A new method of imaging particle tracks in solid state nuclear track detectors

Solid state nuclear track detectors are used to determine the concentration of α particles in the environment. The standard method for assessing exposed detectors involves 2D image analysis. However 3D imaging has the potential to provide additional information relating to angle as well as to differentiate clustered hit sequences and possibly energy of α particles but this could be time consuming. Here we describe a new method for rapid high-resolution 3D imaging of solid state nuclear track detectors. A 'LEXT' OLS3100 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) was used in confocal mode to successfully obtain 3D image data on four CR-39 plastic detectors. Three-dimensional visualization and image analysis enabled characterization of track features. This method may provide a means of rapid and detailed 3D analysis of solid state nuclear track detectors.     

Three-dimensional image restoration and super-resolution in fluorescence confocal microscopy

Confocal scanning laser microscopy (CSLM) provides optical sectioning of a fluorescent sample and improved resolution with respect to conventional optical microscopy. As a result, three-dimensional (3-D) imaging of biological objects becomes possible. A difficulty is that the lateral resolution is better than the axial resolution and, thus, the microscope provides orientation-dependent images. However, a theoretical investigation of the process of image formation in CSLM shows that it must be possible to improve the resolution obtained in practice. We present two methods for achieving such a result in the case of 3-D fluorescent objects. The first method applies to conventional CSLM, where the image is detected only on the optical axis for any scanning position. Since the resulting 3-D image is the convolution of the object with the impulse-response function of the instrument, the problem of image restoration is a deconvolution problem and is affected by numerical instability. A short introduction to the linear methods developed for obtaining stable solutions of these problems (the so-called regularization theory of ill-posed problems) is given and an application to a real image is discussed. The second method applies to a new version of CSLM proposed in recent years. In such a case the full image must be measured by a suitable array of detectors. For each scanning position the data are not single numbers but vectors. Then, in order to recover the object, one must solve a Fredholm integral equation of the first kind. A method for the solution of this equation is presented and the possibility of achieving super-resolution is demonstrated. More precisely, we show that it is possible to improve by about a factor of 2 the resolution of conventional CSLM both in the lateral and axial directions.     

Microfractography of granitic rocks under confocal scanning laser microscopy

Scanning laser microscopy, in the confocal mode (CSLM) has been applied to a granitic rock to characterize its fissure space. The technique provides a unique three-dimensional picture of the rock microfractography. CSLM is unique in observing fine details of the fractographic network (connectivity, tortuosity, etc.), its geometry and its relation to other rock-forming components. The fractographic images with standard fluorescence microscopy are compared with those obtained with CSLM. The examples presented emphasize the advantages of CSLM: three-dimensional visualization of the microfractographic network, crack connectivity, automatic evaluation of direction and slope of fissures. These studies are related to the migration of radionuclides in the geosphere. The relations between potentially water-conducting open fissures, and the rock-forming minerals provide a means of modelling the ‘radionuclide retardation mechanism’, a security factor in their definitive storage in rock masses.     

Determination of the water droplet size distribution of fat spreads using confocal scanning laser microscopy

Knowledge of the water droplet size distribution of fat spreads is necessary for the development and production of high quality microbiological safe products. Fat spreads are water‐in‐oil emulsions. The water droplet size distribution can be determined by confocal scanning laser microscopy (CSLM) after staining the fat with Nile Red. The profiles of the non‐fluorescent water droplets in the 2D images are identified and measured using image analysis. The ‘true’ water droplet size distribution is calculated from the distribution of the measured profile diameters using a Wicksell transformation of log‐normal distributions. The influence of the fluorescent staining and CSLM parameters on the information were studied. The CSLM method was tested on fat spreads with a fat content ranging from 40% to 80%. The results were compared with those obtained by nuclear magnetic resonance spectroscopy (NMR). The distribution parameters [volume weighed geometric mean diameter (D?_3,3) and the standard deviation (σ) of the logarithm of the droplet diameter] calculated for 80% fat spreads are in good agreement with those obtained by NMR (within ± 7% relative). Small differences were found for 65% fat spreads and large differences were identified for 40% fat spreads. The precision for the determination of the D?_3,3 value by CSLM is worse than that of NMR, even when three images were used to calculate this parameter [3–11% and 1–6% relative standard deviation (RSD), respectively]. The precision for the determination of exp(σ) by CSLM is comparable or better than that of NMR (1–5% and 3–6% RSD, respectively). CSLM proved to be a reliable method for the determination of the water droplet size distribution of margarines (80% fat). The advantage of CSLM compared to NMR is that visual information is given about the water droplet size distribution in the sample.     

