Ultrastructural histopathology of human olfactory dysfunction.

This paper presents electron-microscopic observations on biopsies of the olfactory mucosae of several classes of patients with smell disorders: 1) patients with loss of smell function following head injury (post-traumatic anosmics or hyposmics); 2) patients with loss of smell function following severe head colds and/or sinus infections (post-viral olfactory dysfunction, or PVOD); and 3) patients that have lacked smell function since birth (congenital anosmics). Of these, the traumatic anosmics' olfactory epithelia were quite disorganized; the orderly arrangement of supporting cells, ciliated olfactory receptor neurons, microvillar cells, and basal cells was disrupted. Although many somata of ciliated olfactory receptors were present, few of their dendrites reached the epithelial surface. The few olfactory vesicles present usually lacked olfactory cilia. The post-viral anosmics, too, had a greatly reduced number of intact ciliated olfactory receptor neurons, and most of those present were aciliate. The post-viral hyposmics had a larger population of intact, ciliated olfactory receptor cells. In the seven cases of congenital anosmia studied, no biopsies of olfactory epithelium were obtained, indicating the olfactory epithelium is either absent--or greatly reduced in area--in these individuals.     

Use of an automated image analyser to quantitate cellular hyperplasia in urinary bladder epithelium.

The feasibility of using an automated image analyser to evaluate quantitatively hyperplasia caused by N-butyl-N-(4-hydroxybutyl) nitrosamine in bladder epithelium was studied. The number of cells per unit length of epithelium was counted manually, and compared with automated measurements of: (1) the number of nuclei, (2) the number of positive tangents to the lower edge of nuclei, (3) the nuclear cross-sectional area, and (4) the epithelial cross-sectional area per unit length respectively. Regression of each of the automated measurements on the manual counts all revealed close linear relationships with correlation coefficients in excess of 0-9. Coefficients of variation for repetitive automated measurements were less than or equal to 0-06 in each of the four modes. The automated system resulted in a great saving in time over manual counting. It is concluded that the automated image analyser provides an accurate, precise, and efficient tool for estimating epithelial cell numbers in normal and hyperplastic bladder epithelia.     

Application of stereologic methods to stratified gingival epithelia

Stereologic point-counting procedures were applied to estimate quantitative tissue and cytoplasm parameters of the oral and the junctional epithelium of the human gingiva. Three gingival biopsies of female children, obtained and processed under standardized conditions, served (1) to determine the minimal sample size of cytoplasmic area required for estimating representative volumetric parameters in different strata of both epithelia, and (2) to compare average volume and surface density data derived from standardized samples in epithelial cross sections cut at two planes orientated perpendicular to each other. Sampling of electron micrographs, performed at two levels of magnifications according to a systematic stratified random sampling procedure consisted of recording field strips parallel to the epithelial or tooth surface within each of the various strata. The optimal sample size required varied for different organelles and epithelial strata. Minimal sample size of cytoplasmic area per biopsy were 300–440 μm² for basal, and 450 μm² for stratum spinosum cells of oral epithelium, and 130–185 μm² for basal cells of the junctional epithelium. Average morphometric parameters for gross tissue components (cells, nuclei, cytoplasm, intercellular space) which resulted from analysing 4900 μm² tissue area in each of the epithelia in each biopsy, were almost identical when determined in two different section planes. Volume and surface density data of cytoplasmic organelles (mitochondria, rough and smooth endoplasmic reticulum, cytoplasmic filaments) resulting from a sample of 280 μm² cytoplasmic area per stratum and biopsy, revealed differences of varying magnitude, when determined in the two section planes. These differences were markedly smaller than those between comparable strata of both epithelia. Discussion of sampling procedures concluded that the type of sampling used for the present study is suitable for comparative morphometric studies.     

