Tomographic imaging with a scanning laser ophtalmoscop

Retinal imaging with a confocal scaning laser Ophthalmoscope (cSLO) involves scanning a small laser beam over the retina and constructing an image from the reflected light. By applying the confocal principle, tomographic images can be produced by measuring a sequence of slices at different depths. However, the thickness of such slices, when compared with the retinal thickness, is too large to give useful 3D retinal images, if no processing is done. In this work, a prototype cSLO was modified in terms hardware and software to give the ability of doing the tomographic measurements with the maximum theoretical axial resolution possible. A model eye was built to test the performance of the system. A novel algorithm has been developed which fits a double Gaussian curve to the axial intensity profiles generated from a stack of images slices. The underlying assumption is that the laser light has mainly been reflected by two structures in the retina, the internal limiting membrane and the retinal pigment epithelium. From the fitted curve topographic images and novel thickness images of the retina can be generated. Deconvolution algorithms have also been developed to improve the axial resolution of the system, using a theoretically predicted cSLO point spread function. The technique was evaluated using measurements made on a model eye, four normal eyes and seven eyes containing retinal pathology. The reproducibility, accuracy and physiological measurements obtained, were compared with available published data, and showed good agreement. The difference in the measurements when using a double rather than a single Gaussian model was also analysed.

Evolution of a debris flow channel monitored using a 3D terrestrial laser scanner

Like numerous torrents in mountainous regions, the Illgraben creek (canton of Wallis, SW Switzerland) produces almost every year several debris flows. The total area of the active catchment is only 4.7 km², but large events ranging from 50'000 to 400'000 m³ are common (Zimmermann 2000). Consequently, the pathway of the main channel often changes suddenly. One single event can for instance fill the whole river bed and dig new several-meters-deep channels somewhere else (Bardou et al. 2003). The quantification of both, the rhythm and the magnitude of these changes, is very important to assess the variability of the bed's cross section and long profile. These parameters are indispensable for numerical modelling, as they should be considered as initial conditions. To monitor the channel evolution an Optech ILRIS 3D terrestrial laser scanner (LIDAR) was used. LIDAR permits to make a complete high precision 3D model of the channel and its surroundings by scanning it from different view points. The 3D data are treated and interpreted with the software Polyworks from Innovmetric Software Inc. Sequential 3D models allow for the determination of the variation in the bed's cross section and long profile. These data will afterwards be used to quantify the erosion and the deposition in the torrent reaches. To complete the chronological evolution of the landforms, precise digital terrain models, obtained by high resolution photogrammetry based on old aerial photographs, will be used. A 500 m long section of the Illgraben channel was scanned on 18th of August 2005 and on 7th of April 2006. These two data sets permit identifying the changes of the channel that occurred during the winter season. An upcoming scanning campaign in September 2006 will allow for the determination of the changes during this summer. Preliminary results show huge variations in the pathway of the Illgraben channel, as well as important vertical and lateral erosion of the river bed. Here we present the results of a river bank on the left (north-western) flank of the channel (Figure 1). For the August 2005 model the scans from 3 viewpoints were superposed, whereas the April 2006 3D image was obtained by combining 5 separate scans. The bank was eroded. The bank got eroded essentially on its left part (up to 6.3 m), where it is hit by the river and the debris flows (Figures 2 and 3). A debris cone has also formed (Figure 3), which suggests that a part of the bank erosion is due to shallow landslides. They probably occur when the river erosion creates an undercut slope. These geometrical data allow for the monitoring of the alluvial dynamics (i.e. aggradation and degradation) on different time scales and the influence of debris flows occurrence on these changes. Finally, the resistance against erosion of the bed's cross section and long profile will be analysed to assess the variability of these two key parameters. This information may then be used in debris flow simulation.

