Allan S. Jones

Visualizing lipid structure and raft domains in living cells with two-photon microscopy

The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell–cell contacts and cover 10–15% of the cell surface at 37°C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 μm2), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis.

3-Dimensional imaging of collagen using second harmonic generation

Collagen is the most important structural protein of the animal body. Its unique triple-helix structure and extremely high level of crystallinity make it exceptionally efficient in generating the second harmonic of incident light, and we show here how this leads to a novel mode of microscopy of immediate practical significance in medicine and biology. In particular, it provides sensitive and high-resolution information on collagen distribution, discriminates between type I and type III collagen, and allows both a greater understanding of and a sensitive test for cirrhosis of the liver. Future research applications could include wound healing and hereditary collagen diseases such as osteogenesis imperfecta.

The relationship between bone, hemopoietic stem cells, and vasculature

A large body of evidence suggests hemopoietic stem cells (HSCs) exist in an endosteal niche close to bone, whereas others suggest that the HSC niche is intimately associated with vasculature. In this study, we show that transplanted hemopoietic stem and progenitor cells (HSPCs) home preferentially to the trabecular-rich metaphysis of the femurs in nonablated mice at all time points from 15 minutes to 15 hours after transplantation. Within this region, they exist in an endosteal niche in close association with blood vessels. The preferential homing of HSPCs to the metaphysis occurs rapidly after transplantation, suggesting that blood vessels within this region may express a unique repertoire of endothelial adhesive molecules. One candidate is hyaluronan (HA), which is highly expressed on the blood vessel endothelium in the metaphysis. Analysis of the early stages of homing and the spatial dis-tribution of transplanted HSPCs at the single-cell level in mice devoid of Has3-synthesized HA, provides evidence for a previously undescribed role for HA expressed on endothelial cells in directing the homing of HSPCs to the metaphysis.

Physical properties of root cementum: Part 10. Comparison of the effects of invisible removable thermoplastic appliances with light and heavy orthodontic forces on premolar cementum. A microcomputed-tomography study

INTRODUCTION Orthodontic treatment with clear sequential removable thermoplastic appliances (TAs) is gaining popularity as an alternative to treatment with fixed appliances. The amount of orthodontically induced inflammatory root resorption generated by such appliances has not been investigated. In this prospective randomized clinical trial, we used x-ray microtomography to quantify resorption generated by treatment with ClearSmile appliances (ClearSmile, Woollongong, Australia) and compared the effects with those of heavy and light conventional orthodontic forces and no force. METHODS The sample consisted of 54 maxillary first premolars in 27 patients who required bilateral extractions as part of their planned orthodontic treatment. The subjects were randomly assigned to 3 groups, each with 9 subjects. A split-mouth design was used, and forces were applied to the first premolars. In group 1, TAs were used to move teeth on 1 side in a buccal direction at a rate of 0.5 mm every 2 weeks (TA movement); the contralateral teeth were not moved and served at controls. In group 2, TA movement was used on 1 side. A buccal force of 225 g from a beta-titanium alloy cantilever spring (heavy force) was used on the contralateral side. In group 3, TA movement was used on 1 side. A buccal force of 25 g from a cantilever spring (light force) was used on the contralateral side. The treatment duration was 8 weeks (56 days +/- 1 day). The TAs were changed every 14 days, and each patient used 4 appliances. The springs were not reactivated. At the end of the study period, the teeth were extracted according to a strict protocol to prevent root damage. Resorption was measured with an x-ray microtomograph (1072, SkyScan, Aartselaar, Belgium). Software analysis determined quantity, location, and distribution of root resorption craters. RESULTS The control teeth had the least amount of resorption. The light-force teeth had approximately 5 times more resorption than the control teeth (P <.001). The TA teeth had similar but slightly greater resorption than the light-force teeth, or approximately 6 times greater than the control teeth (P <.001). The heavy-force teeth had the most resporption, about 9 times greater than the controls (P <.001). CONCLUSIONS Clear removable TAs have similar effects on root cementum as light (25 g) orthodontic forces with fixed appliances.

