brackets.

In this review, I ask the question of what is the relationship between growth and the orientations of microtubules and cellulose microfibrils in plant cells. This should be a relatively simple question to answer considering that text books commonly describe microtubules and cellulose microfibrils as hoops that drive expansion perpendicular to their orientation. However, recent live imaging techniques, which allow microtubules and cellulose synthase dynamics to be imaged simultaneously with cell elongation, show that cells can elongate with nonperpendicular microtubule arrays. In this review, I look at the significance of these different microtubule arrangements for growth and cell wall architecture and how these resultant walls differ from those derived from perpendicular arrays. I also discuss how these divergent arrays in stems may be important for coordinating growth between the different cell layers. This role reveals some general features of microtubule alignment that can be used to predict the growth status of organs. In conclusion, nonperpendicular arrays demonstrate alternative ways of cell elongation that do not require hooped arrays of microtubules and cellulose microfibrils. Such nonperpendicular arrays may be required for optimal growth and strengthening of tissues.

Plant cells are directly connected by plasmodesmata that form channels through the cell wall and enable the intercellular movement of cytosolic solutes, membrane lipids and signalling molecules. Transport through plasmodesmata is regulated not only by a fixed size-exclusion limit, but also by physiological and pathological adaptation. To understand plant cell communication, carbon allocation and pathogen attack, the capacities for a specific molecule to pass a specific cell–wall interface is an essential parameter. So far, the degree of cell coupling was derived from frequency and diameter of plasmodesmata in relevant tissues as assessed by electron microscopy of fixed material. However, plasmodesmata functionality and capacity can only be determined in live material, not from electron microscopy, which is static and prone to fixation artefacts. Plasmodesmata functionality was a few times assessed using fluorescent tracers with diffusion properties similar to cytosolic solutes. Here, we used three-dimensional photoactivation microscopy to quantify plasmodesmata-mediated cell–wall permeability between living Cucurbita maxima leaf mesophyll cells with caged fluorescein as tracer. For the first time, all necessary functional and anatomical data were gathered for each individual cell from three-dimensional time series. This approach utilized a confocal microscope equipped with resonant scanner, which provides the high acquisition speed necessary to record optical sections of whole cells and offers time resolution high enough to follow the kinetics of photoactivation. The results were compared to two-dimensional measurements, which are shown to give a good estimate of cell coupling adequate for homogenous tissues. The two-dimensional approach is limited whenever tissues interfaces are studied that couple different cell types with diverse cell geometries.

Effect of aging on the morphology of bitumen was investigated. Two bitumens were aged according to the thin film oven test (TFOT), pressure aging vessel (PAV) test and ultraviolet (UV) radiation, respectively. The morphology of the binders before and after aging was characterized by atomic force microscopy. The physical properties and chemical compositions of the binders were also measured. The results showed that aging affected the bitumen morphology significantly. Aging increased the overall surface stiffness of the bitumen and made the bitumen surface more solid-like. The extent of these changes was dependent on aging conditions. TFOT decreased the contrast between the dispersed domains and the matrix, which contributed to the single-phase trend of the binders. The effect of PAV aging on morphology of the binders was dependent on the base bitumen. In one case, it further accelerated the single-phase trend of bitumen in comparison with that after TFOT. In the other case, it caused the phase separation of bitumen. In both cases, PAV aging increased the surface roughness of the binders obviously. As a result of UV aging, the contrast between the matrix phase and dispersed phase was increased due to the difference in sensitivity to UV radiation of the bitumen molecules, which caused or further promoted the phase separation in the binders. Regardless of the aging procedure carried out, a strong correlation was observed between the changes in morphology and physical properties as well as chemical compositions of the binders before and after aging.

Na-CMC or sodium carboxylmethyl cellulose is a water soluble anionic polymer obtained by introducing carboxymethyl groups along the cellulose chain. Na-CMC is usually synthesized by the alkali catalyzed reaction of cellulose with monochloroacetic acid. The functional properties of Na-CMC depend on the degree of substitution of the cellulose structure (i.e. how many of the hydroxyl groups are substituted per monomer unit), and also on the chain length of the cellulose backbone. The degree of substitution of Na-CMC is usually determined according to ASTM D1439 which evolves the conversion of the Na-CMC to free acid then again forming Na-CMC by adding excess alkali and finally titrating the excess alkali with standard hydrochloric acid (0.3 N). The used volume of the standard alkali determines the degree of substitution. These existing chemical methods for determining the degree of substitution are not very convenient and very time-consuming involving the use of hazardous chemicals. In this research, we have evaluated that the scanning electron microscope equipped with Energy Dispersive X-Ray Analysis can be used to directly determine the degree of substitution.