Laser stripe peak detector for 3D scanners: a FIR filter approach

The accuracy of a 3D reconstruction using laser scanners is significantly determined by the detection of the laser stripe. Since the energy pattern of such a stripe corresponds to a Gaussian profile, it makes sense to detect the point of maximum light intensity (or peak) by computing the zero-crossing point of the first derivative of such Gaussian profile. However, because noise is present in every physical process, such as electronic image formation, it is not sensitive to perform the derivative of the image of the stripe in almost any situation, unless a previous filtering stage is done. Considering that stripe scanning is an inherently row-parallel process, every row of a given image must be processed independently in order to compute its corresponding peak position in the row. This paper reports on the use of digital filtering techniques in order to cope with the scanning of different surfaces with different optical properties and different noise levels, leading to the proposal of a more accurate numerical peak detector, even at very low signal-to-noise ratios

Laser shock peening without absorbent coating (LSPwC) effect on 3D surface topography and mechanical properties of 6082-T651 Al alloy

The influence of nanosecond laser pulses applied by laser shock peening without absorbent coating (LSPwC) with a Q-switched Nd:YAG laser operating at a wavelength of λ = 1064 nm on 6082-T651 Al alloy has been investigated. The first portion of the present study assesses laser shock peening effect at two pulse densities on three-dimensional (3D) surface topography characteristics. In the second part of the study, the peening effect on surface texture orientation and micro-structure modification, i.e. the effect of surface craters due to plasma and shock waves, were investigated in both longitudinal (L) and transverse (T) directions of the laser-beam movement. In the final portion of the study, the changes of mechanical properties were evaluated with a residual stress profile and Vickers micro-hardness through depth variation in the near surface layer, whereas factorial design with a response surface methodology (RSM) was applied. The surface topographic and micro-structural effect of laser shock peening were characterised with optical microscopy, InfiniteFocus® microscopy and scanning electron microscopy (SEM). Residual stress evaluation based on a hole-drilling integral method confirmed higher compression at the near surface layer (33 μm) in the transverse direction (σmin) of laser-beam movement, i.e. − 407 ± 81 MPa and − 346 ± 124 MPa, after 900 and 2500 pulses/cm2, respectively. Moreover, RSM analysis of micro-hardness through depth distribution confirmed an increase at both pulse densities, whereas LSPwC-generated shock waves showed the impact effect of up to 800 μm below the surface. Furthermore, ANOVA results confirmed the insignificant influence of LSPwC treatment direction on micro-hardness distribution indicating essentially homogeneous conditions, in both L and T directions.

Comparison of the GDx VCC scanning laser polarimeter and the stratus optical coherence tomograph in the detection of band atrophy of the optic nerve

Aim To compare the ability of scanning laser polarimeter (SLP) with variable corneal compensation (GDx VCC) and optical coherence tomograph (Stratus OCT) to discriminate between eyes with band atrophy (BA) of the optic nerve and healthy eyes. Methods The study included 37 eyes with BA and temporal visual field (VF) defects from chiasmal compression, and 29 normal eyes. Subjects underwent standard automated perimetry (SAP) and retinal nerve fibre layer (RNFL) scans using GDx VCC and Stratus OCT. The severity of the VF defects was evaluated by the temporal mean defect (TMD), calculated as the average of 22 values of the temporal total deviation plot on SAP. Receiver operating characteristic (ROC) curves were calculated. Pearson`s correlation coefficients were used to evaluate the relationship between RNFL thickness parameters and the TMD. Results No significant difference was found between the ROC curves areas (AUCs) for the GDx VCC and Stratus OCT with regard to average RNFL thickness (0.98 and 0.99, respectively) and the superior (0.94; 0.95), inferior (0.96; 0.97), and nasal (0.92; 0.96) quadrants. However, the AUC in the temporal quadrant (0.77) was significantly smaller (P < 0.001) with GDx VCC than with Stratus OCT (0.98). Lower TMD values were associated with smaller RNFL thickness in most parameters from both equipments. Conclusion Adding VCC resulted in improved performance in SLP when evaluating eyes with BA, and both technologies are sensitive in detecting average, superior, inferior, and nasal quadrant RNFL loss. However, GDx VCC still poorly discriminates RNFL loss in the temporal quadrant when compared with Stratus OCT.