Measurement and characterization of cockroach allergens detected during normal domestic activity

BACKGROUND Cockroach allergen is recognized as a causal factor for asthma. However, airborne cockroach allergen has not been detected in undisturbed conditions, and therefore the behavior and properties of airborne cockroach allergen have been poorly characterized. A new aeroallergen sampling method and sensitive system of immunoassay have been used to examine cockroach allergen exposure. OBJECTIVE Our purpose was to measure and characterize airborne cockroach allergens during normal domestic exposure in the homes of Sydney, Australia. METHODS Air sampling with Institute of Occupational Medicine, Edinburgh (IOM) samplers was performed in the living rooms of 10 houses during low- and no-disturbance environments. In addition, inhaled particles were collected by each home occupant during low domestic exposure with use of intra-nasal samplers that impact particles onto an adhesive surface. The particles collected on the IOMs and the intra-nasal samplers were immunostained with Bla g 1 monoclonal antibodies. Particle size, morphologic characteristics, and the relative Bla g 1 content of particles were estimated. Reservoir dust samples from the kitchen, living room, and bedroom were assayed by an ELISA. Two forms of repeatability of IOM air sampling were examined. The first measure tested the repeatability of 2 IOM samples collected simultaneously in the same room during low- and no-disturbance activities. The second measure examined the repeatability of IOM sampling over time on 10 consecutive days. RESULTS Bla g 1 was detected in reservoir dust samples taken from all homes (geometric mean 1.5 U/g, range 0.2-9.4 U/g). Inhaled particles containing Bla g 1 were detected during 1 hour of intra-nasal sampling in 8 of 10 homes during low disturbance. Cockroach particles were detected on all of the IOM samples collected for both 4-hour low-disturbance and overnight no-disturbance sampling environments. Particles containing Bla g 1 collected with the IOM samplers during low disturbance ranged in size from 3 to 350 microm. These particles are amorphous and irregular in shape, and a majority of the large particles were described as flakes (flat, transparent particles) and fibers (threadlike). A relationship was demonstrated between the allergen content of cockroach particles and their particle size. The larger particles elute more Bla g 1. The coefficient of repeatability for measurements made during low and no disturbance was 3.62 and 2.09, respectively. For measurements repeated over time at the same site, the coefficient of repeatability was 2.61. This represents the fold range within which 95% of pairs of measurements made at an interval of 1 day would be expected to lie. CONCLUSIONS Airborne cockroach allergen is present in both undisturbed and low-disturbance environments in homes with relatively low reservoir levels of Bla g 1. In agreement with previous reports, airborne particles containing cockroach allergen (Bla g 1) are mainly associated with particles >10 microm. These particles are amorphous and irregular in shape and can be described as flakes and fibers.

Attachment of the utricular and saccular maculae to the temporal bone.

The present investigation concerns the true morphology of the attachment of the two otolith receptor organs the utricular and the saccular maculae in two and three dimensions. By applying a new visualization method, which utilized the application of X-ray microtomography and a method of contrast enhancement based on en-bloc staining in osmium tetroxide, we were able to overcome problems of artefact production such as tissue distortion and loss of valuable information that was present in previous studies. A series of more than 1000 axial sections were obtained for each of the specimens, which subsequently formed the basis for detailed 2D and 3D visualizations. Our interpretations of these data reveal that the saccular maculae are closely attached to the curved bony surface of the temporal bone as traditionally believed, but the utricular macula is attached to the temporal bone only at the anterior region of the macula.