Histological changes in rat nasal epithelia after unilateral neonatal naris occlusion

The authors studied the extent of the different epithelia lining the nasal fossae of the albino rat after neonatal closure of one naris. Newborn pups were anesthetized by hypothermia and the external opening of their right naris cauterized, while littermates served as controls. Animals were sacrificed at 3 months, and the occluded (OCF) and nonoccluded (NOF) fossae of experimental animals as well as both fossae of control animals (CTF) were histologically studied. In both control and experimental animals, nasal fossae were lined by five different types of epithelia: squamous stratified, transitional, metaplastic, respiratory, and olfactory epithelia. It was found that closure of one naris provokes reorganization of the epithelial lining in both the occluded and nonoccluded side. In CTF airflow, physical conditions as well as pollutants and biological agents irritate the epithelial lining, causing squamous metaplasia as well as metaplastic epithelium showing inflammation in rostral levels. In CTF caudal levels, the metaplastic epithelium appears to a lesser degree and the respiratory epithelium prevails, except for the most caudal level where the olfactory epithelium is prevalent. In OCF, the protected environment created prevents the occurrence of metaplastic epithelium, the transitional, respiratory, and olfactory epithelia developing in the corresponding area instead. In NOF, where the airflow is double, the same pattern occurs as in CTF, although metaplastic epithelium values are approximately double, suggesting a clear linear effect. An outstanding feature observed was the increased extent of the olfactory epithelium in OCF regarding NOF, although changes in its morphological structure were not found. Airflow properties, including pressure, coldness, velocity, and turbulence, as well as biological and chemical hazards present in inflow, cause histological reorganization of the nasal epithelium lining during postnatal development. Results prove the need to consider airflow changes in nasal fossae surgery and point to the protective value of naris closure in ENT clinics, supporting it as a treatment of atrophic rhinitis.     

A simple and efficient method for objective discrimination between pseudostratified,metaplastic and dysplastic nasal epithelium

Morphologic features may sometimes be interpreted differently by different pathologists. By introducing objective methods in the histological evaluation the subjectivity of the judgement is reduced and a higher degree of consistency achieved. In the present study three linear parameters of nasal epithelial basal cells: the largest transversal nuclear diameter, the sum of the longitudinal and transversal nucleolar axes and the basal cell width, i.e. the width of the attachment face to the basement membrane, were classified by means of three-class rulers. From this classification weighted indices were made which were then given equal weight and transformed, so that when added up their sum, the joint index (Q) to the largest possible extent was consistent with pseudostratified/stratified cuboidal, stratified squamous and dysplastic epithelium when Q ≤ 1, 1 < Q ≤ 2 and Q > 2 respectively. Tested prospectively we found 91% agreement between the morphometric and histological classification. The method presented is extremely simple and rapid to perform, and appears to have diagnostic potential in distinguishing various types of epithelial alterations of the nasal mucosa. The method may also be useful in approaching similar problems in other epithelia.     

Association of limited joint mobility and increased plantar hardness in diabetic foot ulceration in north Asian Indian: a preliminary study

The aim of this article is to investigate the association of limited joint mobility and foot sole hardness in north Asian Indian type 2 diabetic patients. Limited joint mobility and hardness of the foot sole were measured for 39 subjects attending the AIIMS Endocrinology & Metabolism Clinic. The total subject divided into three groups: 13 control subjects (nondiabetic), 13 diabetic patients without neuropathy and 13 diabetic neuropathy patients. Neuropathy status was assessed using 10 gm Semen's Weinstein monofilament. Joint mobility parameters, such as ankle dorsiflexion/plantar flexion and metatarsophalangeal-1 dorsiflexion/plantar flexion, are measured using a goniometer. Foot sole hardness was measured using a durometer or shore meter. We found that diabetic patients with a neuropathic foot had significantly reduced joint mobility and increased foot sole hardness, placing them at risk for subsequent ulceration. Metatarsophalangeal-1 dorsiflexion/plantar flexion of both feet of diabetic patients had significant correlation (at p < 0.05, p < 0.001, p < 0.001 level) over age and body mass index. Also ankle plantar flexion/dorsiflexion and metatarsophalangeal-1 dorsiflexion/plantar flexion has a significant correlations (at p < 0.01, p < 0.05, p < 0.001, p < 0.001 level) with foot sole hardness in both feet of diabetic neuropathy subjects. Also linear regression analysis showed that duration of diabetes was significantly associated with the joint mobility parameters. In this study we conclude that joint mobility had reduced further if neuropathy and increased foot sole hardness coexisted owing to high plantar pressures. Hence, both limited joint mobility and increased foot sole hardness appears to be important determinants of foot sole ulceration in diabetic neuropathic subject.     

