Automated noninvasive epithelial cell counting in phase contrast microscopy images with automated parameter selection.

Cell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a 'destructive' process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy-based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal. The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.

Potential role of periodontal pathogens in compromising epithelial barrier function by inducing epithelial-mesenchymal transition.

BACKGROUND AND OBJECTIVE: Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire a mesenchymal-like phenotype and this may be induced by exposure to gram-negative bacteria. It has been proposed that EMT is responsible for compromising epithelial barrier function in the pathogenesis of several diseases. However, the possible role of EMT in the pathogenesis of periodontitis has not previously been investigated. The aim of this study therefore was to investigate whether gram-negative, anaerobic periodontal pathogens could trigger EMT in primary oral keratinocytes in vitro. MATERIAL AND METHODS: Primary oral keratinocytes were harvested from labial mandibular mucosa of Wistar Han rats. Cells were exposed to heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis (100 bacteria/epithelial cell) and to 20 µg/mL of Escherichia coli lipopolysaccharide over an 8-day period. Exposure to bacteria did not significantly change epithelial cell number or vitality in comparison with unstimulated controls at the majority of time-points examined. Expression of EMT marker genes was determined by semiquantitative RT-PCR at 1, 5, and 8 days following stimulation. The expression of EMT markers was also assessed by immunofluorescence (E-cadherin and vimentin) and using immunocytochemistry to determine Snail activation. The loss of epithelial monolayer coherence, in response to bacterial challenge, was determined by measuring trans-epithelial electrical resistance. The induction of a migratory phenotype was investigated using scratch-wound and transwell migration assays. RESULTS: Exposure of primary epithelial cell cultures to periodontal pathogens was associated with a significant decrease in transcription (~3-fold) of E-cadherin and the upregulation of N-cadherin, vimentin, Snail, matrix metalloproteinase-2 (~3-5 fold) and toll-like receptor 4. Bacterial stimulation (for 8 days) also resulted in an increased percentage of vimentin-positive cells (an increase of 20% after stimulation with P. gingivalis and an increase of 30% after stimulation with F. nucleatum, compared with controls). Furthermore, periodontal pathogens significantly increased the activation of Snail (60%) and cultures exhibited a decrease in electrical impedance (P < .001) in comparison with unexposed controls. The migratory ability of the cells increased significantly in response to bacterial stimulation, as shown by both the number of migrated cells and scratch-wound closure rates. CONCLUSION: Prolonged exposure of primary rat oral keratinocyte cultures to periodontal pathogens generated EMT-like features, which introduces the possibility that this process may be involved in loss of epithelial integrity during periodontitis.

Periodontal pathogens promote epithelial-mesenchymal transition in oral squamous carcinoma cells in vitro.

Epithelial-mesenchymal transition (EMT) is potentially involved in increasing metastasis of oral squamous cell carcinoma (OSCC). Periodontal pathogens are well-known for their ability to induce intense immune responses and here we investigated whether they are involved in inducing EMT. Cultures of OSCC cell line (H400) were treated separately with heat-killed periodontal pathogens F. nucleatum, or P. gingivalis or E. coli LPS for 8 d. EMT-associated features were assayed using sq-PCR and PCR-arrays, for EMT-related markers, and ELISAs for TGF-ß1, TNF-α, and EGF. The migratory ability of cells was investigated using scratch and transwell migration assays. E-cadherin and vimentin expression was assessed using immunofluorescence while Snail activation was detected with immunocytochemistry. In addition, the integrity of the cultured epithelial layer was investigated using transepithelial electrical resistance (TEER). PCR data showed significant upregulation after 1, 5, and 8 d in transcription of mesenchymal markers and downregulation of epithelial ones compared with unstimulated controls, which were confirmed by immunofluorescence. Periodontal pathogens also caused a significant increase in level of all cytokines investigated which could be involved in EMT-induction and Snail activation. Exposure of cells to the bacteria increased migration and the rate of wound closure. Downregulation of epithelial markers also resulted in a significant decrease in impedance resistance of cell monolayers to passage of electrical current. These results suggested that EMT was likely induced in OSCC cells in response to stimulation by periodontal pathogens.

Automatic thresholding from the gradients of region boundaries.