Confocal microscopy in the analysis of the etched nuclear particle tracks in polymers

The possibility of the morphometric analysis of etched tracks, induced by protons and alpha particles in the organic polymer allyl diglycol carbonate (CR-39), using the confocal scanning laser microscope (CSLM), was studied. The detectors were investigated in two groups of irradiation experiments, namely: (a) irradiated with mono-energetic neutrons of energy 1–2 MeV, (b) exposed to the alpha radiation from ²²²Rn and its progeny. Both groups were irradiated at normal incidence. Radiation-induced latent tracks were electrochemically etched, and their morphometric parameters were investigated in the reflection mode by using the 488-nm spectral line of an argon ion laser. A constant number of up to 200 optical sections in Z-scan mode was taken through each selected etched track at vertical spacings of 0·642 μm. Successive reconstructions of Z-sections were used to determine the following parameters: the mean radius of the opening channel, the maximum diameter and the length of the track, and the angle of the track wall to the surface of the sample. The results show that tracks produced by alpha particles differ from those induced by protons. The radius of the opening channel of alpha-particle-induced tracks ranges from 7·9 to 11 μm, whereas for protons the same parameter ranges between 2·0 and 3·8 μm for a specific electrochemical etch procedure.     

Applications of confocal microscopy in industrial solid materials: some examples and a first evaluation

We report here the first results of the application of confocal scanning laser microscopy (CSLM) for the study of the microstructure of solid industrial materials. Glass-fibre-reinforced composites, heterogeneous and conductive polymers, homogeneous as well as heterogeneous catalyst (precursor) specimens and soils were examined. We conclude that both the fluorescence and reflection modes of CSLM can yield valuable information. In particular, the optical sectioning capability of CSLM appears to be of great value as it enables one to access the 3-D organization of the specimen without the need for a difficult and time-consuming specimen preparation procedure. However, local obscuration may be an important factor in confocal image formation, limiting the penetration capabilities of the technique for industrial materials.     

Fluorescence photobleaching recovery in the confocal scanning light microscope

Measurement of mobilities of species in liquid systems is of great importance for understanding a number of dynamic phenomena. A well known method for measuring mobilities driven by diffusion is fluorescence photobleaching recovery (FPR), also known as fluorescence recovery after photobleaching (FRAP). New FPR recovery equations for three-dimensional (3-D) apertured scanning using a Gaussian approximation for the axial beam profile have been successfully developed and found to provide a solid basis for extraction of the lateral diffusion coefficient from confocal scanning light microscopy (CSLM)-FPR experimental data. The 2-D diffusion coefficients of fluorescent species can be successfully measured by FPR in the CSLM, which has the great advantage that bleaching can be targeted at a well-defined volume element in bulk samples. Two-dimensional diffusion coefficients of 45-nm latex spheres, of FITC molecules and of a 2·45-nm protein-FITC complex in water-glycerol mixtures, measured by FPR in the CSLM, are in close agreement with those calculated from the size of the diffusing species and viscosity of the medium. Diffusion coefficients as high as 2 times 10^?6 cm²/s can be measured.     

Alpha particle spectroscopy using FNTD and SIM super‐resolution microscopy

Structured illumination microscopy (SIM) for the imaging of alpha particle tracks in fluorescent nuclear track detectors (FNTD) was evaluated and compared to confocal laser scanning microscopy (CLSM). FNTDs were irradiated with an external alpha source and imaged using both methodologies. SIM imaging resulted in improved resolution, without increase in scan time. Alpha particle energy estimation based on the track length, direction and intensity produced results in good agreement with the expected alpha particle energy distribution. A pronounced difference was seen in the spatial scattering of alpha particles in the detectors, where SIM showed an almost 50% reduction compared to CLSM. The improved resolution of SIM allows for more detailed studies of the tracks induced by ionising particles. The combination of SIM and FNTDs for alpha radiation paves the way for affordable and fast alpha spectroscopy and dosimetry.     

Dynamic confocal scanning laser microscopy methods for studying milk protein gelation and cheese melting

Dynamic confocal scanning laser microscopy (CSLM) methods were developed to enable observation of milk protein gelation and cheese melting. Protein aggregation and the formation of gel networks in renneted full-fat and low-fat milks and glucono-δ-lactone (GDL)-acidified skim milks were observed by CSLM and observations correlated with increases in shear modulus (G′) and dynamic viscosity (η*) as determined by dynamic amplitude oscillatory rheology. Confocal scanning laser microscopy observation of low-fat and full-fat cheeses showed changes in fat distribution and an increase in staining intensity during cheese melting.     

