Sphingolipids are found in essentially all animals, plants and fungi, and some prokaryotic organisms and viruses. Sphingolipids function as structural components of membranes, lipoproteins, and as cell signaling modulators and mediators. To complicate matters further, sphingolipids often vary in type in different regions of tissues, and even in single cells, the subcellular localization of sphingolipids and their metabolic enzymes, transport proteins and targets may influence their functions. It is important to study sphingolipids spatial distribution within living organisms to understand how sphingolipids are involved in complex biochemical processes. As part of this thesis, procedures were optimized for the use of matrix assisted laser desorption/ionization (MALDI) tissue mass spectrometry (TIMS) to visualize the location of several types of lipids including sulfatides (ST), gangliosides and phosphoglycerolipids in brains from a mouse model for Tay-Sachs/Sandhoff disease. MALDI-TIMS was next applied to human ovarian carcinoma tissue to detect sulfatide location and established that ST are associated specifically with the regions of the ovarian tissue that bear the carcinoma. Electrospray ionization tandem mass spectrometry (ESI-MS-MS) was also used to confirm that ST and galactosylceramide (GalCer) are elevated in ovarian cancer. Gene expression data using tumor cells collected using laser capture microdissection revealed greater expression of mRNAs for GalCer synthase, GalCer sulfotransferase (Gal3ST1) and other enzymes of ST biosynthesis in epithelial ovarian carcinoma cells. This is a unique combination of two complementary, profiling technologies--mass spectrometry (metabolomic approach) with analysis of gene expression to study complex cancer pathology. The next study focused on the subcellular location of sphingolipids. In comparison with wild type Hek293 cells, a Hek293 cell line stably overexpressing serine palmitoyltransferase (SPT1/2 cells) was found to have elevated amounts of all subspecies of ceramide (Cer), but produces disproportionately higher amounts of C18-Cer and GalCer. Since Cer is known to inhibit protein ER/Golgi trafficking, these studies found that the higher production of Cer caused impairment of ER/Golgi trafficking of Ceramide synthase 1 (CerS1), thus increased C18-Cer. In addition, since GalCer is only synthesized in the lumen of the ER, this impairement of ER/Golgi trafficking also gave GalCer synthase access to its substrate and increased GalCer biosynthesis. These studies illustrate the complexity of sphingolipid biology and the usefulness of multiple tools to understand sphingolipid complex biological processes.

No description available.

ToF-SIMS imaging has been drawing attention due to the wide range of applications in the biological and biomedical fields. These applications include the acquisition of quantitative and qualitative data that ranges in scale from single cells to organs, image visualisation and interpretation of biomarkers for diagnosis and development of pharmaceutics. This study focused on molecular imaging of mouse brain tissue sections using cluster primary ion beams. First, cluster ion beams were applied to comparative background studies of biomolecules and brain total lipid extract. Enhancement of the secondary ion signal was observed using water-containing cluster primary ion beams, especially for [M+H]+ type secondary ions. Water-containing clusters were then used to acquire ToF-SIMS images from the cerebellar area of serial mouse brain tissue sections. Again, water-containing cluster beams produced the highest secondary ion yields in both grey and white matter, gaining a new level of insight into the lipid compositions of both types of tissue in the brain. A clinical case was also evaluated with ToF-SIMS imaging, using cluster beams for the analysis of 3xTg-AD mouse brain tissue. SIMS images were registered with fluorescence microscopy images for the in situ identification and co-localisation of the Amyloid-β plaques on the SIMS images. Spectra from regions of interest were analysed to identify possible ion fragments derived from the Aβ protein. The co-localisation of cholesterol was also studied from images obtained with different primary ion beams. The results presented show that cluster ToF-SIMS can be successfully applied to brain tissue imaging. New primary ion beam technologies allow us to acquire data with more useful secondary ion yield for clinical applications and biological research. Nevertheless, future technological improvements are required for specialised applications e.g. cellular imaging. Moreover, processing the data obtained is still challenging and more data processing tools are also needed for interpretation.