We present an approach for automatic threshold segmentation of greyscale images. The procedure is inspired by a reinterpretation of the strategy observed in human operators when adjusting thresholds manually and interactively by means of 'slider' controls. The approach translates into two methods. The first one is suitable for single or multiple global thresholds to be applied globally to images and consists of searching for a threshold value that generates a phase whose boundary coincides with the largest gradients in the original image. The second method is a variation, implemented to operate on the discrete connected components of the thresholded phase (i.e. the binary regions) independently. Consequently, this becomes an adaptive local threshold procedure, which operates relative to regions, rather than to local image subsets as is the case in most local thresholding methods previously published. Adding constraints for specifying certain classes of expected objects in the images can improve the output of the method over the traditional 'segmenting first, then classify' approach.

Efficacy, safety and feasibility of intravenous iloprost in the domiciliary treatment of patients with ischemic disease of the lower limbs.

OBJECTIVE: Intravenous iloprost is an important option in the treatment of ischemic disease of the lower limbs; however, the administration of therapy is frequently compromised because of the need for long cycles of infusion in a hospital setting. The aim of the study is to evaluate the efficacy, safety, feasibility, and the economic impact of infusion therapy in the outpatient setting. PATIENTS AND METHODS: Twenty-four consecutive patients were treated with iloprost at their homes where they were administered a slow rate of infusion for 24 hours a day, during 9.9 ± 2.3 days, with a portable syringe pump (Infonde®). RESULTS: The clinical condition of patients evaluated with the modified SVS/ISCVS scale significantly improved after treatment (+1.29 ± 1.04 points vs. baseline, p<0.001). The drug was well tolerated; neither significant adverse events associated with medication nor problems related to venous access were recorded at home. Ninety-six percent of patients successfully completed the entire treatment cycle, and the evaluation questionnaire showed a high acceptance of the therapy. From the perspective of the hospital authority, lower direct medical costs were estimated for the domiciliary infusion process compared with the inpatient infusion setting. CONCLUSIONS: Treatment with iloprost in the outpatient setting is effective, safe, feasible, and more acceptable to patients than infusion at the hospital. In addition, it has a favorable economic and organizational impact on the medical ward.

X-ray Study of Human Dental Tissues Affected by Erythroblastosis Fetalis.

Numerous diseases are known to cause microstructural alteration of dental tissues structure. One type in particular is associated with neonatal jaundice and circulation of bilirubin in blood at high concentration due to increased hemolysis in conditions such as erythroblastosis fetalis, septicemia, biliary atresia, and other causes of hyperbilirubinemia. In those conditions, the products of the catabolism of hemoglobin end up deposited in various tissues, including teeth, where they can present clinically as visibly stained brown/green teeth. There is almost no information on the nature or extent of the structural changes taking place in these conditions. Here, advanced nondestructive wide-angle synchrotron X-ray scattering techniques combined with scanning microscopy methods were used to investigate for the first time the ultrastructure of the dental hard tissues in an archival case of intrinsically pigmented green teeth. Despite no obvious elemental variation across the pigmented tissue region, the high-resolution crystallographic properties probed by wide-angle synchrotron X-ray scattering revealed an ultrastructural variation (orientation, particle size, and lattice parameter of hydroxyapatite crystallites) associated with a pigmentation line in dentine and with a distinct neonatal line in enamel.

Fractals in microscopy.

Fractal geometry, developed by B. Mandelbrot, has provided new key concepts necessary to the understanding and quantification of some aspects of pattern and shape randomness, irregularity, complexity and self-similarity. In the field of microscopy, fractals have profound implications in relation to the effects of magnification and scaling on morphology and to the methodological approaches necessary to measure self-similar structures. In this article are reviewed the fundamental concepts on which fractal geometry is based, their relevance to the microscopy field as well as a number of technical details that can help improving the robustness of morphological analyses when applied to microscopy problems.

Cellular automata and integrodifferential equation models for cell renewal in mosaic tissues.