Ultrastructural investigation of neurons identified and localized using the confocal scanning laser microscope

Studies of labeled neurons at the light-microscopic level often pinpoint a substructure of particular interest, i.e., a synapse or a spine. An ultrastructural investigation would explain a lot about how these structures arose, how they function, and how they are regulated. Finding a small region in a large block can require constant checking during sectioning, until past the structure. In our pursuit of the synaptic structure of varicosities on the axons of neurons identified physiologically and morphologically at the light level, we have combined confocal scanning laser microscopy (CSLM) with conventional and high-voltage electron microscopy (EM). CSLM images were collected in the reflection mode to view neurons filled with horseradish peroxidase and stained with nickel-intensified diaminobenzidine, which is compatible with EM. The CSLM optical sections provided a record of what one should expect to see at regular intervals throughout the depth of the tissue block. We have shown that the CSLM greatly simplified the task of localizing small structures in brain tissue prepared for EM.     

Confocal scanning light microscopy with high aperture immersion lenses

The imaging characteristics of a confocal scanning light microscope (CSLM) with high aperture, immersion type, lenses (N.A. = 1·3) are investigated. In the confocal arrangement the images of the illumination and detector pinholes are made to coincide in a common point, through which the object is scanned mechanically. Results show that for point objects the theoretically expected improved response by a factor of 1·4 in comparison with standard microscopy can indeed be realized. Low side lobe intensity and absence of glare permits the imaging at high resolution of weak details close to strong features. A further improvement by a factor of 1·25 in point resolution in CSLM is found after apodization with an annular aperture. Due to the scanning approach all possibilities of electronic image processing become available in light microscopy.     

Measurements of fibre direction in reinforced polymer composites

High-quality data on the three-dimensional (3-D) spatial distributions of glass and carbon fibres in fibre-reinforced polymer composites are important for both process control and the modelling of the mechanical and thermal properties of these composites. The advent of economical, high-speed, image analyser systems has enabled numerous research groups to measure directional distributions of fibre samples. Specimens are microtomed and polished and, using optical reflection microscopy, thousands of elliptical fibre images may be analysed within a short period of time. From the eccentricity of the fibre images, estimation of the angles (θ, φ) of each fibre relative to the vertical axis and within the measurement plane is deduced. However, this measurement is subject to considerable error. The confocal scanning laser microscope (CSLM), operating in fluorescence mode or reflection mode, is capable of improving the angular resolution (δθ, δφ) for all fibre directions. The ability of the CSLM to optically section glass and carbon fibre-reinforced polymer composites down to depths of 20 or 30 μm allows the user to determine accurate fibre directions from the apparent movement of fibre profiles. The CSLM has the potential for standardizing measurements of 3-D fibre directions in polymer composites and providing the quality directional data which are required for the theoretical modelling of composite processing and composite strength.     

In situ analysis of microbial consortia in activated sludge using fluorescently labelled,rRNA-targeted oligonucleotide probes and confocal scanning laser microscopy

Activated sludge flocs are complex consortia of various micro-organisms. The community structures of samples taken from municipal sewage treatment plants were characterized using fluorescently labelled, 16S and 23S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM). In comparison with conventional epifluorescence microscopy, CSLM considerably improved the capability to visualize directly the spatial distribution of defined bacterial populations inside the sludge flocs. Analyses could be performed at high resolution undisturbed by problems such as autofluorescence or limited spatial resolution in thick samples. In addition, CSLM was used to analyse some structural properties of paraformaldehyde-fixed activated sludge flocs, such as floc size and homogeneity. Typical floc sizes were found to be in the range between 5 and 50 μm. Whereas most of the flocs were completely colonized by bacteria, there were also examples of flocs containing gas bubbles or particles in the interior.     

Application of confocal scanning laser microscopy in experimental pathology

Confocal scanning laser microscopy (CSLM) represents an exciting new tool for scientific disciplines which focus on mechanistic studies such as experimental pathology. Enhanced resolution in the specimen plane and rejection of out-of-focus fluorescence flare allow analysis of specific nucleic acid sequences, enzymes, structural macromolecules, and cellular homeostasis utilizing fluorescent probes. Four different experimental applications are discussed which utilize CSLM to evaluate pathological processes at the subcellular, cellular, and tissue levels. Programmed cell death, or apoptosis, is a natural process of significance both during development and as a response to toxic stimuli. CSLM-imaging of nuclei of human B lymphoblastoid cells following exposure to a monofunctional alkylating agent suggests that the degradation of chromatin characteristic of apoptosis may occur in asymmetric patterns. Surfactant apoprotein-A is the major non-serum protein component of pulmonary surfactant and is essential for the extracellular function of surfactant. CSLM of alveolar type II cells suggests that apoprotein-A is present in both the cytoplasm, predominantly in lamellar bodies, and in the nucleus. The tumor promoter, phorbol myristate acetate, rapidly stimulated the formation of vacuoles in human neutrophils. CSLM using Lucifer Yellow as a probe suggests that cylindrical vacuoles are formed by fluid-phase pinocytosis. The blood-nerve barrier (BNB) in peripheral nerves may be an important target during toxin-induced neuropathies. Ricin-induced permeability of the BNB in the rat was rapidly visualized by CSLM as leakage of fluorescein isothiocynate (FITC)-dextran into the endoneurial compartment.     