Scanning laser polarimetry with enhanced corneal compensation for detection of axonal loss in band atrophy of the optic nerve

PURPOSE: To compare the abilities of scanning laser polarimetry (SLP) with enhanced corneal compensation (ECC) and variable corneal compensation (VCC) modes for detection of retinal nerve fiber layer (RNFL) loss in eyes with band atrophy (BA) of the optic nerve. DESIGN. Cross-sectional study. METHODS: Thirty-seven eyes from 37 patients with BA and temporal visual field defect from chiasmal compression and 40 eyes from 40 healthy subjects were studied. Subjects underwent standard automated perimetry and RNFL measurements using an SLP device equipped with VCC and ECC. Receiver operating characteristic (ROC) curves were calculated for each parameter. Pearson correlation coefficients were obtained to evaluate the relationship between RNFL thickness parameters and severity of visual field loss, as assessed by the temporal mean defect. RESULTS: All RNFL thickness parameters were significantly lower in eyes with BA compared with normal eyes with both compensation modes. However, no statistically significant differences were observed in the areas under the ROC curves for the different parameters between GDx VCC and ECC (Carl Zeiss Meditec, Inc, Dublin, California, USA). Structure-function relationships also were similar for both compensation modes. CONCLUSIONS: No significant differences were found between the diagnostic accuracy of GDx ECC and that of VCC for detection of BA of the optic nerve. The use of GDx ECC does not seem to provide a better evaluation of RNFL loss on the temporal and nasal sectors of the peripapillary retina in subjects with BA of the optic nerve.

Comparative analysis of root surface smear layer removal by different etching modalities or erbium:yttrium-aluminum-garnet laser irradiation. A scanning electron microscopy study

The purpose of this study was to evaluate the effect of erbium:yttrium-aluminum-garnet (Er:YAG) laser (2.94 mu m) irradiation on the removal of root surface smear layer of extracted human teeth and to compare its efficacy with that of citric acid, ethylenediamine tetra-acetic acid (EDTA), or a gel containing a mixture of tetracycline hydrochloride (HCl) and citric acid, using scanning electron microscopy (SEM). Thirty human dentin specimens were randomly divided into six groups: G1 (control group), irrigated with 10 ml of physiologic saline solution; G2, conditioned with 24% citric acid gel; G3, conditioned with 24% EDTA gel; G4, conditioned with a 50% citric acid and tetracycline gel; G5, irradiated with Er:YAG laser (47 mJ/10 Hz/5.8 J/cm(2)/pulse); G6, irradiated with Er:YAG laser (83 mJ/10 Hz/10.3 J/cm(2)/pulse). Electron micrographs were obtained and analyzed according to a rating system. Statistical analysis was conducted with Kruskal-Wallis and Mann-Whitney tests (P < 0.05). G1 was statistically different from all the other groups; no statistically significant differences were observed between the Er:YAG laser groups and those undergoing the other treatment modalities. When the two Er:YAG laser groups were compared, the fluency of G6 was statistically more effective in smear layer removal than the one used in G5 (Mann-Whitney test, P < 0.01). Root surfaces irradiated by Er:YAG laser had more irregular contours than those treated by chemical agents. It can be concluded that all treatment modalities were effective in smear layer removal. The results of our study suggest that the Er:YAG laser can be safely used to condition diseased root surfaces effectively. Furthermore, the effect of Er:YAG laser irradiation on root surfaces should be evaluated in vivo so that its potential to enhance the healing of periodontal tissues can be assessed.

Adhesives bonded to erbium:yttrium-aluminum-garnet laser-irradiated dentin: transmission electron microscopy, scanning electron microscopy and tensile bond strength analyses

The aim of this in vitro study was to investigate the effect of erbium:yttrium-aluminum-garnet (Er:YAG) laser irradiation on dentinal collagen by transmission electron microscopy and to analyze the resin-dentin interface by scanning electron microscopy. A tensile bond strength test was also applied. Specimens from 69 sound human third molars were randomly divided into three groups: control (no laser), and two irradiated groups, laser 250 (250 mJ/2 Hz) and laser 400 (400 mJ/4 Hz). Then, specimens were restored with two adhesive systems, an etch-and-rinse or a self-etch system. Although ultrastructural examination showed a modified surface in the irradiated dentin, there was no statistical difference in bond strength values between the laser groups and controls (P < 0.05). In conclusion, the use of Er:YAG laser for ablating human dentin did not alter the main adhesion parameters when compared with those obtained by conventional methods, thus reinforcing its use in restorative dentistry.