Background: It is well known that performance of the right ventricle (RV) determines exercise capacity and may confer prognostic information in different cardiopulmonary diseases. To allow optimal patient management, ideal methods to assess right heart function are therefore important. Echocardiography is an attractive investigation for that purpose, although limited by the anatomical and functional complexities of the RV. The aim of the present thesis was to present applicable methods useful in clinical practice by traditional 2D/Doppler echocardiography and Doppler tissue imaging (DTI) in the assessment of global and regional RV function in both health and disease. Methods: The studies were performed on 4 different groups; (1) 255 healthy subjects (125 females), (2) 92 consecutive patients with different cardiac diseases (36 females), (3) 26 patients with systemic sclerosis, (SSc) (21 females) and (4) 26 consecutive patients with heart failure (8 females) undergoing cardiac catheterisation. Results: RV outflow tract fractional shortening (RVOT fs), which is a new method in the assessment of RV function, correlated significantly with RV systolic long axis motion (r= 0.66, p< 0.001), pulmonary artery acceleration time (r= 0.80, p< 0.001) and RV-right atrial peak systolic pressure drop gradient (r= -0.53, p< 0.001). Furthermore, RVOT fs was reduced in patients with pulmonary hypertension whereas RV systolic long axis motion was not in difference. This finding was confirmed after comparing RV function with invasive pressures. In healthy subjects, while the systolic myocardial velocities were preserved over age, the peak isovolumic contraction velocity (IVCv) was weakly increased with advanced age (r= 0.34, p< 0.01). Furthermore, both global and regional E/A ratios were reduced (r= -0.57, r= -0.67, p< 0.001 for both) with age whereas no alteration was found in the myocardial isovolumic relaxation time (IVRt). In patients with systemic sclerosis (SSc) both global (64± 23 vs. 39± 12 ms, p< 0.001) as well as regional (83± 40 vs. 46± 24 ms, p< 0.001) IVRt were prolonged. After evaluating echocardiographic parameters with invasive pressures we found a significant correlation between DTI derived IVRt and pulmonary artery systolic pressures (r= 0.83, p< 0.01) while the IVCv was related to the state of contractility (r= 0.77, p< 0.001). Furthermore, an IVCv below 6 cm/s was shown to be an accurate marker of increased right atrial pressure (>6 mm Hg). In conclusion, RVOT fs can be used as a complementary measurement of RV systolic function, being more sensitive to elevated pulmonary artery systolic pressures than the systolic longitudinal RV motion. Right heart function, mainly the diastolic function, is relatively weakly influenced by age compared to the left heart function. In patients with SSc, we found diastolic disturbances, including a prolonged IVRt and proposed the findings to be early markers related to intermittent pulmonary hypertension. This observation was strengthened after evaluating IVRt against invasive pulmonary artery systolic pressures. IVCv can be used to determine the state of RV contractility and also be used to identify patients with elevated filling pressures. The presented methods can be used to detect early signs of RV dysfunction which might prohibit right heart failure. All presented methods are non-invasive, reproducible, easy obtainable, and thus useful in clinical practice.

Background: The ageing process not only increases the risk of coronary artery disease (CAD) but also complicates its diagnosis and treatment. It is therefore important to understand the newer concepts of cardiovascular ageing physiology as well as methods of predicting the outcomes of therapeutic options available for the elderly with severe CAD. Studies of atrioventricular (AV) ring or plane motion have attracted considerable interest in the last few years as a means of assessing ventricular and atrial function. As the displacement of AV rings towards the ventricular apex is a direct reflection of longitudinal fibre contraction, its measurement by echocardiography provides additional information regarding global and regional systolic and diastolic function. Left ventricular (LV) long axis amplitude of motion, referred to as mitral valve annular (MA) motion, is reduced in CAD and to some extent in the elderly as part of the normal ageing process. Objectives & Methods: The aim of the present study was two-fold. First, to investigate the relationship between the timing of MA motion and transmitral and pulmonary venous flow in healthy subjects, and to define the physiological significance of that relationship including its potential diagnostic utility. Second, to investigate the relationship between the clinical outcome and the behaviour of long axis function in patients with severe ischaemic LV dysfunction (SLVD) after percutaneous coronary angioplasty (PTCA). Transmitral early (E) and late (A) filling, and pulmonary venous flow reversal (Ar) were studied by Doppler echocardiography, while at the left lateral AV ring, the MA motion in early (Em) and late (Am) diastole were recorded by Doppler tissue imaging (DTI) and M-mode echocardiography. Results: Healthy subjects – In early diastole the onsets of LV filling (E) and relaxation (Em) were simultaneous, and peak Em preceded peak E by 26 msec in all age groups, constituting a time interval referred to as early diastolic temporal discordance (EDTD). Similarly, the onsets of Am, A and Ar were simultaneous at onset and began approximately 84 msec after the electrocardiographic P wave. Peak Am preceded peak A by 23 msec in the young and by 13 msec in the elderly, a time interval referred to as late diastolic temporal discordance (LDTD). Peak Ar, on the other hand, coincided with peak Am in all age groups. With increasing age and sequential prolongation of isovolumic relaxation time, the peaks of Am, Ar and A converged. This point of convergence is described as atrial mechanical alignment (AMA). Patients – MA total amplitude of motion, rates of shortening and lengthening were all reduced in patients with SLVD. At mid-term, 3-6 months after PTCA, there was improvement in all these variables. A pre-procedure long axis cut off value of ≥5 mm was associated with favourable symptomatic outcome. Overall angiographic success was 95.2%, and event-free survival was 78.4% at one month and declined steadily to 62.3% at one year with 2.5% mortality. Conclusions: EDTD, which reflects ventricular restoring forces (suction) is age independent while the narrowing of LDTD leading to AMA provides a novel method to identify healthy subjects at increased dependency on left atrial contraction for late diastolic filling. Peak atrial contraction (Am) coincides with peak Ar, thus the timing of regional atrial contraction by DTI can be used to estimate corresponding measurements of Ar, which is often difficult to image by transthoracic echocardiography. In patients with SLVD long axis total amplitude of at least 5 mm at the left MA suggests a significant potential for segmental function recovery after PTCA. Keywords: Echocardiography, Doppler tissue imaging, ageing, coronary disease, left ventricular dysfunction, atrial contraction, electromechanical function, coronary angioplasty.

