Stiffness tomography exploration of living and fixed macrophages.

Stiffness tomography is a new atomic force microscopy imaging technique that allows highlighting structures located underneath the surface of the sample. In this imaging mode, such structures are identified by investigating their mechanical properties. We present here, for the first time, a description of the use of this technique to acquire detailed stiffness maps of fixed and living macrophages. Indeed, the mechanical properties of several macrophages were studied through stiffness tomography imaging, allowing some insight of the structures lying below the cell's surface. Through these investigations, we were able to evidence the presence and properties of stiff column-like features located underneath the cell membrane. To our knowledge, this is the first evidence of the presence, underneath the cell membrane, of such stiff features, which are in dimension and form compatible with phagosomes. Moreover, by exposing the cells to cytochalasin, we were able to study the induced modifications, obtaining an indication of the location and mechanical properties of the actin cytoskeleton. Copyright © 2012 John Wiley & Sons, Ltd.

Force volume and stiffness tomography investigation on the dynamics of stiff material under bacterial membranes.

The determination of the characteristics of micro-organisms in clinical specimens is essential for the rapid diagnosis and treatment of infections. A thorough investigation of the nanoscale properties of bacteria can prove to be a fundamental tool. Indeed, in the latest years, the importance of high resolution analysis of the properties of microbial cell surfaces has been increasingly recognized. Among the techniques available to observe at high resolution specific properties of microscopic samples, the Atomic Force Microscope (AFM) is the most widely used instrument capable to perform morphological and mechanical characterizations of living biological systems. Indeed, AFM can routinely study single cells in physiological conditions and can determine their mechanical properties with a nanometric resolution. Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work, we exploit the capabilities and peculiarities of AFM to analyze the mechanical properties of Escherichia coli in order to evidence with a high spatial resolution the mechanical properties of its structure. In particular, we will show that the bacterial membrane is not mechanically uniform, but contains stiffer areas. The force volume investigations presented in this work evidence for the first time the presence and dynamics of such structures. Such information is also coupled with a novel stiffness tomography technique, suggesting the presence of stiffer structures present underneath the membrane layer that could be associated with bacterial nucleoids.

Atomic Force Microscopy Stiffness Tomography on Living Arabidopsis thaliana Cells Reveals the Mechanical Properties of Surface and Deep Cell-Wall Layers during Growth.

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

Stiffness tomography by atomic force microscopy.

The atomic force microscope is a convenient tool to probe living samples at the nanometric scale. Among its numerous capabilities, the instrument can be operated as a nano-indenter to gather information about the mechanical properties of the sample. In this operating mode, the deformation of the cantilever is displayed as a function of the indentation depth of the tip into the sample. Fitting this curve with different theoretical models permits us to estimate the Young's modulus of the sample at the indentation spot. We describe what to our knowledge is a new technique to process these curves to distinguish structures of different stiffness buried into the bulk of the sample. The working principle of this new imaging technique has been verified by finite element models and successfully applied to living cells.

New methods for detecting antibiotic resistant bacteria and preventing their spread

