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Biomedical subjects

A Boyde

Publications and source records attributed to A Boyde.

285 records · Page 16Linked to original sources
PubMedDate not supplied

Vital confocal microscopy in bone.

We wished to exploit confocal microscopy for high spatial and temporal resolution vital microscopy in bone. To this end, we evolved implants with glass windows supported in titanium, which were placed in the medial proximal tibial plateau of the rabbit, and special small, self-focussing objectives (dry 10/0.25, water immersion 20/0.45, and oil immersion 45/0.65 and 120/1.0) which mated and matched to the conical window entrance section of the metal components. At intervals of up to 21 months after implant healing, these lenses were used to study live tissue using two genera of confocal microscope: multiple aperture disc, tandem scanning, microscopes for observation in reflection, and video rate confocal laser scanning microscopes for recording, mainly in the fluorescence mode. The latter allowed the study of a variety of intravenously administered substances, including fluorescein, fluorescein-dextrans, fluorescent microspheres, acridine orange, DASPMI, calcein, and tetracycline. We were able to remove blood, stain cells with fluorescent markers, and replace them into the circulation. Calcein and tetracycline bind to the mineral front in bone: this labelling was studied in progress. We observed that both substances partition and remain for long periods (at least days) in adipocytes. Further characterisation of the system used both confocal fluorescence and scanning electron microscopy methods in the study of retrieved implants. These studies showed that the subimplant cortical bone remodelled to a less compact structure with a rich microvasculature extremely close to bone. The points of attachment of bone to glass were found to involve coarse fibres, with the matrix containing large numbers of large cells: some of this tissue was cartilage and some immature bone. An amorphous, mineralised matrix was in immediate contact with glass. The results provide further confirmation of the general utility of high-scan speed confocal methodology in physiology.

Adipocytes↗
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Surface roughness of preparations for backscattered electron-scanning electron microscopy: the image differences and their Monte Carlo simulation.

Patterns and levels of mineralisation in the biological hard tissues have been studied using the backscattered electron (BSE) mode in the scanning electron microscope (SEM). To prevent gross topographic detail overwhelming changes in signal from composition, samples are embedded in polymethylmethacrylate (PMMA) and a flat block surface produced by polishing or micromilling. This study was undertaken to establish the degree of residual topography achieved in these finishing processes. A sample of human rib was embedded in PMMA and prepared, as for examination in the SEM, by polishing on graded abrasives and pre- and, finally, ultramilling. After each preparation step, the block face was imaged using a confocal reflection microscope surface mapping facility. The recorded topographies were used in a Monte Carlo simulation to model the surface interface and thus, for each of the sample preparation techniques, to calculate predicted variations in BSE signal. The latter were compared with experimental data derived under standard operating conditions in the SEM. Micromilling produced block faces with typical peak-trough relief of 80 nm, while hand polishing left occasional scratches 1.5 microns deep with a general undulation of 150-250 nm. Monte Carlo simulations of a rough surface of bone using these data predicted that additional contrast levels of 5% could be expected from micromilled surfaces and > 10% for hand polished samples of bone. Thus, micromilling is the best preparation method for bone, since this tissue develops a collagen orientation-related relief on polishing, which may be largely responsible for the (incorrect) supposition that lamellation in bone is related to changes in net degree of mineralisation.

Humans↗
PubMedDate not supplied

Correlative light and backscattered electron microscopy of bone--part II: automated image analysis.

Detailed studies of biological phenomena often involve multiple microscopy and imaging modes and media. For bone biology, various forms of light and electron microscopy are used to study the microscopic structure of bone. Integrating information from the different sources is necessary to understand how different aspects of the bone structure interact. To accomplish this, methods were developed to prepare and image thin sections for correlative light microscopy (LM) and backscattered electron imaging in the scanning electron microscope (BSE-SEM). Images of the same fields of view may then be analyzed for degrees of relationships between specimen features not observed by LM or SEM alone. These methods are applied here to study possible associations between the degree of bone mineralization and pattern of collagen fiber orientation in the mid-shaft of the human femur. The "relational images" obtained allow us to examine the relationship between these two variables, both objectively and quantitatively.

Femur↗
PubMedDate not supplied

Light collection efficiency and light transport in backscattered electron scintillator detectors in scanning electron microscopy.

Experimentally, scintillator detectors used in scanning electron microscopy (SEM) to record backscattered electrons (BSE) show a noticeable difference in detection efficiency in different parts of their active zones due to light losses transport in the optical part of the detector. A model is proposed that calculates the local efficiency of the active parts of scintillator detectors of arbitrary shapes. The results of these calculations for various designs are presented.

Journal Article↗
PubMedDate not supplied

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.

Animals↗
PubMedDate not supplied

Back-scattered electron imaging of skeletal tissues.

The use of solid-state back-scattered electron (BSE) detectors in the scanning electron microscopic study of skeletal tissues has been investigated. To minimize the topographic element in the image, flat samples and a ring detector configuration with the sample at normal incidence to the beam and the detector are used. Very flat samples are prepared by diamond micromilling or diamond polishing plastic-embedded tissue. Density discrimination in the image is so good that different density phases within mineralized bone can be imaged. For unembedded spongy bone, cut surfaces can be discriminated from natural surfaces by a topographic contrast mechanism. BSE imaging also presents advantages for unembedded samples with rough topography, such as anorganic preparations of the mineralization zone in cartilage, which give rise to severe charging problems with conventional secondary electron imaging.

