Quantification and optimization of clot retraction in washed human platelets by Sonoclot coagulation analysis.

INTRODUCTION: Clot retraction is a pivotal process for haemostasis, where platelets develop a contractile force in fibrin meshwork and lead to the increased rigidity of clot. The pathophysiological alteration in contractile forces generated by the platelet-fibrin meshwork can lead to haemostatic disorders. Regardless of its utter significance, clot retraction remains a limited understood process owing to lack of quantification methodology. Sonoclot analysis is a point-of-care technique used in clinical laboratories for whole blood analysis that provides in vitro qualitative as well as quantitative assessment of coagulation process from initial fibrin formation to clot retraction. METHODS: Human washed platelets were isolated by differential centrifugation method and analysed via optical imaging, microscopy and Sonoclot analysis using 1-2 × 108 /mL of washed platelets, 1 U/mL of thrombin, 1 mg/mL of fibrinogen and 1 mM of calcium chloride. RESULTS: In this study, we demonstrate the novelty of this instrument in the quantitative evaluation of clot retraction in washed platelets and attempted to optimize the reference range of Sonoclot parameters including ACT - 87.3 ± 20.997, CR - 16.23 ± 3.538 and PF - 3.57 ± 0.629, (n = 10). DISCUSSION: Sonoclot analysis provides a simple and quantitative method to better understand in vitro clot retraction and its modulation by retraction components including platelet count, fibrinogen and platelet-fibrin interaction compared with existing conventional methods. Sonoclot may prove to be a valuable tool in thrombus biology research to understand fundamental basis of blood clot retraction.

An improved ring removal procedure for in-line x-ray phase contrast tomography.

The suppression of ring artifacts in x-ray computed tomography (CT) is a required step in practical applications; it can be addressed by introducing refined digital low pass filters within the reconstruction process. However, these filters may introduce additional ringing artifacts when simultaneously imaging pure phase objects and elements having a non-negligible absorption coefficient. Ringing originates at sharp interfaces, due to the truncation of spatial high frequencies, and severely affects qualitative and quantitative analysis of the reconstructed slices. In this work, we discuss the causes of ringing artifacts, and present a general compensation procedure to account for it. The proposed procedure has been tested with CT datasets of the mouse central nervous system acquired at different synchrotron radiation facilities. The results demonstrate that the proposed method compensates for ringing artifacts induced by low pass ring removal filters. The effectiveness of the ring suppression filters is not altered; the proposed method can thus be considered as a framework to improve the ring removal step, regardless of the specific filter adopted or the imaged sample.

Assessment of occupational exposure to asbestos fibers: Contribution of analytical transmission electron microscopy analysis and comparison with phase-contrast microscopy.

From November 2009 to October 2010, the French general directorate for labor organized a large field-study using analytical transmission electron microscopy (ATEM) to characterize occupational exposure to asbestos fibers during work on asbestos containing materials (ACM). The primary objective of this study was to establish a method and to validate the feasibility of using ATEM for the analysis of airborne asbestos of individual filters sampled in various occupational environments. For each sampling event, ATEM data were compared to those obtained by phase-contrast optical microscopy (PCOM), the WHO-recommended reference technique. A total of 265 results were obtained from 29 construction sites where workers were in contact with ACM. Data were sorted depending on the combination of the ACM type and the removal technique. For each "ACM-removal technique" combination, ATEM data were used to compute statistical indicators on short, fine and WHO asbestos fibers. Moreover, exposure was assessed taking into account the use of respiratory protective devices (RPD). As in previous studies, no simple relationship was found between results by PCOM and ATEM counting methods. Some ACM, such as asbestos-containing plasters, generated very high dust levels, and some techniques generated considerable levels of dust whatever the ACM treated. On the basis of these observations, recommendations were made to measure and control the occupational exposure limit. General prevention measures to be taken during work with ACM are also suggested. Finally, it is necessary to continue acquiring knowledge, in particular regarding RPD and the dust levels measured by ATEM for the activities not evaluated during this study.

Axial Phase-Darkfield-Contrast (APDC), a new technique for variable optical contrasting in light microscopy.

Axial phase-darkfield-contrast (APDC) has been developed as an illumination technique in light microscopy which promises significant improvements and a higher variability in imaging of several transparent 'problem specimens'. With this method, a phase contrast image is optically superimposed on an axial darkfield image so that a partial image based on the principal zeroth order maximum (phase contrast) interferes with an image, which is based on the secondary maxima (axial darkfield). The background brightness and character of the resulting image can be continuously modulated from a phase contrast-dominated to a darkfield-dominated character. In order to achieve this illumination mode, normal objectives for phase contrast have to be fitted with an additional central light stopper needed for axial (central) darkfield illumination. In corresponding condenser light masks, a small perforation has to be added in the centre of the phase contrast providing light annulus. These light modulating elements are properly aligned when the central perforation is congruent with the objective's light stop and the light annulus is conjugate with the phase ring. The breadth of the condenser light annulus and thus the intensity of the phase contrast partial image can be regulated with the aperture diaphragm. Additional contrast effects can be achieved when both illuminating light components are filtered at different colours. In this technique, the axial resolution (depth of field) is significantly enhanced and the specimen's three-dimensional appearance is accentuated with improved clarity as well as fine details at the given resolution limit. Typical artefacts associated with phase contrast and darkfield illumination are reduced in our methods.