Image registration is a process of aligning two or more images taken at different times or using different sensors by transforming the same area into one coordinate system. Imaging conditions, image and area deteriorations from repeated sectioning, are serious impediments to successful image registration. The application of artificial neural networks for feature recognition is introduced to the field of metallurgy to assist in an automated approach to image registration of metallurgical microstructures. Low susceptibility to feature deterioration, often occurring during serial sectioning, is demonstrated and assessed. The process of image registration using an artificial neural network to aid in feature segmentation is performed using computer generated shapes and a metallurgical microstructure.

This work describes the characean internodal cell as a model system for the study of wound healing and compares wounds induced by certain chemicals and UV irradiation with wounds occurring in the natural environment. We review the existing literature and define three types of wound response: (1) cortical window formation characterised by disassembly of microtubules, transient inhibition of actin-dependent cytoplasmic streaming and chloroplast detachment, (2) fibrillar wound walls characterised by exocytosis of vesicles carrying wall polysaccharides and membrane-bound cellulose synthase complexes coupled with endocytosis of surplus membrane and (3) amorphous, callose- and membrane-containing wound walls characterised by exocytosis of vesicles and endoplasmic reticulum cisternae in the absence of membrane recycling. We hypothesize that these three wound responses reflect the extent of damage, probably Ca²⁺ influx, and that the secretion of Ca²⁺-loaded endoplasmic reticulum cisternae is an emergency reaction in case of severe Ca²⁺ load. Microtubules are not required for wound healing but their disassembly could have a signalling function. Transient reorganisation of the actin cytoskeleton into a meshwork of randomly oriented filaments is required for the migration of wound wall forming organelles, just as occurs in tip-growing plant cells. New data presented in this study show that during the deposition of an amorphous wound wall numerous actin rings are present, which may indicate specific ion fluxes and/or a storage form for actin. In addition, we present new evidence for the exocytosis of FM1–43-stained organelles, putative endosomes, required for plasma membrane repair during wound healing. Finally, we show that quickly growing fibrillar wound walls, even when deposited in the absence of microtubules, have a highly ordered helical structure of consistent handedness comprised of cellulose microfibrils.

Thirty years ago, in December 1981, The Journal of Microscopy published a very short paper entitled ‘Vitrification of pure water for electron microscopy’. It turned out to be important for the development of cryo-electron microscopy and it contributed to reverse, from foe to friend, the status of water in electron microscopists’ minds. This change has brought obvious gains. The future will tell how many more are still to come.

In this paper we report stimulated emission depletion (STED) and two-photon excitation (2PE) fluorescence microscopy with continuous wave (CW) laser beam using a new generation laser scanning confocal microscope equipped for STED-CW (TCS STED-CW, Leica Microsystems, Mannheim, Germany). We show the possibility to achieve CW-2PE with the very same beam used for STED-CW. This feature extends the performance of the microscope allowing multimodal imaging (CW-2PE, STED-CW, confocal).

KillerRed, a bright red fluorescent protein, is a genetically encoded photosensitizer, which generates radicals and hydrogen peroxide upon green light illumination. The protein is a potentially powerful tool for selective light-induced protein inactivation and cell killing, and can also be used to study downstream effects of locally increased levels of reactive oxygen species. The initial aim of this study was to investigate whether or not KillerRed-mediated reactive oxygen species production inside peroxisomes could trigger the sequestration of these organelles into autophagosomes. Green fluorescent protein-tagged microtubule-associated protein 1 light chain 3 was used as autophagosome marker. We observed that KillerRed also emits weak green fluorescence upon excitation at 480 nm, and this may lead to erroneous data interpretation in conditions where green fluorophores are used. We discuss this potential pitfall of KillerRed for biological imaging and formulate recommendations to avoid misinterpretation of the data.