Animals↗
PubMedDate not supplied

Age- and sex-dependent cancellous bone changes in a 4000y BP population.

We studied cancellous bone loss in a 4000y BP population, using several methods designed to detect age-related changes, in order to investigate the pattern of cancellous bone loss in this ancient population and to compare the results deriving from different methods used on identical specimens. We used 10-mm sections of fourth lumbar vertebral bodies and left femoral necks of 18 individuals of both sexes with estimated ages from 20 to 60 years of a 4000y BP bronze-age population. Stereoscopic photographs were used for three-dimensional analysis and trabecular number (TN) counting. After embedding, the following parameters were measured in different image analysis systems using plane parallel block samples: bone mineral density (BMD) in water by DEXA, and by evaluation of standardized radiographic images; fractional bone volume (BV/TV) in backscattered electron images of the trabecular surface layer and in optical images of trabeculae in a surface-stained layer; and trabecular bone pattern factor (TBPf) in the latter images. There was a high correlation between the results of morphological methods for measuring fractional bone volume. Reasonable correlations were found between the x-ray photon methods and poor correlations between these and the morphological methods. These poor correlations may be due to the diagenetic substitution occurring in archaeological skeletons, which would strongly influence x-ray-based density measurements. However, all the methods demonstrated that the most dramatic loss of quantity and quality in cancellous bone occurred in females between 40 and 60 years.(ABSTRACT TRUNCATED AT 250 WORDS)

Austria↗
PubMedDate not supplied

Monte Carlo simulations of electron scattering in bone.

Relative changes in the mineralization level within bone can be studied using backscattered electron (BSE) imaging in a scanning electron microscope (SEM). We calculated the size and shape of the volume element studied, choosing conditions which are typical for practical experimental work with polymethylmethacrylate (PMMA)-embedded bone. Absolutely flat surfaces of embedded bone blocks cannot be generated, and a further aim was to examine the effect of the surface topography on the detected BSE signal level. For normal beam incidence, 20 kV, and modeling an annular detector by collecting BSE with take-off angles of between 45 degrees and 75 degrees to the flat sample surface, it was found that the collectable BSE signal intensity peaks for electrons which leave the specimen surface at a radial distance of approximately 1 micron from the beam impact point. The layered structure of the bone generates topographic relief on polishing. Modeling this by a sinusoidal profile of wavelength 5.0 and amplitude 0.5 micron and again for 20 kV, it was found that the signal derived from the troughs is reduced by 14.4% and that from the crests is increased by 17.2%. The two effects may add constructively to generate the frequently observed strong contrast correlating with the distribution of bone lamellae. No net change in mineral packing density would be expected from a change in collagen orientation, and the lamellar contrast observed in practice can be explained solely by the topographic contrast.

Bone Density↗
PubMedDate not supplied

Conventional and confocal epi-reflection and fluorescence microscopy of the rat kidney in vivo.

To visualize superficial and accessible renal tubule cells functioning in situ and to relate what we can 'see' to what we know of their function from more invasive in vivo or less direct in vitro studies means applying and adapting recent advances in epifluorescence and confocal microscopy to improve image resolution and to combine this with the use of fluorescent labels to monitor the handling of specific molecules by the proximal and distal renal tubule cells in vivo. Doing this in living tissue is novel, especially in the kidney. Application of confocal microscopy to the imaging of living tissue, as opposed to isolated cells, has not been widely reported. The kidney surface has been imaged before using the confocal microscope and in preliminary studies we have extended this by using a different confocal system with and without fluorescence. While the studies published up to now have been morphological, comparing standard renal (structural) histology of surface glomeruli and renal tubules with the corresponding in vivo confocal images, more dynamic, real-time studies have been limited. Individual red blood cells can be seen flowing around the peritubule capillary network and nucleated white blood cells can also be distinguished. Tubule cells, endothelial cells, the proximal tubule cell brush border and cell mitochondria can be visualized. Filtration and secretion can be observed, and the early and late parts of the proximal tubule distinguished, and the distal tubule recognized. Localization of fluorescently labeled insulin to the luminal brush border and progressive uptake of label and distribution within proximal tubule cells toward the basolateral (blood side) membrane can be demonstrated. The possibility of monitoring hemodynamic changes and tracking the filtration, uptake, secretion and absorption of fluorescently tagged molecules, as well as intracellular fluorescence, e.g. calcium or pH, is an exciting prospect and is ripe for detailed exploration.

Animals↗
PubMedDate not supplied

Tandem-scanning (confocal) microscopy of the full-thickness cornea.

We have utilized a radically new type of optical scanning microscope to study the full-thickness morphology of the intact cornea in an excised human eye bank eye and in freshly sacrificed rabbit eyes in situ. This technology enables one to study corneal morphology layer by layer in extremely thin sections, only disturbing the tissue with an applanating tip. We have demonstrated the cells of the corneal surface, subsurface cells, the topography of Bowman's membrane, corneal lamellae, stromal keratocytes, and the corneal endothelium. The application of this technology lends itself to the in vivo examination of the human cornea. This should aid us greatly in the study of normal morphology, disease states, and the reaction of the cornea in wound healing.

Animals↗