Dans cette thèse, nous proposons l'utilisation d'un nouveau type de fibre à cristal photonique, la fibre Kagomé à coeur creux, pour la livraison d'impulsions ultra-courtes en endoscopie non linéaire. Ces fibres permettent la livraison d'impulsions sans distorsion sur une large bande spectrale, avec un faible bruit de fond, grâce à la propagation dans le cœur creux. Nous avons résolu le problème de la résolution spatiale, à l'aide d'une microbille en silice, insérée dans le cœur de la fibre Kagomé. Nous avons développé un système d'imagerie compacte, qui utilise un tube piézo-électrique pour le balayage du faisceau, un système achromatiques de microlentilles et une fibre Kagomé double gaine, spécialement conçue pour l'endoscopie. Avec ce système, nous avons réussi à imager des tissus biologiques, à l'extrémité distale de la fibre (endoscopie), en utilisant des différentes techniques tels que TPEF, SHG et CARS, un résultat qui ne trouve pas d'égal dans la littérature actuelle. L'intégration dans une sonde portable (4,2 mm de diamètre) montre le potentiel de ce système pour de futures applications en endoscopie multimodale in-vivo. / In this thesis, we propose the use of a novel type of photonic crystal fiber, the Kagomé lattice hollow core fiber, for the delivery of ultra-short pulses in nonlinear endoscopy. These fibers allow undistorted pulse delivery, over a broad transmission window, with minimum background signal generated in the fiber, thanks to the propagation in a hollow-core. We solved the problem of spatial resolution, by means of a silica micro-bead inserted in the Kagomé fiber large core. We have developed a miniature imaging system, based on a piezo-electric tube scanner, an achromatic micro-lenses assembly and a specifically designed Kagomé double-clad fiber. With this system we were able to image biological tissues, in endoscope modality, activating different contrasts such as TPEF, SHG and CARS, at the distal end of the fiber, a result which finds no equal in current literature. The integration in a portable probe (4.2 mm in diameter) shows the potential of this system for future in-vivo multimodal endoscopy.

Intravital microscopy (IVM) is a well-established imaging technique for real-time monitoring of microscale lung tissue dynamics. Although accepted as a gold standard in respiratory research, its characteristic image features are scarcely understood, especially when trying to determine the actual position of alveolar walls. To allow correct interpretation of these images with respect to the true geometry of the lung parenchyma, we analyzed IVM data of alveoli in a mouse model in comparison with simultaneously acquired optical coherence tomography images. Several IVM characteristics, such as double ring structures or disappearing alveoli in regions of liquid filling, could be identified and related to the position of alveoli relative to each other. Utilizing a ray tracing approach based on an idealized geometry of the mouse lung parenchyma, two major reflection processes could be attributed to the IVM image formation: partial reflection and total internal reflection between adjacent alveoli. Considering the origin of the reflexes, a model was developed to determine the true position of alveolar walls within IVM images. These results allow thorough understanding of IVM data and may serve as a basis for the correction of alveolar sizes for more accurate quantitative analysis within future studies of lung tissue dynamics.

No description available.