Surgical planning for resection of an ameloblastoma and reconstruction of the mandible using a selective laser sintering 3D biomodel

Ameloblastoma is a benign locally aggressive infiltrative odontogenic lesion. It is characterized by slow growth and painless swelling. The treatment for ameloblastoma varies from curettage to en bloc resection, and the reported recurrence rates after treatment are high; the safety margin of resection is important to avoid recurrence. Advances in technology brought about great benefits in dentistry; a new generation of computed tomography scanners and 3-dimensional images enhance the surgical planning and management of maxillofacial tumors. The development of new prototyping systems provides accurate 3D biomodels on which surgery can be simulated, especially in cases of ameloblastoma, in which the safety margin is important for treatment success. A case of mandibular follicular ameloblastoma is reported where a 3D biomodel was used before and during surgery.

Use of confocal scanning laser microscopy to measure the concentrations of aerial and penetrative hyphae during growth of Rhizopus oligosporus on a solid surface

In order to develop a method for use in investigations of spatial biomass distribution in solid-state fermentation systems, confocal scanning laser microscopy was used to determine the concentrations of aerial and penetrative biomass against height and depth above and below the substrate surface, during growth of Rhizopus oligosporus on potato dextrose agar. Penetrative hyphae had penetrated to a depth of 0.445 cm by 64 h and showed rhizoid morphology, in which the maximum biomass concentration, of 4.45 mg dry wt cm(-3), occurred at a depth of 0.075 cm. For aerial biomass the maximum density of 39.54 mg dry wt(-3) occurred at the substrate surface. For both aerial and penetrative biomass, there were two distinct regions in which the biomass concentration decayed exponentially with distance from the surface. For aerial biomass, the first exponential decay region was up to 0.1 cm height. The second region above the height of 0.1 cm corresponded to that in which sporangiophores dominated. This work lays the foundation for deeper studies into what controls the growth of fungal hyphae above and below the surfaces of solid substrates. (C) Wiley Periodicals, Inc.

Comparison of fundus autofluorescence images acquired by the confocal scanning laser ophthalmoscope (488 nm excitation) and the modified Topcon fundus camera (580 nm excitation).

To compare autofluorescence (AF) images obtained with the confocal scanning laser ophthalmoscope (using the Heidelberg retina angiograph; HRA) and the modified Topcon fundus camera, in a routine clinical setting. A prospective comparative study conducted at the Jules-Gonin Eye Hospital. Fifty-six patients from the medical retina clinic. All patients had complete ophthalmic slit-lamp and fundus examinations, colour and red-free fundus photography, AF imaging with both instruments, and fluorescein angiography. Cataract and fixation were graded clinically. AF patterns were analyzed for healthy and pathological features. Differences of image noise were analyzed by cataract grading and fixation. A total of 105 eyes were included. AF patterns discovered by the retina angiograph and the fundus camera images, respectively, were a dark optic disc in 72 % versus 15 %, a dark fovea in 92 % versus 4 %, sub- and intraretinal fluid visible as hyperautofluorescence on HRA images only, lipid exudates visible as hypoautofluorescence on HRA images only. The same autofluorescent pattern was found on both images for geographic atrophy, retinal pigment changes, drusen and haemorrhage. Image noise was significantly associated with the degree of cataract and/or poor fixation, favouring the fundus camera. Images acquired by the fundus camera before and after fluorescein angiography were identical. Fundus AF images differ according to the technical differences of the instruments used. Knowledge of these differences is important not only for correctly interpreting images, but also for selecting the most appropriate instrument for the clinical situation.