Trunk and lower extremity muscle activity in seated weight-moving tasks: Three-dimensional analyses of intersubject and intertask differences

A three-dimensional musculoskeletal model was used to predict the trunk and lower extremity muscle activity required to stabilize the body while performing dynamic tasks in the seated posture. We studied seven subjects performing four tasks consisting of cyclic or single-directional movements of a hand-held weight in the sagittal plane. Five different optimization schemes involving the minimization of muscle forces, muscle stresses, or joint force components were used to predict the 64 muscle force-time histories. A quantitative method was used to compare prediction schemes by correlating predicted muscle forces with measured myoelectric data and ranking the efficacy of each scheme for different tasks and subjects. The results showed that (1) the trunk and lower extremity muscles play an important role in stabilizing the seated posture in these tasks. (2) the most successful muscle force prediction scheme was not the same for all seven subjects performing a given task, or for a given subject performing all four tasks, and (3) these linear optimization techniques successfully predicted activity in up to 10 of the 15 muscles whose myoelectric signals were actually monitored.     

Trabecular bone microarchitecture is related to the number of risk factors and etiology in osteoporotic men

Microarchitecture of trabecular bone is a very important component of bone quality in osteoporosis and a determinant of vertebral fracture in men with low bone mineral density (BMD). In contrast to women, male osteoporosis is, in most cases, secondary. The relationships between microarchitecture and different risk factors have never been evaluated in men. About 152 men with low BMD at the lumbar spine or hip (BMD, T-score < -2.5) were included in this study. Risk factors were: age, BMI, alcohol intake, corticosteroid therapy, hypogonadism, and chronic diseases. Transiliac bone biopsies were obtained and histomorphometry was done on an image analyzer; the following parameters were measured: cortical thickness (Ct.Th), trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), separation (Tb.Sp) and number (Tb.N), interconnectivity Index (ICI), star volume of the bone marrow, and strut analysis with node and free-end count. The 50 men with two risk factors had a lower BMD, lower Ct.Th and a significant higher star volume than those with one factor or idiopathic osteoporosis. The 26 men with at least three risk factors, had a lower BMD, a reduction of BV/TV and Ct.Th and a marked disorganization of the trabecular network (increased Tb.Sp, ICI, star volume, and free-end to free-end struts). The prevalence of vertebral fractures was higher in these patients. When the main risk factor was considered, a marked decrease in trabecular bone connectivity was observed in hypogonadic men. In osteoporotic men, higher the number of risk factors, lower the connectivity of trabecular network and higher the vertebral fracture risk.     

Measurement by Quantimet 720 of the proportion of actively growing cells in tissue cultures of human lymphocytes.

The size-distribution of normal human lymphocytes growing in tissue culture was measured with a Quantimet 720 image-analysing computer. The proportion of cells undergoing volumetric growth after phytohaemagglutinin (PHA) stimulation was dervived from a simple mathematical model describing the growth patterns of the cells up to the first mitotic division. The approach may have general application in biology as a method for measuring the proportion of growing cells by microscopy.     

Morphological polarity in oral epithelial cells revealed by boric acid partial dissociation

Layers of epithelium were stripped with adhesive tape from the surface of rat palatal mucosa after that tissue had been fixed in OsO₄, treated in 0.2 M boric acid and critical point or freeze dried. Cell layers stripped off evenly with very few intracellular fractures. The oral surface of cells in the stratum corneum was characterized by small pits and raised cell boundaries. By contrast the deep aspect of these cells displayed microvilli and linear grooves which exactly matched the pits and raised boundaries on the opposing cell beneath. Partial dissociation with boric acid after OsO₄ fixation greatly assists the demonstration of complementary cell surfaces deep in the epithelium.     

The application of an image analyzing computer (Quantimet 720) for quantitation of biological structures--the automatic counting of mast cells.