An inter-laboratory study to determine the effectiveness of procedures for discriminating amphibole asbestos fibers from amphibole cleavage fragments in fiber counting by phase-contrast microscopy.

The US Occupational Safety and Health Administration (OSHA) and Mine Safety and Health Administration do not regulate cleavage fragments of amphibole and serpentine minerals as asbestos, even when particles meet the dimensional criteria for counting under standard phase-contrast microscopy methods. The OSHA ID-160 method cautions that discriminatory counting is difficult and should not be attempted unless necessary and no procedure is provided for differentiation. A standard published by the American Society for Testing and Materials (ASTM International D7200-06) includes an attempt to codify a procedure but recognizes that the procedure should be validated in an inter-laboratory study. The US National Institute for Occupational Safety and Health has carried out such a study with multiple laboratories using slides made from riebeckite and crocidolite, grunerite and amosite, tremolite and tremolite asbestos, and actinolite and actinolite asbestos using two different measurement aids (graticules). The asbestos fibers had dimensions consistent with those reported for air samples from actual amphibole asbestos operations, and the cleavage fragments were also dimensionally consistent with those found in non-asbestos mining and milling operations. The procedure for discriminating asbestos fibers from other mineral particles in the ASTM Standard calls for the recognition of characteristics supposedly common to asbestos. For the asbestos fibers created in this study, these characteristics were found not to be common and generally a function of length. More importantly, different laboratories did not recognize these features consistently. Laboratories were much more consistent in measuring dimensions, but excessive overlap in the lengths of asbestos fibers and cleavage fragments rendered length a poor criterion for discrimination. The ASTM discrimination procedure as written could not be supported on the basis of this study. Width was a much more consistent parameter for distinguishing the asbestos and non-asbestos fibers in this study and inclusion of aspect ratio, while considered important by some researchers, did not refine the discrimination further. The ability of the majority of microscopists in this study to discriminate fibers and cleavage fragments through measurement of particle widths was determined and found to be within limits of uncertainty typical for air sampling measurements. A width criterion might be a very simple and useful aid where discrimination between asbestos and non-asbestos fibers in fiber counting by phase-contrast microscopy is required for further investigation. Recognition of asbestos features can also be retained as excessive recognition by some laboratories will lead to a conservative decision for additional investigation.

Combined use of hard X-ray phase contrast imaging and X-ray fluorescence microscopy for sub-cellular metal quantification.

Hard X-ray fluorescence microscopy and magnified phase contrast imaging are combined to obtain quantitative maps of the projected metal concentration in whole cells. The experiments were performed on freeze dried cells at the nano-imaging station ID22NI of the European Synchrotron Radiation Facility (ESRF). X-ray fluorescence analysis gives the areal mass of most major, minor and trace elements; it is validated using a biological standard of known composition. Quantitative phase contrast imaging provides maps of the projected mass and is validated using calibration samples and through comparison with Atomic Force Microscopy and Scanning Transmission Ion Microscopy. Up to now, absolute quantification at the sub-cellular level was impossible using X-ray fluorescence microscopy but can be reached with the use of the proposed approach.

Calibrating differential interference contrast microscopy with dual-focus fluorescence correlation spectroscopy.

We present a novel calibration technique for determining the shear distance of a Nomarski Differential Interference Contrast prism, which is used in Differential Interference Contrast microscopy as well as for the recently developed dual-focus fluorescence correlation spectroscopy. In both applications, an exact knowledge of the shear distance induced by the Nomarski prism is important for a quantitative data evaluation. In Differential Interference Contrast microscopy, the shear distance determines the spatial resolution of imaging, in dual-focus fluorescence correlation spectroscopy, it represents the extrinsic length scale for determining diffusion coefficients. The presented calibration technique is itself based on a combination of fluorescence correlation spectroscopy and dynamic light scattering. The method is easy to implement and allows for determining the shear distance with nanometer accuracy.

Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function.