Registration of confocal scanning laser microscopy and quantitative backscattered electron images for the temporospatial quantification of mineralization density in 18-month old thoroughbred racehorse articular calcified cartilage

Combined backscattered electron scanning electron microscopy (BSE SEM) and confocal scanning laser microscopy (CSLM) have been used to put tissue mineralization data into the context of soft tissue histology and fluorescent label information. Mineralization density (Dm) and linear accretion rate (LAR) are quantifiable parameters associated with mineralizing fronts within calcified tissues. Quantitative BSE (qBSE) may be used to determine Dm, while CSLM may be used to detect label fluorescence from which LAR is calculated. Eighteen-month old Thoroughbred horses received single calcein injections 19 and 8 days prior to euthanasia, labeling sites of active mineralization with fluorescent bands. Confocal scanning laser microscopy images of articular calcified cartilage (ACC) from distal third metacarpal condyles were registered to qBSE images of the same sites using an in-house program. ImageJ and Sync Windows enabled the simultaneous collection of LAR and Dm data. The repeatability of the registration and measurement protocols was determined. Dm profiles between calcein labels were explored for an association with time. Dm was 119.7 +/- 24.5 (mean +/- standard deviation) gray levels (where 0 = backscattering from monobrominated and 255 from monoiodinated dimethacrylate standards, respectively), while modal and maximum LAR were 0.45 and 3.45 microm/day, respectively. Coefficients of variation (CV) for Dm were 0.70 and 0.77% with and without repeat registration, respectively; CVs for LAR were 1.90 and 2.26% with and without repeat registration, respectively. No relationship was identified between Dm and time in the 11-day interlabel interval. Registration of CSLM to qBSE images is sufficiently repeatable for quantitative studies of equine ACC.     

Preliminary confocal scanning laser microscopy study of fluid inclusions in quartz

The initial results of the first dedicated confocal scanning laser microscopy (CSLM) study of fluid inclusions in quartz are presented. CSLM imaging of a large inclusion shows the quartz crystal to contain numerous small (< 1 μm), highly reflective inclusions arranged along planes in at least two directions that are not readily visible in transmitted light. The technique allows measurements to be made of the angular intersection and orientation of the planes in both two and three dimensions. Results suggest that larger inclusions (> 10 μm) occur where two planes of small inclusions intersect, and that the shape of the large inclusions is controlled by the angular relationship between intersecting planes.     

Increasing the energy dynamic range of solid-state nuclear track detectors using multiple surfaces

Solid-state nuclear track detectors, such as CR-39, are widely used in physics and in many inertial confinement fusion (ICF) experiments. In the ICF experiments, the particles of interest, such as D(3)He-protons, have ranges of order of the detector thickness. In this case, the dynamic range of the detector can be extended by recording data on both the front and back sides of the detector. Higher energy particles which are undetectable on the front surface can then be measured on the back of the detector. Studies of track formation under the conditions on the front and back of the detector reveal significant differences. Distinct front and back energy calibrations of CR-39 are therefore necessary and are presented for protons. Utilizing multiple surfaces with additional calibrations can extend the range of detectable energies on a single piece of CR-39 by up to 7-8 MeV. The track formation process is explored with a Monte Carlo code, which shows that the track formation difference between front and back is due to the non-uniform ion energy deposition in matter.     

Imaging modes in confocal scanning light microscopy (CSLM)

The optical arrangement for confocal scanning light microscopy can be incorporated in various imaging modes. Light microscopical specimens can be imaged with contrast enhanced, under γ-control, inverted, etc. In interference, conditions can be set such that either pure phase or pure amplitude images result. Stereoscopic images at arbitrary aspect ratios can be realized in CSLM by electronic processing of the data obtained when the specimen is sampled with more than one confocal point concurrently. Also forms of differential imaging either amplitude or phase are possible. The coupling of these imaging modes with the improved resolving powers of CSLM results in some unique imaging opportunities, especially of value for high resolution light microscopy of living specimens.     

Three-dimensional visualization methods for confocal microscopy

Three-dimensional images of microscopic objects can be obtained by confocal scanning laser microscopy (CSLM). The imaging process in a CSLM consists of sampling a specific volume in the object and storing the result in a three-dimensional memory array of a digital computer. Methods are needed to visualize these images. In this paper three methods are discussed, each suitable in a specific area of application. For purposes where realistic rendering of solid or semi-transparent objects is required, an algorithm based on simulation of a fluorescence process is most suitable. When speed is essential, as for interactive purposes, a simple procedure to generate anaglyphs can be used. Both methods have in common that they require no previous interpretation or analysis of the image. When the study of an object imaged by CSLM involves analysis in terms of a geometrical model, sophisticated graphics techniques can be used to display the results of the analysis.