Methicillin resistant Staphylococcus aureus (MRSA) bacteria have emerged in the early 1980's in numerous health care institutions around the world. The main transmission mechanism within hospitals and healthcare facilities is through the hands of health care workers. Resistant to several antibiotics, the MRSA is one of the most feared pathogens in the hospital setting since it is very difficult to eradicate with the standard treatments. There are still a limited number of anti-MRSA antibiotics but the first cases of resistance to these compounds have already been reported and their frequency is likely to increase in the coming years. Every year, the MRSA infections result in major human and financial costs, due to the high associated mortality and expenses related to the required care. Measures towards a faster detection of resistant bacteria and establishment of appropriate antibiotic treatment parameters are fundamental. Also as part as infection prevention, diminution of bacteria present on the commonly touched surfaces could also limit the spread and selection of antibiotic resistant bacteria. During my thesis, projects were developed around MRSA and antibiotic resistance investigation using innovative technologies. The thesis was subdivided in three main parts with the use of atomic force microscopy AFM for antibiotic resistance detection in part 1, the importance of the bacterial inoculum size in the selection of antibiotic resistance in part 2 and the testing of antimicrobial surfaces creating by sputtering copper onto polyester in part 3. In part 1 the AFM was used two different ways, first for the measurement of stiffness (elasticity) of bacteria and second as a nanosensor for antibiotic susceptibility testing. The stiffness of MRSA with different susceptibility profiles to vancomycin was investigated using the stiffness tomography mode of the AFM and results have demonstrated and increased stiffness in the vancomycin resistant strains that also paralleled with increased thickness of the bacterial cell wall. Parts of the AFM were also used to build a new antibiotic susceptibility-testing device. This nano sensor was able to measure vibrations emitted from living bacteria that ceased definitively upon antibiotic exposure to which they were susceptible but restarted after antibiotic removal to which they were resistant, allowing in a matter of minute the assessment of antibiotic susceptibility determination. In part 2 the inoculum effect (IE) of vancomycin, daptomycin and linezolid and its importance in antibiotic resistance selection was investigated with MRSA during a 15 days of cycling experiment. Results indicated that a high bacterial inoculum and a prolonged antibiotic exposure were two key factors in the in vitro antibiotic resistance selection in MRSA and should be taken into consideration when choosing the drug treatment. Finally in part 3 bactericidal textile surfaces were investigated against MRSA. Polyesters coated after 160 seconds of copper sputtering have demonstrated a high bactericidal activity reducing the bacterial load of at least 3 logio after one hour of contact. -- Au cours des dernières décennies, des bactéries multirésistantes aux antibiotiques (BMR) ont émergé dans les hôpitaux du monde entier. Depuis lors, le nombre de BMR et la prévalence des infections liées aux soins (IAS) continuent de croître et sont associés à une augmentation des taux de morbidité et de mortalité ainsi qu'à des coûts élevés. De plus, le nombre de résistance à différentes classes d'antibiotiques a également augmenté parmi les BMR, limitant ainsi les options thérapeutiques disponibles lorsqu'elles ont liées a des infections. Des mesures visant une détection plus rapide des bactéries résistantes ainsi que l'établissement des paramètres de traitement antibiotiques adéquats sont primordiales lors d'infections déjà présentes. Dans une optique de prévention, la diminution des bactéries présentes sur les surfaces communément touchées pourrait aussi freiner la dissémination et l'évolution des bactéries résistantes. Durant ma thèse, différents projets incluant des nouvelles technologies et évoluant autour de la résistance antibiotique ont été traités. Des nouvelles technologies telles que le microscope à force atomique (AFM) et la pulvérisation cathodique de cuivre (PCC) ont été utilisées, et le Staphylococcus aureus résistant à la méticilline (SARM) a été la principale BMR étudiée. Deux grandes lignes de recherche ont été développées; la première visant à détecter la résistance antibiotique plus rapidement avec l'AFM et la seconde visant à prévenir la dissémination des BMR avec des surfaces crées grâce à la PCC. L'AFM a tout d'abord été utilisé en tant que microscope à sonde locale afin d'investiguer la résistance à la vancomycine chez les SARMs. Les résultats ont démontré que la rigidité de la paroi augmentait avec la résistance à la vancomycine et que celle-ci corrélait aussi avec une augmentation de l'épaisseur des parois, vérifiée grâce à la microscopie électronique. Des parties d'un AFM ont été ensuite utilisées afin de créer un nouveau dispositif de test de sensibilité aux antibiotiques, un nanocapteur. Ce nanocapteur mesure des vibrations produites par les bactéries vivantes. Après l'ajout d'antibiotique, les vibrations cessent définitivement chez les bactéries sensibles à l'antibiotique. En revanche pour les bactéries résistantes, les vibrations reprennent après le retrait de l'antibiotique dans le milieu permettant ainsi, en l'espace de minutes de détecter la sensibilité de la bactérie à un antibiotique. La PCC a été utilisée afin de créer des surfaces bactéricides pour la prévention de la viabilité des BMR sur des surfaces inertes. Des polyesters finement recouverts de cuivre (Cu), connu pour ses propriétés bactéricides, ont été produits et testés contre des SARMs. Une méthode de détection de viabilité des bactéries sur ces surfaces a été mise au point, et les polyesters obtenus après 160 secondes de pulvérisation au Cu ont démontré une excellente activité bactéricide, diminuant la charge bactérienne d'au moins 3 logio après une heure de contact. En conclusion, l'utilisation de nouvelles technologies nous a permis d'évoluer vers de méthodes de détection de la résistance antibiotique plus rapides ainsi que vers le développement d'un nouveau type de surface bactéricide, dans le but d'améliorer le diagnostic et la gestion des BMR.