Mosaic tissues are composed of two or more genetically distinct cell types. They occur naturally, and are also a useful experimental method for exploring tissue growth and maintenance. By marking the different cell types, one can study the patterns formed by proliferation, renewal and migration. Here, we present mathematical modelling suggesting that small changes in the type of interaction that cells have with their local cellular environment can lead to very different outcomes for the composition of mosaics. In cell renewal, proliferation of each cell type may depend linearly or nonlinearly on the local proportion of cells of that type, and these two possibilities produce very different patterns. We study two variations of a cellular automaton model based on simple rules for renewal. We then propose an integrodifferential equation model, and again consider two different forms of cellular interaction. The results of the continuous and cellular automata models are qualitatively the same, and we observe that changes in local environment interaction affect the dynamics for both. Furthermore, we demonstrate that the models reproduce some of the patterns seen in actual mosaic tissues. In particular, our results suggest that the differing patterns seen in organ parenchymas may be driven purely by the process of cell replacement under different interaction scenarios.

Reconstruction of dental ultrasonic scaler 3D vibration patterns from phase-related data.

Ultrasonic scalers are used in dentistry for removing mineralised plaque, known as calculus, from tooth surfaces. Though there is much information relating to the longitudinal vibrations of scaler probes, corresponding lateral data is limited. Understanding the lateral motion of ultrasonic probes is essential as, when used correctly, this motion will contribute to the cleaning process as well as to any damage caused to tooth surfaces. In this work we demonstrate the use of a single-axis scanning laser vibrometer, in conjunction with a mirror, to evaluate simultaneously the longitudinal and lateral motion of dental scaler probes oscillating at ultrasonic frequencies (approximately 30 kHz). Node/antinode patterns along the probe length were observed, as was an elliptical motion along the length of the probe. Application of a load to the tip of the instrument modified the vibration pattern of the whole probe. This technique seems an important step towards better characterisation of the three-dimensional movement of oscillating ultrasonic scaler probes, particularly when probes are contacted against teeth. Understanding the three-dimensional probe motion and how this is affected by contact with tooth surfaces may lead to future instrument designs with improved cleaning efficiency whilst minimising potential tooth damage.

Digital enhancement of haematoxylin- and eosin-stained histological images for red-green colour-blind observers.

Individuals with red-green colour-blindness (CB) commonly experience great difficulty differentiating between certain histological stain pairs, notably haematoxylin-eosin (H&E). The prevalence of red-green CB is high (6-10% of males), including among medical and laboratory personnel, and raises two major concerns: first, accessibility and equity issues during the education and training of individuals with this disability, and second, the likelihood of errors in critical tasks such as interpreting histological images. Here we show two methods to enhance images of H&E-stained samples so the differently stained tissues can be well discriminated by red-green CBs while remaining usable by people with normal vision. Method 1 involves rotating and stretching the range of H&E hues in the image to span the perceptual range of the CB observers. Method 2 digitally unmixes the original dyes using colour deconvolution into two separate images and repositions the information into hues that are more distinctly perceived. The benefits of these methods were tested in 36 volunteers with normal vision and 11 with red-green CB using a variety of H&E stained tissue sections paired with their enhanced versions. CB subjects reported they could better perceive the different stains using the enhanced images for 85% of preparations (method 1: 90%, method 2: 73%), compared to the H&E-stained original images. Many subjects with normal vision also preferred the enhanced images to the original H&E. The results suggest that these colour manipulations confer considerable advantage for those with red-green colour vision deficiency while not disadvantaging people with normal colour vision.

Ultrasonic scaler oscillations and tooth-surface defects.

Damage to tooth root surfaces may occur during ultrasonic cleaning with both piezoelectric and magnetostrictive ultrasonic scalers. It is unclear which mechanism causes more damage or how their mechanism of action leads to such damage. Our null hypothesis is that tooth-surface defect dimensions, resulting from instrumentation with ultrasonic scalers, are independent of whether the scaler probe is magnetostrictive or piezoelectric. Piezoelectric and magnetostrictive ultrasonic scaler probes were placed into contact against polished dentin samples (100 g/200 g). Resulting tooth surfaces were evaluated with a laser metrology system. Ultrasonic instrumentation produced an indentation directly related to the bodily movement of the probe as it made an impact on the surface. Load, generator power, and probe cross-section significantly affected probe vibration and defect depth/volume. Defect dimensions were independent of generator type. Magnetostrictive probes oscillated with greater displacement amplitudes than piezoelectric probes, but produced similar defects. This may be due to the cross-sectional shape of the probes.