BACKGROUND: Despite advances in imaging techniques, real-time visualization of the structure and dynamics of tissues and organs inside small living animals has remained elusive. Recently, we have been using synchrotron x-rays to visualize the internal anatomy of millimeter-sized opaque, living animals. This technique takes advantage of partially-coherent x-rays and diffraction to enable clear visualization of internal soft tissue not viewable via conventional absorption radiography. However, because higher quality images require greater x-ray fluxes, there exists an inherent tradeoff between image quality and tissue damage. RESULTS: We evaluated the tradeoff between image quality and harm to the animal by determining the impact of targeted synchrotron x-rays on insect physiology, behavior and survival. Using 25 keV x-rays at a flux density of 80 microW/mm-2, high quality video-rate images can be obtained without major detrimental effects on the insects for multiple minutes, a duration sufficient for many physiological studies. At this setting, insects do not heat up. Additionally, we demonstrate the range of uses of synchrotron phase-contrast imaging by showing high-resolution images of internal anatomy and observations of labeled food movement during ingestion and digestion. CONCLUSION: Synchrotron x-ray phase contrast imaging has the potential to revolutionize the study of physiology and internal biomechanics in small animals. This is the only generally applicable technique that has the necessary spatial and temporal resolutions, penetrating power, and sensitivity to soft tissue that is required to visualize the internal physiology of living animals on the scale from millimeters to microns.

Quantitative phase microscopy: a new tool for investigating the structure and function of unstained live cells.

1. The optical transparency of unstained live cell specimens limits the extent to which information can be recovered from bright-field microscopic images because these specimens generally lack visible amplitude-modulating components. However, visualization of the phase modulation that occurs when light traverses these specimens can provide additional information. 2. Optical phase microscopy and derivatives of this technique, such as differential interference contrast (DIC) and Hoffman modulation contrast (HMC), have been used widely in the study of cellular materials. With these techniques, enhanced contrast is achieved, which is useful in viewing specimens, but does not allow quantitative information to be extracted from the phase content available in the images. 3. An innovative computational approach to phase microscopy, which provides mathematically derived information about specimen phase-modulating characteristics, has been described recently. Known as quantitative phase microscopy (QPM), this method derives quantitative phase measurements from images captured using a bright-field microscope without phase- or interference-contrast optics. 4. The phase map generated from the bright-field images by the QPM method can be used to emulate other contrast image modes (including DIC and HMC) for qualitative viewing. Quantitative phase microscopy achieves improved discrimination of cellular detail, which permits more rigorous image analysis procedures to be undertaken compared with conventional optical methods. 5. The phase map contains information about cell thickness and refractive index and can allow quantification of cellular morphology under experimental conditions. As an example, the proliferative properties of smooth muscle cells have been evaluated using QPM to track growth and confluency of cell cultures. Quantitative phase microscopy has also been used to investigate erythrocyte cell volume and morphology in different osmotic environments. 6. Quantitative phase microscopy is a valuable, new, non-destructive, non-interventional experimental tool for structural and functional cellular investigations.

The effects of stains and investigators on assessment of morphology of canine spermatozoa.

Percentage and types of morphological abnormalities found in canine spermatozoa were evaluated by three investigators using three stains (Giemsa-Wright stain [Diff-Quik], eosin Y/nigrosin [Hancock], and eosin B/nigrosin [Society for Theriogenology morphology stain] with conventional light microscopy, compared to phase contrast microscopy on unstained samples. The percentage of spermatozoa with abnormal heads, midpieces, and tails varied by technique and by investigator. Average percentages of morphologically normal spermatozoa were significantly higher in samples stained with Diff-Quik and samples examined by phase contrast microscopy than in samples stained with Hancock or Society for Theriogenology morphology stains. No effect of investigator on the percentage of morphologically normal spermatozoa was assessed. Results suggest that staining or preparation technique may alter the morphology of canine spermatozoa artifactually.

Diagnosis of urinary tract infection in children: fresh urine microscopy or culture?

Fresh unspun and unstained urine specimens from 342 children with previous urinary tract infection (UTI) or symptoms compatible with a UTI were examined by microscopy at a magnification of x 400 in a mirrored counting chamber by a clinician, and sent for culture in a microbiology laboratory; 200 samples were also plated onto dip-slides. When microscopy and culture results were discrepant, further urine samples were collected until a diagnosis of UTI (24) or sterile urine (318) could be confirmed. Initial microscopy correctly identified 23 of 24 UTIs and 286 of 318 sterile urines; 1 false-positive result was caused by vaginal contamination with lactobacilli. 32 specimens (9%) gave an equivocal result on microscopy; the 1 other true-positive result was identified correctly on microscopy of the next urine specimen obtained. Culture of the initial urines correctly identified all 24 UTIs, but only 82% of the negative samples. Of the samples from uninfected children, 35 (11%) showed a mixed growth which was sterile on repeat sampling, and 21 (6.6%) initially grew a false-positive pure growth of more than 10(5) colony-forming units/ml of one organism. True UTIs were associated with bacterial counts above 10(7)/ml. Microscopy by a clinician represents a cheaper, quicker, and more reliable screening test for UTI in children than does routine culture in a microbiology laboratory.