The fixation of the cemented femoral component. Effects of stem stiffness, cement thickness and roughness of the cement-bone surface.

After cemented total hip arthroplasty (THA) there may be failure at either the cement-stem or the cement-bone interface. This results from the occurrence of abnormally high shear and compressive stresses within the cement and excessive relative micromovement. We therefore evaluated micromovement and stress at the cement-bone and cement-stem interfaces for a titanium and a chromium-cobalt stem. The behaviour of both implants was similar and no substantial differences were found in the size and distribution of micromovement on either interface with respect to the stiffness of the stem. Micromovement was minimal with a cement mantle 3 to 4 mm thick but then increased with greater thickness of the cement. Abnormally high micromovement occurred when the cement was thinner than 2 mm and the stem was made of titanium. The relative decrease in surface roughness augmented slipping but decreased debonding at the cement-bone interface. Shear stress at this site did not vary significantly for the different coefficients of cement-bone friction while compressive and hoop stresses within the cement increased slightly.

Diagnosing pulmonary embolism in outpatients with clinical assessment, D-dimer measurement, venous ultrasound, and helical computed tomography: a multicenter management study.

PURPOSE: To evaluate a diagnostic strategy for pulmonary embolism that combined clinical assessment, plasma D-dimer measurement, lower limb venous ultrasonography, and helical computed tomography (CT). METHODS: A cohort of 965 consecutive patients presenting to the emergency departments of three general and teaching hospitals with clinically suspected pulmonary embolism underwent sequential noninvasive testing. Clinical probability was assessed by a prediction rule combined with implicit judgment. All patients were followed for 3 months. RESULTS: A normal D-dimer level (<500 microg/L by a rapid enzyme-linked immunosorbent assay) ruled out venous thromboembolism in 280 patients (29%), and finding a deep vein thrombosis by ultrasonography established the diagnosis in 92 patients (9.5%). Helical CT was required in only 593 patients (61%) and showed pulmonary embolism in 124 patients (12.8%). Pulmonary embolism was considered ruled out in the 450 patients (46.6%) with a negative ultrasound and CT scan and a low-to-intermediate clinical probability. The 8 patients with a negative ultrasound and CT scan despite a high clinical probability proceeded to pulmonary angiography (positive: 2; negative: 6). Helical CT was inconclusive in 11 patients (pulmonary embolism: 4; no pulmonary embolism: 7). The overall prevalence of pulmonary embolism was 23%. Patients classified as not having pulmonary embolism were not anticoagulated during follow-up and had a 3-month thromboembolic risk of 1.0% (95% confidence interval: 0.5% to 2.1%). CONCLUSION: A noninvasive diagnostic strategy combining clinical assessment, D-dimer measurement, ultrasonography, and helical CT yielded a diagnosis in 99% of outpatients suspected of pulmonary embolism, and appeared to be safe, provided that CT was combined with ultrasonography to rule out the disease.

Octopus Standard Automated Perimetry, Pulsar Perimetry, Moorfields Motion Displacement Test and Heidelberg Retinal Tomography in Glaucoma Detection - A Comparison of Diagnostic Accuracy

Purpose: To investigate the accuracy of 4 clinical instruments in the detection of glaucomatous damage. Methods: 102 eyes of 55 test subjects (Age mean = 66.5yrs, range = [39; 89]) underwent Heidelberg Retinal Tomography (HRTIII), (disc area<2.43); and standard automated perimetry (SAP) using Octopus (Dynamic); Pulsar (TOP); and Moorfields Motion Displacement Test (MDT) (ESTA strategy). Eyes were separated into three groups 1) Healthy (H): IOP<21mmHg and healthy discs (clinical examination), 39 subjects, 78 eyes; 2) Glaucoma suspect (GS): Suspicious discs (clinical examination), 12 subjects, 15 eyes; 3) Glaucoma (G): progressive structural or functional loss, 14 subjects, 20 eyes. Clinical diagnostic precision was examined using the cut-off associated with the p<5% normative limit of MD (Octopus/Pulsar), PTD (MDT) and MRA (HRT) analysis. The sensitivity, specificity and accuracy were calculated for each instrument. Results: See table Conclusions: Despite the advantage of defining glaucoma suspects using clinical optic disc examination, the HRT did not yield significantly higher accuracy than functional measures. HRT, MDT and Octopus SAP yielded higher accuracy than Pulsar perimetry, although results did not reach statistical significance. Further studies are required to investigate the structure-function correlations between these instruments.