Airway dimensions measured from micro-computed tomography and high-resolution computed tomography

Volume averaging results in both over- and underestimation of airway dimensions when they are measured by high-resolution computed tomography (HRCT). The current authors calibrated computerised measurements of airway dimensions from HRCT against a novel three-dimensional micro-computed tomography (CT) standard, which has a 50-fold greater resolution, as well as against traditional morphometry. Inflation-fixed porcine lung cubes were scanned by HRCT and micro-CT. A total of 59 lumen area (Ai), 30 wall area (Aaw) and 11 lumen volume (Vi) measurements were made. Ai was measured from the cut surface of 11 airways by morphometry. Airways in scanned images were matched using branching points. After calibration, the errors of Ai, Aaw and Vi HRCT measurements were determined. The current authors found a systematic, size-dependent underestimation of Ai and overestimation of Aaw from HRCT measurements. This was used to calibrate an HRCT measurement algorithm. The 95% limits of agreement of subsequent measurements were ±3.2 mm2 for Ai, ±4.3 mm2 for Aaw, and ±11.2 mm3 for Vi with no systematic error. Morphometric measurements agreed with micro-CT (±2.5 mm2) without systematic error. In conclusion, micro-computed tomography image data from inflation-fixed airways can be used as calibration standards for three-dimensional lumen volume measurements from high-resolution computed tomography, while morphometry is acceptable for two-dimensional measurements. The image dataset could be used to validate other developmental three-dimensional segmentation algorithms.

A new approach to visualizing the membranous structures of the inner ear - high resolution X-ray micro-tomography.

Conclusion. Through the application of high resolution X-ray micro-tomography and a method of contrast enhancement based on en bloc staining in osmium tetroxide (OsO4), we report an approach that facilitates accurate three-dimensional (3D) reconstruction to reveal the fine structure of the inner ear. Objectives. To overcome the problems of artefacts, including tissue distortion and loss of 3D context that are inherent in existing methods that rely on manual dissection and/or histological sectioning. Materials and methods. A staining protocol was developed that involved the en bloc application of the OsO4solution (2% w/v) for an extended period of time. The samples were then scanned using an X-ray micro-tomography platform and subsequent 3D visualizations were constructed. Results. The digital nature of the data allowed a complete 3D contextual visualization to be constructed whereby the individual sensory structures could be seen in relation to other inner ear structures. This included a detailed anatomy of the membranous labyrinth and nerve supply including the spatial configuration of the utricular and saccular maculae. This is a new way of undertaking temporal bone histology.

Direct neural network application for Automated cell recognition

Automated cell recognition from histologic images is a very complex task. Traditionally, the image is segmented by some methods chosen to suit the image type, the objects are measured, and then a classifier is used to determine cell type from the objects measurements. Different classifiers have been used with reasonable success, including neural networks working with data from morphometric analysis.

Effect of Low Magnitude and High Frequency Mechanical Stimuli on Defects Healing in Cranial Bones

PURPOSE The aim of this investigation was to assess the effect of low magnitude high frequency (LMHF) mechanical vibrated stimulation on healing the defects surgically imposed on craniofacial bones. MATERIALS AND METHODS Forty 12-week-old C3H strain mice were separated into surgical and non-surgical groups. The surgical groups had a reproducible surgical bony lesion prepared in their right parietal bone. Both groups were further subdivided into vibration (experimental) and non-vibration (control) groups at 3 time points (zero hours, 2 weeks, and 4 weeks). The vibration groups were subjected to LMHF mechanical stimuli (30 Hz with peak strain 5 microepsilon) via a vibration machine for 20 minutes a day for 5 days a week for a total of 28 days. The specimens were analyzed using micro-computer tomography (micro-CT). RESULTS Micro-CT volumetric measurement showed that in the surgical defects groups there was a significant decrease in the volume of the healing lesion with time (P < .001) and the linear decrease was significantly more pronounced in the vibration-treated group than the nontreated group (P < .001). Micro-CT histomorphometric measurement showed that in the nonsurgical groups there was no significant difference in microstructures of bony trabeculae between vibration-treated and nontreated groups. CONCLUSION It is suggested that the introduction of LMHF mechanical stimuli in a healing bony lesion in the non-weight bearing bone significantly increases its healing capacity.