Comparison of fundus autofluorescence between fundus camera (Topcon) and confocal scanning laser ophthalmoscope (HRA) in various pathologies

Purpose: To investigate the differences between Fundus Camera (Topcon TRC-50X) and Confocal Scanning Laser Ophthalmoscope (Heidelberg retina angiogram (HRA)) on the fundus autofluorescence (FAF) imaging (resolution and FAF characteristics). Methods: 105 eyes of 56 patients with various retinal diseases underwent FAF imaging with HRA (488nm exciter/500nm barrier filter) before fluorescein angiography (FFA) and Topcon Fundus Camera (580nm exciter/695nm barrier filter) before and after FFA. The quality of the FAF images was compared for their resolution and analysed for the influence of fixation stability and cataracts. Hypo-and hyper-FAF behaviour was analysed for the healthy disc, healthy fovea, and a variety of pathological features. Results: HRA images were found to be of superior resolution in 18, while Topcon images were estimated superior in 29 eyes. No difference was found in 58 eyes. Both poor fixation (p=0.009) and more advanced cataract (p=0.013) were found associated with better image quality by Topcon. Images acquired by Topcon before and after FFA were identical (100%). The healthy disc was usually dark on HRA (72%), but showed mild autofluorescence on Topcon (85%). The healthy fovea showed in 100% Hypo-FAF on HRA, while Topcon showed in 53% Iso-FAF, in 43% mild Hypo-FAF, and in 4% Hypo-FAF as on HRA. No difference of FAF was found for geographic atrophy, pigment changes, and drusen, although Topcon images were often more detailed. Hyper-FAF due to serous exudation showed better on HRA. Cystic edema was visible only on HRA in a petaloid hyper-FAF pattern in 83%, while only two eyes (17%) showed similar behavior in both HRA- and Topcon images. Hard exudates caused Hypo-FAF only on HRA, hardly visible on Topcon. Blockage phenomenon by blood however was identical. Conclusions: The filter set of Topcon and the single image acquisition appear to be an advantage for patients with cataract and poor fixation respectively. Preceding FFA does not alter the Topcon FAF image. Regarding the FAF behavior, there are differences between the 2 systems which need to be taken into account when interpreting the images.

Comparison of Fundus Autofluorescence Between Fundus Camera (Topcon) and Confocal Scanning Laser Ophthalmoscope (HRA) in Various Pathologies

Purpose: To investigate the differences between the Fundus Camera (Topcon TRC-50X) and Confocal Scanning Laser Ophthalmoscope (Heidelberg retina angiogram (HRA)) on the fundus autofluorescence (FAF) imaging (resolution and FAF characteristics). Methods: Eighty nine eyes of 46 patients with various retinal diseases underwent FAF imaging with HRA (488nm exciter / 500nm barrier filter) before fluorescein angiography (FFA) and Topcon Fundus Camera (580nm exciter / 695nm barrier filter) before and after FFA. The quality of the FAF images was estimated, compared for their resolution and analysed for the influence of fixation stability and cataracts. Hypo- and hyper-FAF behaviour was analysed for the healthy disc, healthy fovea, and a variety of pathological features. Results: HRA images were found to be of superior quality in 18 eyes, while Topcon images were estimated superior in 21 eyes. No difference was found in 50 eyes. Both poor fixation (p=0.009) and more advanced cataract (p=0.013) were found to strongly increase the likelihood of better image quality by Topcon. Images acquired by Topcon before and after FFA were identical (100%). The healthy disc was usually dark on HRA (71%), but showed mild autofluorescence on Topcon (88%). The healthy fovea showed in 100% Hypo-FAF on HRA, while Topcon showed in 52% Iso-FAF, in 43% mild Hypo-FAF, and in 5% Hypo-FAF as on HRA. No difference of FAF was found for geographic atrophy, pigment changes, and drusen, although Topcon images were often more detailed. Hyper-FAF due to exudation showed better on HRA. Pigment epithelium detachment showed identical FAF behaviour on the border, but reduced FAF with Topcon in the center. Cystic edema was visible only on HRA in a petaloid pattern. Hard exsudates caused Hypo-FAF only on HRA, hardly visible on Topcon. Blocage phenomenon by blood however was identical. Conclusions: The filter set of Topcon and the single image acquisition appear to be an advantage for patients with cataract or poor fixation. Preceding FFA does not alter the Topcon FAF image. Regarding the FAF behaviour, there are differences between the two systems which need to be taken into account when interpreting the images.