Influence of age, risk factors, and cardiovascular and renal disease on arterial stiffness: clinical applications.

Age is the main clinical determinant of large artery stiffness. Central arteries stiffen progressively with age, whereas peripheral muscular arteries change little with age. A number of clinical studies have analyzed the effects of age on aortic stiffness. Increase of central artery stiffness with age is responsible for earlier wave reflections and changes in pressure wave contours. The stiffening of aorta and other central arteries is a potential risk factor for increased cardiovascular morbidity and mortality. Arterial stiffening with aging is accompanied by an elevation in systolic blood pressure (BP) and pulse pressure (PP). Although arterial stiffening with age is a common situation, it has now been confirmed that older subjects with increased arterial stiffness and elevated PP have higher cardiovascular morbidity and mortality. Increase in aortic stiffness with age occurs gradually and continuously, similarly for men and women. Cross-sectional studies have shown that aortic and carotid stiffness (evaluated by the pulse wave velocity) increase with age by approximately 10% to 15% during a period of 10 years. Women always have 5% to 10% lower stiffness than men of the same age. Although large artery stiffness increases with age independently of the presence of cardiovascular risk factors or other associated conditions, the extent of this increase may depend on several environmental or genetic factors. Hypertension may increase arterial stiffness, especially in older subjects. Among other cardiovascular risk factors, diabetes type 1 and 2 accelerates arterial stiffness, whereas the role of dyslipidemia and tobacco smoking is unclear. Arterial stiffness is also present in several cardiovascular and renal diseases. Patients with heart failure, end stage renal disease, and those with atherosclerotic lesions often develop central artery stiffness. Decreased carotid distensibility, increased arterial thickness, and presence of calcifications and plaques often coexist in the same subject. However, relationships between these three alterations of the arterial wall remain to be explored.

Postmortem computed tomography angiography, contrast medium administration and toxicological analyses in urine.

Postmortem angiography methods that use water soluble or lipid soluble liquid contrast compounds may potentially modify the composition of fluid-based biological samples and thus influence toxicological findings. In this study, we investigated whether toxicological investigations performed in urine collected prior to and post angiography using Angiofil? mixed with paraffin oil are characterized by different qualitative or quantitative results. In addition, we studied whether diluting samples with 1% and 3% contrast medium solution may modify molecule concentration. A postmortem angiography group consisting of 50 cases and a postmortem group without angiography consisting of 50 cases were formed. In the first group, toxicological investigations were performed in urine samples collected prior to and post angiography as well as in undiluted and diluted samples. In the second group, analyses were performed in undiluted and diluted urine, bile, gastric content, cerebrospinal and pericardial fluids collected during autopsy. The preliminary results indicate that differences may be observed between urine samples collected prior to and post angiography in the number of identified molecules in relation to specific cases. Analyses performed in diluted samples failed to reveal differences that might potentially alter the interpretation of toxicological results in all analyzed specimens for nearly all molecules, except for tetrahydrocannabinol and its metabolites. Though these findings suggest that toxicology might be effectively performed, in very special cases and for a large number of molecules, in biological samples collected after angiography, it remains recommendable to collect biological fluids for toxicology prior to contrast medium injection.

Increased plasticity of the stiffness of melanoma cells correlates with their acquisition of metastatic properties.

The stiffness of tumor cells varies during cancer progression. In particular, metastatic carcinoma cells analyzed by Atomic Force Microscopy (AFM) appear softer than non-invasive and normal cells. Here we examined by AFM how the stiffness of melanoma cells varies during progression from non-invasive Radial Growth Phase (RGP) to invasive Vertical Growth Phase (VGP) and to metastatic tumors. We show that transformation of melanocytes to RGP and to VGP cells is characterized by decreased cell stiffness. However, further progression to metastatic melanoma is accompanied by increased cell stiffness and the acquisition of higher plasticity by tumor cells, which is manifested by their ability to greatly augment or reduce their stiffness in response to diverse adhesion conditions. We conclude that increased plasticity, rather than decreased stiffness as suggested for other tumor types, is a marker of melanoma malignancy. These findings advise caution about the potential use of AFM for melanoma diagnosis. FROM THE CLINICAL EDITOR: This study investigates the changes to cellular stiffness in metastatic melanoma cells examined via atomic force microscopy. The results demonstrate that increased plasticity is a marker of melanoma malignancy, as opposed to decreased stiffness.