The challenge of mapping the human connectome based on diffusion tractography

Tractography based on non-invasive diffusion imaging is central to the study of human brain connectivity. To date, the approach has not been systematically validated in ground truth studies. Based on a simulated human brain data set with ground truth tracts, we organized an open international tractography challenge, which resulted in 96 distinct submissions from 20 research groups. Here, we report the encouraging finding that most state-of-the-art algorithms produce tractograms containing 90% of the ground truth bundles (to at least some extent). However, the same tractograms contain many more invalid than valid bundles, and half of these invalid bundles occur systematically across research groups. Taken together, our results demonstrate and confirm fundamental ambiguities inherent in tract reconstruction based on orientation information alone, which need to be considered when interpreting tractography and connectivity results. Our approach provides a novel framework for estimating reliability of tractography and encourages innovation to address its current limitations

Microscopie de Génération de Seconde Harmonique Multimodale et Interférométriquepour une Caractérisation Améliorée des Biopolymères dans les Cellules et Tissus

Multiphoton microscopy is a paramount paradigm for tissue imaging and characterization in biology,and more generally in material sciences. Within it, second harmonic generation (SHG) has become thegold standard for in-situ 3D visualization of tissues containing a widespread biopolymer: fibrillar collagen.SHG’s intrinsic properties, being the result of a highly coherent exciting wave being inelastically scattered,allows these types of materials to be specifically revealed, among which are also myosin in muscles, andvarious nonlinear crystals.However, the optical coherence of SHG also means that the images contain the result of complexinterferences, and not the actual structure of the material. Partly because of this problem, manytechniques have been proposed to enhance SHG, and to fully benefit of its numerous properties: thedirection of generation can be probed, its sensitivity to polarization, or its relative phase.Interferometric Second-Harmonic Generation microscopy (I-SHG), and its application to biologicaltissues, have allowed for the relative phase to be measured. This technique has been increasingly bettercontrolled, but lacked a real incorporation with other multiphoton techniques. It was also quite long andcomplicated to use, and measured only the phase, without taking advantage of other parameters.This dissertation first portrays the general context around SHG, before detailing the process itself.Directional and polarization-resolved SHG are then presented and applied to the analysis of a complexcollagen-rich tissue: the equine meniscus in a joint. A property of Gaussian beams being focused, theGouy phase-shift, which explains some SHG imaging artifacts in stacked structures, is then reported tobe measurable with I-SHG’s phase retrieval. Afterwards, I-SHG was used to decouple these artefactualinterferences from the real structure of samples containing alternating polarities, while also enhancingthe structure’s visibility.I-SHG was subsequently made compatible with laser-scanning schemes, which greatly enhanced itsspeed. This was applied to in-situ imaging of the microtubules’ polarity during an embryo mitosis.Because this "fast I-SHG" still presented some experimental latencies, a single-scan paradigm (1S-ISHG)was implemented, using an electro-optic modulator that changes the relative phase of the interferogramswithin the conventional pixels of the image. The complete optical, hardware and software controlsrequired for these improvements are detailed as well.This one order of magnitude speed enhancement remains to be utilized to characterize dynamicprocesses requiring an imaging speed below the Hz scale, or for large-scale studies. Menisci could alsobe further investigated in multimodal microscopy coupled to I-SHG.La microscopie multiphoton est primordiale pour l’imagerie des tissus biologiques et de certains matériaux.La Génération de Seconde Harmonique (SHG) est en particulier une référence pour l’imagerie 3Ddes tissus contenant un biopolymère très répandu: le collagène. Les propriétés intrinsèques de la SHG,qui résulte de la diffusion inélastique d’une onde excitatrice très cohérente, permettent de révéler spécifiquementce type de structures, parmi lesquelles figurent aussi la myosine des muscles, ou certainscristaux non-linéaires.Cependant, la cohérence optique de la SHG signifie également que les images contiennent la résultanted’interférences complexes, et non la structure réelle du matériau. Pour cela, de nombreuses techniquesont été proposées pour améliorer la SHG, et tirer pleinement parti de ses nombreuses propriétés: ladirection de la génération, sa sensibilité à la polarisation ou sa phase relative peuvent être sondées. Lamicroscopie de Génération de Seconde Harmonique Interférométrique (I-SHG), et son application auxtissus biologiques, a permis de mesurer la phase. Cette technique a été de mieux en mieux maîtrisée,mais sans être réellement intégrée à d’autres techniques multiphoton. Elle était aussi assez longue etcompliquée à utiliser, et ne mesurait que la phase, sans tirer parti d’autres paramètres.Cette thèse présente d’abord le contexte général autour de la SHG, avant de détailler cette dernière.La SHG directionnelle et résolus en polarisation sont ensuite utilisées pour l’analyse du collagène d’untissu complexe: le ménisque des articulations équines. Une propriété des faisceaux Gaussiens subissantune focalisation, le déphasage de Gouy, est ensuite démontrée mesurable par I-SHG. Cette dernièreexplique notamment certains artefacts d’imagerie par SHG dans des structures en empilement. Ensuite,ces interférences artéfactuelles sont découplées par I-SHG de la structure réelle dans des échantillonscontenant des alternances de polarité, ce qui produit aussi une meilleure visibilité de la structure.La I-SHG est ensuite rendue compatible avec un balayage laser, ce qui améliore considérablementsa vitesse. Ceci est appliqué à l’imagerie in situ de la polarité des microtubules au cours d’une mitoseembryonnaire. Puisque cette "I-SHG rapide" présentait encore quelques latences expérimentales, ellea été ensuite réduite à un seul balayage de l’échantillon (1S-ISHG), via un modulateur électro-optiquequi déphase les interférogrammes au sein même des pixels de l’image. Le contrôle optique, matériel etlogiciel nécessaire à ces améliorations sont également détaillés.Cette amélioration de la vitesse (un ordre de grandeur) pourra servir à caractériser des processus dynamiques,ou pour des études à grande échelle. Le ménisque pourra également bénéficier d’un couplagemicroscopie multimodale - ISHG