Morphometrical differences between pseudo-epitheliomatous hyperplasia in granular cell tumours and squamous cell carcinomas.

AIM: Granular cell tumour (GCT) is a benign tumour which occasionally induces pseudo-epitheliomatous hyperplasia (PEH) of the covering epithelium, mimicking squamous cell carcinoma (SCC) invasion. PEH cells do not show marked atypia, but the presence of pseudoinvasive patterns remains a diagnostic problem since several misdiagnosed cases have been reported. This study investigated objective morphometric criteria to distinguish GCT-PEH from SCC. METHODS AND RESULTS: The complexity of the epithelial connective tissue interface (ECTI) in 57 profiles from nine SCC and 12 GCT-PEH cases was analysed using fractal geometry. Epithelial and non-epithelial cells were segmented using a space partition procedure and analysed morphometrically. GCT ECTI profiles were significantly more complex than those of SCC. The complexity of ECTI quantified by global and local fractal dimensions allowed up to 86% correct discrimination between GCT-PEH and SCC. The cell-wise discrimination between the two entities using cellular morphology was 76% but when the two approaches were combined, the correct discrimination was 96%. CONCLUSIONS: The architectural features of GCT-PEH and SCC show differences which, when quantified, could be used to differentiate the two diagnostic classes. Characterization of these differences may help to elucidate some of the mechanisms of tumour infiltration and metastasis.

A new insight into the oscillation characteristics of endosonic files used in dentistry.

The aim of this study was to assess the oscillation characteristics of unconstrained endosonic files using a scanning laser vibrometer (SLV). Factors investigated included file vibration frequency and node/antinode location as well as the variation in file displacement amplitude due to increasing generator power setting. A 30 kHz Mini Piezon generator (Electro-Medical Systems, Switzerland) was used in conjunction with a #15 and #35 K-file. Each file was fixed in position with the long axis of the file perpendicular to the SLV camera head. The laser from the SLV was scanned over the length of the oscillating file for generator power settings 1 to 5 (minimum to half power). Measurements were repeated ten times. The fundamental vibration frequency for both files was 27.50 kHz. Scans of each file showed the positions of nodes/anti-nodes along the file length. The #15 file demonstrated no significant variation in its mean maximum displacement amplitude with increasing generator power, except at power setting 5, where a decrease in displacement amplitude was observed. The #35 file showed a general increase in mean maximum displacement amplitude with increasing power setting, except at power setting 4 where a 65% decrease in displacement amplitude occurred. In conclusion, scanning laser vibrometry is an effective method for assessing endosonic file vibration characteristics. The SLV was able to demonstrate that (unloaded) file vibration displacement amplitude does not increase linearly with increasing generator power. Further work is being performed on a greater variety of files and generators. Vibration characteristics of files under various loads and varying degrees of constraint should also be investigated.

Ultrasonic scaler tip performance under various load conditions.

OBJECTIVES: The aim of this investigation was to assess the effect of loading on ultrasonic scaler tip displacement amplitude, using a scanning laser vibrometer. MATERIAL AND METHODS: Four generators were selected for this investigation including: Cavitron Select (Dentsply), Cavitron SPS (Dentsply), Piezon Master 400 (EMS) and Mini Piezon (EMS). The insert designs used with these generators included the TFI-10 and TFI-3 (with the Dentsply generators) and P-tip (with the EMS generators). The insert tips were contacted against tooth surfaces with loads of 0.25, 0.5 and 1.0 N. The scanning laser vibrometer was then used to scan the vibrating scalers at various generator power settings. RESULTS: The tips generally showed an increase in displacement amplitude with increasing power setting, although this increase was often nonlinear. Displacement amplitudes of like tips were generally found to be significantly different from each other at all loads. For individual inserts, loading caused a significant difference in tip response from the unloaded situation and also between loads. CONCLUSIONS: This investigation has demonstrated that there is a high variability associated with dental ultrasonic scaler inserts. Clinicians and researchers should be aware of this variability as it may affect both clinical procedures and future research.

Displacement amplitude of ultrasonic scaler inserts.

