Littérature scientifique sur le sujet « Morpho-functional imaging »

Transillumination with non-ionizing radiation followed by the observation of transmitted and diffused light is the simplest, and probably the oldest method to obtain qualitative information on the internal structure of tissues or body sections. Although scattering precludes formation of high-definition image (unless complex techniques are employed), low resolution pictures complemented by information on the functional condition of the living sample can be extracted. In this context, we have investigated a portable optoelectronic instrumental configuration for efficient transillumination and image detection, even in ambient day-light, of in vivo samples with thickness up to 5 cm, sufficient for visualizing macroscopic structures. Tissue illumination is obtained with an extended source consisting in a matrix of 36 near infrared Vertical Cavity Surface Emitting Lasers (VCSELs) that is powered by a custom designed low-voltage current driver. In addition to the successful acquisition of morphological images of the hand dorsal vein pattern, functional detection of physiological parameters (breath and hearth rate) is achieved non-invasively by means of a monochrome camera, with a Complementary Metal Oxide Semiconductor (CMOS) sensor, turned into a wavelength selective image detector using narrow-band optical filtering.

This review examines whether similarity between the first language (L1) and second language (L2) influences the (morpho)syntactic processing of the L2, using both neural location and temporal processing information. Results from functional magnetic resonance imaging (fMRI) and event-related potential (ERP) studies show that nonnative speakers can exhibit nativelike online L2 (morpho)syntactic processing behavior and neural patterns. These findings are contrary to predictions of the shallow structure hypothesis for syntactic processing (Clahsen & Felser, 2006a, 2006b). The data are in line with predictions of the (morpho)syntactic domain of the unified competition model of L2 acquisition (MacWhinney, 2005): Differences in L2 processing as compared to the L1 (or to native speakers of the L2) were generally associated with constructions that were crosslinguistically dissimilar or unique to the L2. The processing of crosslinguistically similar constructions generally produced no differences in brain activity between the L1 and L2. Overall, the available data suggest that cross-language similarity is an important factor that influences L2 (morpho)syntactic processing.

The aim of this study was to identify new disease-related circulating miRNAs with high diagnostic accuracy for breast cancer (BC) and to correlate their deregulation with the morpho-functional characteristics of the tumour, as assessed in vivo by positron emission tomography/magnetic resonance (PET/MR) imaging. A total of 77 untreated female BC patients underwent same-day PET/MR and blood collection, and 78 healthy donors were recruited as negative controls. The expression profile of 84 human miRNAs was screened by using miRNA PCR arrays and validated by real-time PCR. The validated miRNAs were correlated with the quantitative imaging parameters extracted from the primary BC samples. Circulating miR-125b-5p and miR-143-3p were upregulated in BC plasma and able to discriminate BC patients from healthy subjects (miR-125-5p area under the receiver operating characteristic ROC curve (AUC) = 0.85 and miR-143-3p AUC = 0.80). Circulating CA15-3, a soluble form of the transmembrane glycoprotein Mucin 1 (MUC-1) that is upregulated in epithelial cancer cells of different origins, was combined with miR-125b-5p and improved the diagnostic accuracy from 70% (CA15-3 alone) to 89% (CA15-3 plus miR-125b-5p). MiR-143-3p showed a strong and significant correlation with the stage of the disease, apparent diffusion coefficient (ADCmean), reverse efflux volume transfer constant (Kepmean) and maximum standardized uptake value (SUVmax), and it might represent a biomarker of tumour aggressiveness. Similarly, miR-125b-5p was correlated with stage and grade 2 but inversely correlated with the forward volume transfer constant (Ktransmean) and proliferation index (Ki67), suggesting a potential role as a biomarker of a relatively more favourable prognosis. In situ hybridization (ISH) experiments revealed that miR-143-3p was expressed in endothelial tumour cells, miR-125-5p in cancer-associated fibroblasts, and neither in epithelial tumour cells. Our results suggested that miR-125-5p and miR-143-3p are potential biomarkers for the risk stratification of BC, and Kaplan-Maier plots confirmed this hypothesis. In addition, the combined use of miR-125-b-5p and CA15-3 enhanced the diagnostic accuracy up to 89%. This is the first study that correlates circulating miRNAs with in vivo quantified tumour biology through PET/MR biomarkers. This integration elucidates the link between the plasmatic increase in these two potential circulating biomarkers and the biology of untreated BC. In conclusion, while miR-143-3b and miR-125b-5p provide valuable information for prognosis, a combination of miR-125b-5p with the tumour marker CA15-3 improves sensitivity for BC detection, which warrants consideration by further validation studies.