This paper presents a new high speed vision system using an asynchronous address-event representation camera. Within this framework, an asynchronous event-based real-time Hough circle transform is developed to track microspheres. The technology presented in this paper allows for a robust real-time event-based multiobject position detection at a frequency of several kHz with a low computational cost. Brownian motion is also detected within this context with both high speed and precision. The carried-out work is adapted to the automated or remote-operated microrobotic systems fulfilling their need of an extremely fast vision feedback. It is also a very promising solution to the micro physical phenomena analysis and particularly for the micro/nanoscale force measurement.

The spatial resolution of electron diffraction within the scanning electron microscope (SEM) has progressed from channelling methods capable of measuring crystallographic characteristics from 10 μm regions to electron backscatter diffraction (EBSD) methods capable of measuring 120 nm particles. Here, we report a new form of low-energy transmission Kikuchi diffraction, performed in the SEM. Transmission-EBSD (t-EBSD) makes use of an EBSD detector and software to capture and analyse the angular intensity variation in large-angle forward scattering of electrons in transmission, without postspecimen coils. We collected t-EBSD patterns from Fe–Co nanoparticles of diameter 10 nm and from 40 nm-thick Ni films with in-plane grain size 15 nm. The patterns exhibited contrast similar to that seen in EBSD, but are formed in transmission. Monte Carlo scattering simulations showed that in addition to the order of magnitude improvement in spatial resolution from isolated particles, the energy width of the scattered electrons in t-EBSD is nearly two orders of magnitude narrower than that of conventional EBSD. This new low-energy transmission diffraction approach builds upon recent progress in achieving unprecedented levels of imaging resolution for materials characterization in the SEM by adding high-spatial-resolution analytical capabilities.

Shape from focus is an elegant method that estimates the structure of a 3D object from a video of captured frames using the degree of focus as the principal cue. However, the quality of the estimated structure is vulnerable to scene texture. The effect is particularly pronounced for objects that are smooth relative to the magnification of the optical system. In this paper, the shape estimation process is cast as an inverse problem. We exploit spatial dependencies by modeling the shape of the object with a discontinuity-adaptive Markov random field wherein the focus measure profile is used to judiciously control the degree of smoothness. The 3D information is obtained by minimizing a suitably derived energy function that preserves fine details of the underlying structure. We show by experimentation on several real-world specimens that our method yields state-of-the-art performance.

Gene expression and other cellular processes are stochastic, thus their study requires observing multiple events in multiple cells. Therefore, confocal microscopy cell imaging has recently gained much interest. In time-lapse imaging, adjustments are needed at short intervals to compensate for focus drift. There are several automated methods for this purpose. In general, before acquiring higher resolution images, software-based autofocus algorithms require a set of low-resolution images along the z-axis to determine the plane for which a predefined focusing function is maximized. These algorithms require 10–100 z-slices each time, and there is no fixed number or upper limit of required z-slices that ensures optimal focusing. The higher is this number, the stronger is photo bleaching, hampering the feasibility of long-time series measurements.

 We propose a new focusing strategy in time-lapse imaging. The algorithm relies on the nature and predictability of the focus drift. We first show that the focus drift curve is predictable within a small error bound in standard experimental setups. We, then, exploit the interacting multiple model filter algorithm to predict the drift at time, t, based on the measurement at time t – 1. This allows a drastic reduction of the number of required z-slices for focus drift correction, largely overcoming the problem of photo bleaching. In addition, we propose a new set of functions for focusing in time-lapse imaging, derived from preexisting ones. We demonstrate the method's efficiency in time-lapse imaging of Escherichia coli cells expressing MS2d-GFP tagged RNA molecules.

In this paper, a new toolbox for petrographic thin section analysis is presented. It was designed using ArcGIS ModelBuilder to extract quartz grain boundary and determine its optical axis orientation from a set of thin section images. It can perform these analyses with little or no operator intervention and convert the boundary and optical axis data into a digital database with preserved spatial relationship. In order to test the validity of toolbox's outputs, an assessment was performed using three natural quartzite thin sections. The results indicate a close relationship (>0.8) between manual and model-base extraction of grain boundary and optical axis orientation data. This study demonstrates the feasibility and usefulness of performing thin section image analysis within the GIS framework.