OBJECTIVES: Scanning laser vibrometry is a noninvasive method of measuring the velocity, displacement amplitude and oscillation frequency of vibrating objects. The purpose of this study was to assess, using a scanning laser vibrometer (SLV), the performance of different designs of commercially available ultrasonic scaler generators by measuring the oscillatory characteristics of various scaler tips. METHODS: Four ultrasonic generators were tested (Cavitron SPS and Cavitron Select (Dentsply, York, PA, USA) and Piezon Master 400 and Mini Piezon (Electro-Medical Systems, Switzerland)) with various designs of scaler tip. The tips were positioned with their anterior surface perpendicular to the direction of the laser. A graduated scale, placed over the manufacturer's power dial, enabled incremental power setting selection. For each power setting, the laser beam from the SLV was scanned over the surface of the oscillating tip. RESULTS: The ranges of longitudinal displacement amplitudes (in micrometres) were as follows: Mini Piezon (P-tip): 12.90+/-1.44 to 44.03+/-7.80; Piezon Master 400 (P-tip): 16.02+/-2.66 to 35.85+/-5.29; Cavitron SPS (TFI-10 tip): 7.81+/-0.51 to 29.70+/-1.12; Cavitron Select (TFI-10 tip): 13.13+/-1.44 to 33.77+/-4.27; Cavitron SPS (TFI-3 tip): 5.50+/-0.46 to 31.35+/-3.62. CONCLUSIONS: This study shows that there are differences between commercially available generators and that tip movement varies between tips of the same style as well as between the generator and tip design. Users of ultrasonic scalers should be made aware of this inherent variability that may influence clinical procedures.

Estimation of tissue layer level by sequential morphological reconstruction.

An automated method for labelling layers of clustered elements in two dimensional images based on morphological operations is presented here. Unlike the various distance transforms for regular lattices, the procedure deals successfully with clusters composed of different shaped and sized elements and provides a distance measure, in number of layers, from each element in the cluster to any other reference element (or elements). The method was applied to histological sections of two different types of polystratified epithelia. Local clusters in the different cell profile layers can subsequently be used to define the orientation of the tissue layers relative to each element in it.

Vibration characteristics of ultrasonic scalers assessed with scanning laser vibrometry.

OBJECTIVES: Scanning laser vibrometry is a non-invasive method of accurately measuring the vibratory characteristics of oscillating objects. The aim of this study was to observe, using a scanning laser vibrometer (SLV), the vibration patterns of dental ultrasonic scaler tips and to assess the effects of water flow rate and power setting on these patterns whilst operating the tips in an unloaded environment. METHODS: A 30kHz ultrasonic scaler (TFI-10, Dentsply) was fixed in position and a laser beam from the SLV was focused onto the tip. The laser, guided by a virtual measurement grid, was scanned over the oscillating tip surface. Scans were taken with the laser beam perpendicular to the long axis of the front face of the tip. RESULTS: Oscillation frequencies and the displacement amplitude at the unconstrained end of the tip were measured for various power/water settings. Vibration nodal positions were recorded for the various settings and were found to occur approximately 4mm from the free end of the tip. At low and medium power settings, tip displacement amplitude was reduced by increased water flow. At high power settings, combined with a high flow rate, the water leaves the body of the instrument as a jet. This left the tip relatively unconstrained, allowing it to oscillate at increased displacement amplitudes. CONCLUSIONS: This study shows that the SLV is able to accurately characterise the movement of oscillating ultrasonic scaler tips. The tips are affected by power setting and water flow rates.

Astyanax scabripinnis (Pisces: Characidae) hemoglobins: structure and function

Electrophoretic patterns of hemoglobins, Root effect, Bohr effect in blood and stripped hemoglobin, Hb-O2 affinity GTP modulation of Astyanax scabripinnis (lambari), caught at three different altitudes in Ribeiräo Grande, near Campos do Jordäo (Säo Paulo), are described. All populations showed the same electrophoretic patterns: two cathodal components in starch gel. Normal Bohr effect values were found in these three populations both in blood (phi = -0,11) and stripped hemoglobin (phi = -0,12). Different blood O2 affinities collected in fish of these 3 populations were detected. GTP has a large influence on Hb-O2 binding properties in A. scabripinnis. Stripped hemoglobin shows small Root effect. The addition of triphosphated nucleotides increases this effect. GTP is more effective than ATP on enhancing Root effect. Oxygen availability in water can be the factor responsible for differences found in blood O2 affinity