Over the last decades, significant advances have been achieved in the treatment of coronary artery disease (CAD). Proper non-invasive diagnosis and appropriate management based on functional information and the extension of ischemia or viability remain the cornerstone in the fight against adverse CAD events. Stress echocardiography and single photon emission computed tomography are often used for the evaluation of ischemia. Advancements in non-invasive imaging modalities such as computed tomography (CT) coronary angiography and cardiac magnetic resonance imaging (MRI) have not only allowed non-invasive imaging of coronary artery lumen but also provide additional functional information. Other characteristics regarding the plaque morphology can be further evaluated with the latest modalities achieving a morpho-functional evaluation of CAD. Advances in the utilization of positron emission tomography (PET), as well as software advancements especially regarding cardiac CT, may provide additional prognostic information to a more evidence-based treatment decision. Since the armamentarium on non-invasive imaging modalities has evolved, the knowledge of the capabilities and limitations of each imaging modality should be evaluated in a case-by-case basis to achieve the best diagnosis and treatment decision. In this review article, we present the most recent advances in the noninvasive anatomical and functional evaluation of CAD.

The large use of nonlinear laser scanning microscopy in the past decade paved the way for potential clinical application of this imaging technique. Modern nonlinear microscopy techniques offer promising label-free solutions to improve diagnostic performances on tissues. In particular, the combination of multiple nonlinear imaging techniques in the same microscope allows integrating morphological with functional information in a morpho-functional scheme. Such approach provides a high-resolution label-free alternative to both histological and immunohistochemical examination of tissues and is becoming increasingly popular among the clinical community. Nevertheless, several technical improvements, including automatic scanning and image analysis, are required before the technique represents a standard diagnostic method. In this review paper, we highlight the capabilities of multimodal nonlinear microscopy for tissue imaging, by providing various examples on colon, arterial and skin tissues. The comparison between images acquired using multimodal nonlinear microscopy and histology shows a good agreement between the two methods. The results demonstrate that multimodal nonlinear microscopy is a powerful label-free alternative to standard histopathological methods and has the potential to find a stable place in the clinical setting in the near future.