Study of biomacromolecule conformational changes by Scanning Force Microscopy

L?objectif de ce travail de thèse est l?étude des changements conformationels des biomacromolecules à l?échelle d?une molécule unique. Pour cela on a utilisé la Microscopie à Force Atomique (AFM) appliqué à l?étude des protéines et des acides nucléiques déposés sur une surface. Dans ce type de microscopie, une pointe très fine attachée à l?extrémité d?un levier est balayée au dessus d?une surface. L?interaction de la pointe avec la surface de l?échantillon induit la déflection du levier et ce phénomène permet de reconstruire la topographie de l?échantillon. Très importante dans cette technique est la possibilité de travailler en liquide. Cela permet de étudier les biomolécules en conditions quasi-physiologiques sans qu?elles perdent leur activité. On a étudié GroEL, la chaperonin de E.coli, qui est un homo oligomère avec une structure à double anneau qui joue un rôle très important dans le repliement des protéines dénaturées et celles qui viennent d?être synthétisées. En particulier on a focalisé notre attention sur la stabilité mécanique et sur les changements conformationels qui ont lieu pendant l?activité de GroEL. Une analyse détaillée des changements dans la stabilité mécanique et des effets produits par la liaison et l?hydrolyse de l?ATP est présentée dans ce travail. On a montré que le point le plus faible dans la structure de GroEL est l?interface entre les deux anneaux et que l?étape critique dans l?affaiblissement de la structure est l?hydrolyse de l?ATP. En ce qui concerne le changement conformationel, le passage d?une surface hydrophobe à hydrophile, induit par l?hydrolyse de l?ATP, a été montré. Ensuite on a étudié le changement dans la conformation et dans la topologie de l?ADN résultant de l?interaction avec des molécules spécifiques et en réponse à l?exposition des cellules de E.coli à des conditions de stress. Le niveau de surenroulement est un paramètre très sensible, de façon variée, à tous ces facteurs. Les cellules qui ont crus à de températures plus élevées que leur température optimale ont la tendance à diminuer le nombre de surenroulements négatif pour augmenter la stabilité thermique de leur plasmides. L?interaction avec des agents intercalant induit une transition d?un surenroulement négatif à un surenroulement positif d?une façon dépendante de la température. Finalement, l?effet de l?interaction de l?ADN avec des surfaces différentes a été étudié et une application pratique sur les noeuds d?ADN est présentée.<br/><br/>The aim of the present thesis work is to study the conformational changes of biomacromolecules at the single molecule level. To that end, Atomic Force Microcopy (AFM) imaging was performed on proteins and nucleic acids adsorbed onto a surface. In this microcopy technique a very sharp tip attached at the end of a soft cantilever is scanned over a surface, the interaction of the tip with the sample?s surface will induce the deflection of the cantilever and thus it will make possible to reconstruct the topography. A very important feature of AFM is the possibility to operate in liquid, it means with the sample immersed in a buffer solution. This allows one to study biomolecules in quasi-physiological conditions without loosing their activity. We have studied GroEL, the chaperonin of E.coli, which is a double-ring homooligomer which pays a very important role in the refolding of unfolded and newly synthetized polypeptides. In particular we focus our attention on its mechanical stability and on the conformational change that it undergoes during its activity cycle. A detailed analysis of the change in mechanical stability and how it is affected by the binding and hydrolysis of nucleotides is presented. It has been shown that the weak point of the chaperonin complex is the interface between the two rings and that the critical step to weaken the structure is the hydrolysis of ATP. Concerning the conformational change we have directly measured, with a nanometer scale resolution, the switching from a hydrophobic surface to a hydrophilic one taking place inside its cavity induced by the ATP hydrolysis. We have further studied the change in the DNA conformation and topology as a consequence of the interaction with specific DNA-binding molecules and the exposition of the E.coli cells to stress conditions. The level of supercoiling has been shown to be a very sensitive parameter, even if at different extents, to all these factors. Cells grown at temperatures higher than their optimum one tend to decrease the number of the negative superhelical turns in their plasmids in order to increase their thermal stability. The interaction with intercalating molecules induced a transition from positive to negative supercoiling in a temperature dependent way. The effect of the interaction of the DNA with different surfaces has been investigated and a practical application to DNA complex knots is reported.<br/><br/>Observer les objets biologiques en le touchant Schématiquement le Microscope a Force Atomique (AFM) consiste en une pointe très fine fixée a l?extrémité d?un levier Lors de l?imagerie, la pointe de l?AFM gratte la surface de l?échantillon, la topographie de celui-ci induit des déflections du levier qui sont enregistrées au moyen d?un rayon laser réfléchi par le levier. Ces donnés sont ensuit utilisés par un ordinateur pour reconstituer en 3D la surface de l?échantillon. La résolution de l?instrument est fonction entre autre de la dureté, de la rugosité de l?échantillon et de la forme de la pointe. Selon l?échantillon et la pointe utilisée la résolution de l?AFM peut aller de 0.1 A (sur des cristaux) a quelque dizaine de nanomètres (sur des cellules). Cet instrument est particulierment intéressant en biologie en raison de sa capacité à imager des échantillons immergés dans un liquide, c?est à dire dans des conditions quasiphysiologiques. Dans le cadre de ce travail nous avons étudié les changements conformationels de molécules biologiques soumises à des stimulations externes. Nous avons essentielment concentré notre attention sur des complexes protéiques nommé Chaperons Moléculaires et sur des molécules d?ADN circulaire (plasmides). Les Chaperons sont impliqués entre autre dans la résistance des organismes vivants aux stress thermiques et osmotiques. Leur activité consiste essentielment à aider les autres protéines à être bien pliés dans leur conformation finale et, en conséquence, à eviter que ils soient dénaturées et que ils puissent s?agréger. L?ADN, quant à lui est la molécule qui conserve, dans sa séquence, l?information génétique de tous les organismes vivants. Ce travail a spécifiquement concerné l?étude des changements conformationels des chaperonins suit a leur activation par l?ATP. Ces travaux ont montrés a l?échelle de molécule unique la capacité de ces protéines de changer leur surface de hydrophobique a hydrophilique. Nous avons également utilisé l?AFM pour étudier le changement du nombre des surenroulements des molécules d?ADN circulaire lors d?une exposition à un changement de température et de force ionique. Ces travaux ont permis de montrer comment la cellule regle le nombre de surenroulements dans ces molécules pour répondre et contrôler l?expression génétique même dans de conditions extrêmes. Pour les deux molécules en général, c?était très important d?avoir la possibilité de observer leur transitions d?une conformation a l?autre directement a l?échelle d?une seul molécule et, surtout, avec une résolution largement au dessous des la longueur d?onde de la lumière visible que représente le limite pour l?imagerie optique.