A Quantimet 720 image analyzer was utilized in order to 1) quantify mast cells automatically in gingiva and in doing so to 2) investigate the applicability and accuracy of the machine for quantitative analysis of biological structures. The mast cells of two hundred gingival sections were counted first by means of the Quantimet and then by manual count. Results indicated that a) mean counts of mast cells obtained by means of the Quantimet were comparable to that of the manual counts, b) although the spread was larger than by manual methods, the Quantimet automatic image analyzer performed quantitation more rapidly.     

The microspectrometrical determination of the nucleus-total-cell-protein-ratio: a possibility of objective cell type analysis

Cells from smears of the normal human squamous epithelium of the gingiva, fixed and stained for protein using the tetrazonium method optimized by N?hammer and calibrated by N?hammer et al., were investigated. The extinctions of both the total cells and of the rectangular areas circumscribing the nuclei, were measured microspectrometrically. Altogether 417 cells from 6 healthy persons of both sexes were investigated. 9 distinct subgroups of cells were found showing an exact linear correlation between nuclear and total cell extinctions. In the graph of both the nuclear and the total cell extinctions the 9 subgroups can be seen as 9 distinct linear groups of points, defined exactly by their regression lines. Thus, every squamous epithelial cell within the smear can be typed definitely and objectively in respect to its membership of one of these 9 linear groups of points. The obviously definite, legitimate connection between the extinctions of the total cells and of their nuclei affords a glimpse into the processes of cellular differentiation and allows the definition of the so-called stem cell in terms of protein content of the total cell and of the nucleus.     

Quantitative analysis of intramembranous particles in rapidly frozen 10T1/2 cell monolayers

A simple method for ultrarapid freezing of cell cultures in monolayers was developed. Unfixed and unglycerinated cells were grown on glass substrates. No special treatments of the glass or cells were necessary to facilitate freeze-fracture along the upper plasma membranes. A reliable nonbiased method was developed to detect intramembranous particles (IMP) from the background by totally automatic means using the Cambridge Instruments Quantimet 920 Image Analysis system. Size and density data of IMP from a large number of electron micrographs can be rapidly and objectively quantitated. The automatic determination of locational coordinates for each IMP enables subtle determination of spatial distributional differences by the nearest neighbour function and the differential density distribution function, which are measurements of randomness. Quantitative analysis of the IMP distribution on the fracture face of C3H/10T1/2 mouse embryo fibroblasts upon various drug treatments was demonstrated.     

An assessment of quantimet as an aid in the analysis of wear debris in ferrography

Wear debris extracted from oil samples taken from a twin-disc machine has been analysed using Quantimet to obtain data in terms of the number, size and distribution of wear particles in ferrograms. A correlation between wear and performance in the disc machine is achieved. From optical and stereoscan examination of the wear particles, the nature of the initial running-in wear behaviour of the discs is established.     

Experiments on the characterization of metamorphic textures from a micrograph of an amphibolite

Picture processing using generalized instrumentation with specially developed software, and image analysis by means of a Quantimet 720 image analyser with a linear correlator, were employed in the study of rock textures. The boundaries of all individual grains from a portion of a thin section of a rock were distinguished and hand-drafted for digitization and analysis. From the grey level image of boundaries of the profiles of the grains, obtained by hexagonal scanning of line drawings, binary images were produced for each phase and used for textural measurements. Ink drawings extracted from the boundary pattern were also used for the image analyser experiments. In picture processing experiments, programmed structuring elements of different shapes and two-dimensional autocorrelation functions were used to characterize morphology and spatial distribution of the different phases. Transformations of binary images were programmed at the bit level to reduce storage requirements and computing time. In Quantimet 720 experiments, linear erosion and histogram of sizes were used for computing the mean dimensions of the grains. Periodical arrangements were studied by means of the covariance function. This contribution uses simple texture measures derived from mathematical morphology and picture processing methods, in order to assess the feasibility of two different technical approaches in the analysis of the same material. The extraction of image information from the material analysed, which was not attempted before, is cumbersome and technically complex. This difficulty is partly responsible of why little is known on the quantitative characterization of metamorphic textures from thin sections.     