Although focused ion beam (FIB) microscopy has been used successfully for milling patterns and creating ultra-thin electron and soft X-ray transparent sections of polymers and other soft materials, little has been documented regarding FIB-induced damage of these materials beyond qualitative evaluations of microstructure. In this study, we sought to identify steps in the FIB preparation process that can cause changes in chemical composition and bonding in soft materials. The impact of various parameters in the FIB-scanning electron microscope (SEM) sample preparation process, such as final milling voltage, temperature, ion beam overlap and mechanical stability of soft samples, was evaluated using two test-case materials systems: polyacrylamide, a low melting-point polymer, and Wyodak lignite coal, a refractory organic material. We evaluated changes in carbon bonding in the samples using X-ray absorption near-edge structure spectroscopy (XANES) at the carbon K edge and compared these samples with thin sections that had been prepared mechanically using ultramicrotomy. Minor chemical changes were induced in the coal samples during FIB-SEM preparation, and little effect was observed by changing ion-beam parameters. However, polyacrylamide was particularly sensitive to irradiation by the electron beam, which drastically altered the chemistry of the sample, with the primary damage occurring as an increase in the amount of aromatic carbon bonding (C=C). Changes in temperature, final milling voltage and beam overlap led to small improvements in the quality of the specimens. We outline a series of best practices for preparing electron and soft X-ray transparent samples, with respect to preserving chemical structure and mechanical stability of soft materials using the FIB.

Cortical bone microstructure is an important parameter in the evaluation of bone strength. The aim of this study was to validate the characterization of human cortical bone microarchitecture using microcomputed tomography. In order to do this, microcomputed tomography structural measurements were compared with those obtained through histological examination (the gold standard). Moreover, to calculate structural parameters, microcomputed tomography images have to be binarized with the separation between bone and nonbone structures throughout a global thresholding. As the effect of the surrounding medium on the threshold value is not clear, an easy procedure to find the global uniform threshold for a given acquisition condition is applied. This work also compared the structural parameters of microcomputed tomography cortical sample scan in air or embedded in polymethylmethacrylate; histology was used as a reference. For each acquisition condition, a fixed threshold value was found and was applied on the corresponding microcomputed tomography image for the parameters assessment.

Twenty cortical bone samples were collected from human femur and tibia diaphyses. All samples were microcomputed tomography scanned in air, embedded in polymethylmethacrylate, rescanned by microcomputed tomography, examined by histology and finally compared.

A good correspondence between the microcomputed tomography images and the histological sections was found. Paired comparisons in cortical porosity, Haversian canal diameter and Haversian canal separation between histological sections and microcomputed tomography cross sections, first in air and then embedded in PolyMethylMethAcrylate, were made: no significant differences were found. None of the comparisons showed significant differences for cortical porosity, Haversian canal diameter and Haversian separation over a three-dimensional volume of interest, between microcomputed tomography scans in air and with samples embedded in PolyMethylMethAcrylate.

The very good correlation between bone structural measures obtained from microcomputed tomography datasets and from two-dimensional histological sections confirms that microcomputed tomography may be an efficient tool for the characterization of cortical bone microstructure. Moreover, when the corresponding threshold value for each condition is used, structural parameters determined by microcomputed tomography are not affected by the surrounding medium (PolyMethylMethAcrylate).

Test systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm⁻² and about 300 J cm⁻² for native cells (corresponding to about 250−3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi- or total internal reflection illumination) and decreased to values between 50 J cm⁻² and less than 1 J cm⁻² upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high-resolution wide field or laser scanning microscopy of single cells, typically 10−20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in super-resolution microscopy based on single molecule detection.

We present an algorithm to adjust the contrast of individual dyes from colour (red–green–blue) images of dye mixtures. Our technique is based on first decomposing the colour image into individual dye components, then adjusting each of the dye components and finally mixing the individual dyes to generate colour images. Specifically in this paper, we digitally adjust the staining proportions of hematoxylin and eosin (H&E) chromogenic dyes in tissue images. We formulate the physical dye absorption process as a non-negative mixing equation, and solve the individual components using non-negative matrix factorisation (NMF). Our NMF formulation includes camera dark current in addition to the mixing proportions and the individual H and E components. The novelty of our approach is to adjust the dye proportions while preserving the color of nonlinear dye interactions, such as pigments and red blood cells. In this paper we present results for only H&E images, our technique can easily be extended to other staining techniques.