Diffuse optical tomography is a promising imaging modality that provides functional information of the soft biological tissues, with prime imaging applications including breast and brain tissue in-vivo. This modality uses near infrared light( 600nm-900nm) as the probing media, giving an advantage of being non-ionizing imaging modality. The image reconstruction problem in diffuse optical tomography is typically posed as a least-squares problem that minimizes the difference between experimental and modeled data with respect to optical properties. This problem is non-linear and ill-posed, due to multiple scattering of the near infrared light in the biological tissues, leading to infinitely many possible solutions. The traditional methods employ a regularization term to constrain the solution space as well as stabilize the solution, with Tikhonov type regularization being the most popular one. The choice of this regularization parameter, also known as hyper parameter, dictates the reconstructed optical image quality and is typically chosen empirically or based on prior experience. In this thesis, a simple back projection type image reconstruction algorithm is taken up, as they are known to provide computationally efficient solution compared to regularized solutions. In these algorithms, the hyper parameter becomes equivalent to filter factor and choice of which is typically dependent on the sampling interval used for acquiring data in each projection and the angle of projection. Determining these parameters for diffuse optical tomography is not so straightforward and requires usage of advanced computational models. In this thesis, a computationally efficient simplex Method based optimization scheme for automatically finding this filter factor is proposed and its performances is evaluated through numerical and experimental phantom data. As back projection type algorithms are approximations to traditional methods, the absolute quantitative accuracy of the reconstructed optical properties is poor .In scenarios, like dynamic imaging, where the emphasis is on recovering relative difference in the optical properties, these algorithms are effective in comparison to traditional methods, with an added advantage being highly computationally efficient. In the second part of this thesis, this hyper parameter choice for traditional Tikhonov type regularization is attempted with the help of Least-Squares QR-decompisition (LSQR) method. The established techniques that enable the automated choice of hyper parameters include Generalized Cross-Validation(GCV) and regularized Minimal Residual Method(MRM), where both of them come with higher over head of computation time, making it prohibitive to be used in the real-time. The proposed LSQR algorithm uses bidiagonalization of the system matrix to result in less computational cost. The proposed LSQR-based algorithm for automated choice of hyper parameter is compared with MRM methods and is proven to be computationally optimal technique through numerical and experimental phantom cases.

Lungcancer är en heterogen sjukdom som kräver många histopatologiska analyser för att diagnostisera och behandla effektivt. Dessa analyser utförs för närvarande med singelplex immunohistokemi (IHC) vilket kräver mycket vävnadsprovmaterial om flera markörer behöver färgas för. I den här studien testades två tillvägagångssätt för multiplex immunfluorescens (IF) (indirekt sekventiell IF och multiplex tyramide signal amplification med spektral avblandning) för att se om en panel av 7 markörer, som rutinmässigt används kliniskt, kunde färgas i ett enda experiment på samma vävnadsprov. För att göra detta användes COMET- och LabSat-instrumenten från Lunaphore och de utvärderades utifrån kvalité och användarvänlighet. På COMETen kunde alla sju markörer inkluderas i panelen medan tillvägagångssättet med TSA på LabSat var begränsat till sex markörer på grund av protokollet för spektral separation. Panelen optimerades på COMET och sedan gjordes multiplexa färgningar med båda instrumenten. Färgningarna från LabSat avbildades med mikroskopet på PhenoImager Fusion-instrumentet. Alla färgningar på COMET var av god kvalité medan LabSat hade behövt vidare optimering för att prestera på samma nivå. COMETen fanns också vara det mer användarvänliga instrumentet pga det integrerade mikroskopet och mindre behov av användarintervention. Denna studie visade att det är möjligt att översätta singelplexa IHC-analyser till multiplex IF med vissa optimeringar och byte av några antikroppskloner. / Lung cancer is a heterogeneous group of malignancies that requires many histopathological analyses to diagnose and treat efficiently. These analyses are currently performed with singleplex immunohistochemistry (IHC) which needs a lot of tissue sample material if multiple markers have to be stained for. In this study, two different multiplexed immunofluorescence (IF) approaches (indirect sequential IF and multiplexed tyramide signal amplification with spectral unmixing) were tested to see if a panel of 7 markers routinely used in clinic to could be stained in a single experiment on the same tissue sample. To do this, the COMET and LabSat instruments from Lunaphore were used and evaluated in regards to quality and ease of use. With the COMET all seven markers could be included in the panel while the TSA approach on LabSat was limited to six markers due to the spectral unmixing protocol. The panel was optimized on COMET and then multiplexed stainings were performed on both instruments. The LabSat staining was imaged on the PhenoImager Fusion microscope. All the stainings were of good quality on the COMET while the LabSat would have needed further optimization to perform at the same level. The COMET was also found to be the more user-friendly instrument due to its integrated microscope and lesser need for user intervention. This study showed that it is possible to translate singleplex IHC analyses into multiplexed IF with some optimizations and change of a few antibody clones.