Characterisation of pigment particles by scanning electron microscope and image analysis programs

Coating and filler pigments have strong influence to the properties of the paper. Filler content can be even over 30 % and pigment content in coating is about 85-95 weight percent. The physical and chemical properties of the pigments are different and the knowledge of these properties is important for optimising of optical and printing properties of the paper. The size and shape of pigment particles can be measured by different analysers which can be based on sedimentation, laser diffraction, changes in electric field etc. In this master's thesis was researched particle properties especially by scanning electron microscope (SEM) and image analysis programs. Research included nine pigments with different particle size and shape. Pigments were analysed by two image analysis programs (INCA Feature and Poikki), Coulter LS230 (laser diffraction) and SediGraph 5100 (sedimentation). The results were compared to perceive the effect of particle shape to the performance of the analysers. Only image analysis programs gave parameters of the particle shape. One part of research was also the sample preparation for SEM. Individual particles should be separated and distinct in ideal sample. Analysing methods gave different results but results from image analysis programs corresponded even to sedimentation or to laser diffraction depending on the particle shape. Detailed analysis of the particle shape required high magnification in SEM, but measured parameters described very well the shape of the particles. Large particles (ecd~1 µm) could be used also in 3D-modelling which enabled the measurement of the thickness of the particles. Scanning electron microscope and image analysis programs were effective and multifunctional tools for particle analyses. Development and experience will devise the usability of analysing method in routine use.