Metabolism is the ensemble of biochemical processes allowing a cell to live and build all of its constituents, exert its functions and cooperate to determine, in multicellular organisms, the functions of tissues and organs. Different tissues are characterized by metabolic processes that can differ both qualitatively (for example the type of substrates degraded to obtain chemical energy) and quantitatively (depending on the state of activity). Thus, metabolism is modulated during development and, in adult life, as a function of the level of activity of the whole animal and of each organ. Alterations of the regulation of metabolism can lead to pathologies and a remarkable change in the metabolic asset is characteristic also of cancer cells. At the whole animal level, the measurement of metabolism is largely based on the consumption of oxygen and the production of carbon dioxide; these measurements provide a course indication on the rate of metabolism and allow correlation with physiological or pathological conditions. A more detailed measurement of metabolism, at the cellular level, would provide key insights in the processes of differentiation during embryo development, and possibly clarify the mechanisms of several pathologies. Thus, methods enabling a real time in vivo measurement of metabolism in the whole animal are highly desirable. A fundamental organelle involved in the metabolic activities of the cell is the mitochondrion. Here, ATP is produced with consumption of oxygen and the involvement of coenzymes which are among the most important components contributing to a cell’s autofluorescence. The signal of these molecules (NADH and FAD), as it will be shown in this thesis, represents a great tool to measure metabolism in vivo with cellular resolution. Most of inhaled oxygen is used by mitochondria. Through oxidative phosphorylation, mitochondria oxidize the substrates of the Krebs cycle and produce adenosine triphosphate (ATP) which is the molecule that powers most of the cell's activities that require a source of energy. An increasing number of studies demonstrated that a wide number of human diseases are due to mitochondrial dysfunctions. Mitochondrial diseases are a large group of pathologies associated with defects in mitochondrial energy metabolism, mainly due to anomalies in the mitochondrial respiratory chain and/or in oxidative phosphorylation (OXPHOS)[1]. Oxidative phosphorylation deficiency can cause dysfunctions in the respiratory chain, a heteromultimeric structure embedded in the inner mitochondrial membrane, or it can be associated to a single or multiple defects of the five complexes forming the respiratory chain itself [2]. During my PhD work I tested if it was possible to perform in vivo morpho-functional imaging of zebrafish larvae tissues and organs through nonlinear optical (NLO) microscopy that offers the advantage to obtain images from tissue and organs in vivo without requiring exogenous stains or tissue excision. Moreover, a goal of the thesis has been to setup methods to employ NLO microscopy for characterizing not only the morphology of a tissue but also its metabolism and functionality. NLO microscopy is a high resolution laser scanning imaging technique that allows obtaining images with a good penetration depth into tissues. Cells and extracellular matrix of biological tissues contain a variety of intrinsically fluorescent molecules (NADH, FAD, tryptophan, keratin, melanin, elastin, cholecalciferol and others). These molecules, and their interactions with the surrounding environment, can provide a tool for measuring variations of metabolism and of the cellular and extracellular environmental conditions. One of the parameters commonly utilized to study the mitochondrial health and functions is the redox ratio [1], i.e. the NADH/FAD ratio, which allows obtaining information of the cell’s metabolic activity. In fact, Nicotinamide Adenine Dinucleotide (NADH) transfers electrons to the electron transport chain (ETC) while Flavin Adenine Dinucleotide (FAD) acts as an intermediary acceptor of electrons in the ETC. For this reason, it was decided to take advantage of the endogenous signal produced by these molecules to obtain information on metabolic state using nonlinear microscopy without having to add exogenous probes. The redox ratio can be obtained through the measurement of NADH fluorescence intensity, divided by the FAD fluorescence intensity. As stated above, this ratio is sensitive to changes in the cellular metabolic rate and vascular oxygen supply [1, 3]. An increase in the NADH/FAD redox ratio usually indicates increased cellular metabolic activity [4] and an increase of glycolytic state. The NADH and FAD fluorescence are reliable biomarkers reflecting the mitochondria functions. A complementary method to optically assess cellular metabolism based on NADH fluorescence relies fluorescence lifetime measurements. Combining TPEF and fluorescence-lifetime imaging microscopy (FLIM), it is possible to obtain more information about the molecular microenvironment of a fluorescent molecule. In the case of NADH, this method provides a tool to discriminate free and protein-bound components of NADH. In fact, NADH not bound to proteins has a short lifetime (around 0.3ns), whereas when it is bound to proteins it has long lifetime (2ns – 4.5ns). Therefore, in this work I also developed methods for the in vivo measurement of NADH fluorescence lifetime in zebrafish larvae and compared these measurements with those bases on intensity NADH/FAD ratios for different experimental conditions. To characterize the spectroscopic techniques developed, the zebrafish larvae were treated with compounds known for their effects on metabolism (Rotenone) and metabolic signaling pathways (DMOG). Another test of the method was performed with a transient know-down of the mitochondrial DNA Polymerase by use of morpholinos antisense oligos; in this study it has been used two kinds of morpholinos: splicing morpholinos that is zygotic-specific interfering with the maturation of a primary transcript of the mitochondrial DNA Polymerase; ATG morpholinos that not only interfering with the maturation of a primary transcript but also with mature maternal mRNAs of the mitochondrial DNA Polymerase; (thus inducing a decrease if the density of mitochondria in the developing embryo) In the following sections, the thesis will provide: - a discussion on mitochondrial diseases and different methods that reduce the activity of oxidative phosphorylation or activating the HIF signaling, that mimics the lack of oxygen, and the interference of MOpolG (morpholino against the transcription of mitochondrial DNA polymerase) on mitochondria replication in zebrabrafish larvae; moreover, the physical theories of nonlinear optics, in particular Two-Photon Excited Fluorescence (TPEF) and Fluorescence Lifetime Imaging Microscopy (FLIM) (chapter 1); - a description of the housing and care of zebrafish, the procedures adopted to characterize and calibrate both experimental setups, and the methods applied for data analysis (chapter 2); - a report on the results obtained from each study (chapter 3) - Conclusions (chapter 4).

Medical infrared imaging captures the temperature distribution of the human skin and is employed in various medical applications. Unfortunately, many of the conventional and commercial suites for image processing provide only very basic tools for the processing of medical thermal images which represent a challenging combination of both functional and morpho-structural imaging. In this chapter, several more advanced approaches are discussed which in turn provide tremendous help to the clinician. As an example, it is often useful to cross-reference thermograms with visual images of the patient, either to see which part of the anatomy is affected by a certain disease or to judge the efficacy of the treatment. It is shown that image registration techniques can be effectively used to generate an overlay of visual and thermal images to provide a useful diagnostic visualisation. Image registration can also be performed based on two thermograms and a warping-based method for this is presented. Segmenting the background from the foreground (i.e., the patient) is a crucial task and it is highlighted how this can be accomplished. Finally, it is shown how descriptors, extracted from medical infrared images, can be usefully employed to search through a large database of cases as well as to aid in diagnosis.