Fluorescent human lung macrophages analyzed by spectral confocal laser scanning microscopy and multispectral cytometry

Numerous highly fluorescent macrophages (MPhi), designated "smoker cells," exist in the lungs of smokers and subjects who have quit smoking within 5 years. The brightly fluorescent MPhi, however, are not present in the lungs of never smokers. Some investigators have speculated that the intense fluorescence of the MPhi is due to smoke-induced changes in the autofluorescence of naturally occurring (i.e., endogenous) compounds (e.g., NADP). In contrast, other researchers have theorized that the fluorescence is due to the uptake of tobacco smoke particulates (i.e., "tar"). Studies reported herein were undertaken to test the hypothesis that the origin of the MPhi fluorescence could be profiled with the novel technologies afforded by spectral confocal laser scanning microscopy (sCLSM) and multispectral cytometry (MSC). To this end, spectral emissions were obtained by sCLSM of optical sections of live MPhi isolated from fresh surgically excised human lung tissue and in air-dried lung tissue imprints. Confirmation of spectral profiles of these single cell observations was obtained in population studies with the use of high-throughput MSC in which multispectral analyses were performed with three different lasers. Proof of concept experiments demonstrated that relatively nonfluorescent MPhi from the lungs of nonsmokers became fluorescent upon short-term ex vivo exposure to tobacco smoke tar. Summarily, the studies reported herein document that the fluorescence of human lung MPhi is due to tobacco tar.     

Model order determination and noise removal for modal parameter estimation

Given a noisy impulsive response function (IRF) that has been contributed by an unknown number of modes, this article proposes a different approach from the traditional methods for estimating modal parameters from this noisy IRF. The major difference lies in the way of handling noise and choosing the computational model order. Whereas the traditional approach accommodates noise by purposely increasing the computational model order, the proposed approach uses the actual system order as the computational model order and rejects noise prior to performing the modal parameter estimation. The proposed approach includes three steps: (1) model order (or number of modes) determination from the measured IRF—by finding the rank of a Hankel matrix constructed from the measured IRF, (2) noise removal from the measured IRF to obtain a filtered IRF—by implementing Cadzow's algorithm for the structured low rank approximation (SLRA) on the Hankel matrix, and (3) modal parameters estimation from the filtered IRF—by using the complex exponential method (Prony's method). Numerical studies include both synthesized and experimental data. While measured IRFs with mild and strong noise levels are simulated for a 5 degree-of-freedom mass-spring-dashpot system, the modal parameter estimations based on the filtered IRFs are very good for both noise levels. While experimental data are measured from two accelerometers mounted at a cantilever beam, the modal parameters estimated from the filtered IRFs of the two accelerometers are in excellent agreement.     

Intercomparison of automatic fission track counting systems

The methods of counting fission tracks by two image analyzing computers and a spark counting system are compared with a standard microscopy technique. In this comparison, for automatic fission track counting, the two commercially available image analyzer instruments, the Quantimet 720 and the Leitz-Texture Analyzing System (TAS) and a specially designed spark counter, the UTASC system, have been used. The results showed that the relative counting efficiencies of the Quantimet 720, the Leitz-TAS and the UTASC system are 0.974±0.010, 0.988±0.013 and 0.955±0.010, respectively     

Measurements of critical point shrinkage of glutaraldehyde fixed mouse liver

The dimensional changes of small cubes of glutaraldehyde fixed mouse liver tissue were measured using a light microscope image projected into the Quantimet 720 Image Analysing computer system. The dimensional changes occurring in the critical drying bomb could be followed at all stages when violent turbulence was not occurring. The results show that liver tissue blocks shrink in four stages whilst in the critical point drying bomb: (1) during substitution of the intermediate solvent with the transitional fluid; (2) when the transitional fluid is warmed above the critical temperature; (3) when the transitional fluid, now a gas, is allowed to escape from the CPD bomb – the rate of shrinkage increasing as atmospheric pressure is approached; (4) at atmospheric pressure when all the gas has been allowed to escape from the bomb. Taken together with the authors' previous findings, it would seem that substantial shrinkage of animal soft tissue specimens must occur whilst they are undergoing “critical point drying”. This fact should be taken into account when interpreting SEM images of CPD tissues.