Fast interferometric second harmonic generation microscopy

We report the implementation of fast Interferometric Second Harmonic Generation (I-SHG) microscopy to study the polarity of non-centrosymmetric structures in biological tissues. Using a sample quartz plate, we calibrate the spatially varying phase shift introduced by the laser scanning system. Compensating this phase shift allows us to retrieve the correct phase distribution in periodically poled lithium niobate, used as a model sample. Finally, we used fast interferometric second harmonic generation microscopy to acquire phase images in tendon. Our results show that the method exposed here, using a laser scanning system, allows to recover the polarity of collagen fibrils, similarly to standard I-SHG (using a sample scanning system), but with an imaging time about 40 times shorter. OCIS codes: (180.4315) Nonlinear microscopy, (190.2620) Harmonic generation and mixing, (170.6935) Tissue characterization, (190.4180) Multiphoton processes, (190.4710) Optical nonlinearities in organic material

Maturation of the Meniscal Collagen Structure Revealed by Polarization-Resolved and Directional Second Harmonic Generation Microscopy

International audienceWe report polarization-resolved Second Harmonic Generation (p-SHG) and directional SHG (forward and backward, F/B) measurements of equine foetal and adult collagen in meniscus, over large field-of-views using sample-scanning. Large differences of collagen structure and fibril orientation with maturation are revealed, validating the potential for this novel methodology to track such changes in meniscal structure. The foetal menisci had a non-organized and more random collagen fibrillar structure when compared with adult using P-SHG. For the latter, clusters of homogeneous fibril orientation (inter-fibrillar areas) were revealed, separated by thick fibers. F/B SHG showed numerous different features in adults notably, in thick fibers compared to interfibrillar areas, unlike foetal menisci that showed similar patterns for both directions. This work confirms previous studies and improves the understanding of meniscal collagen structure and its maturation, and makes f/B and p-SHG good candidates for future studies aiming at revealing structural modifications to meniscus due to pathologies. The meniscus is a semilunar fibrocartilaginous structure interposed between the femoral condyle and the tibial plateau in the knee joint. The meniscus is essential for load transmission across the articular surfaces, for femo-rotibial joint stability and for long-term joint health 1. Degradation of the meniscal tissue can increase articular cartilage strain 2 , and may lead to cartilage degeneration and osteoarthritis 3. Knowledge of the complex structure of the meniscal extracellular matrix (ECM) has increased thanks to emerging technologies for in situ imaging of intact specimens, such as Optical Projection Tomography (OPT) 4. In particular the arrangement of meniscal fascicles 4 , its tie-fiber organization 5 , and the menisco-tibial ligament insertion transition have all recently been revealed by investigation of bovine samples 6. SHG microscopy is a recent and powerful technique to image the structure of biological specimens as it provides submicron spatial resolution, has low phototoxicity and a high depth selectivity and penetration. In this respect, SHG imaging is similar to multiphoton-excited fluorescence microscopy 7. However, important differences exist: it is a coherent process sensitive to the phase-matching conditions where the measured signal arises from constructive/destructive interferences, it is also instantaneous and free from photobleaching as the signal conversion is due to a structural arrangement and does not involve electronic transition 8. SHG micros-copy has been used to image fibrillar collagen in specimens including type II collagen in articular cartilage 9-16. Furthermore, because of its coherent nature, the detection of the signal in the direction of propagation (forward-F) provides different imaging features compared to the backward (B) direction 17. The F/B ratio increases with the level of homogeneity of noncentrosymmetric structures within the focal volume and has been related to the size of the collagen fibrils for collagen rich tissues 18,19

Differences between foetal and adult meniscus and cartilage revealed by Polarization Second Harmonic Generation Microscopy

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Removing artifacts in Second Harmonic Generation imaging by interferometry

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Elimination of imaging artifacts in second harmonic generation microscopy using interferometry

International audienceConventional second harmonic generation (SHG) microscopy might not clearly reveal the structure of complex samples if the interference between all scatterers in the focal volume results in artefactual patterns. We report here the use of interferometric second harmonic generation (I-SHG) microscopy to efficiently remove these artifacts from SHG images. Interfaces between two regions of opposite polarity are considered because they are known to produce imaging artifacts in muscle for instance. As a model system, such interfaces are first studied in periodically-poled lithium niobate (PPLN), where an artefactual incoherent SH signal is obtained because of irregularities at the interfaces, that overshadow the sought-after coherent contribution. Using I-SHG allows to remove the incoherent part completely without any spatial filtering. Second, I-SHG is also proven to resolve the doubleband pattern expected in muscle where standard SHG exhibits in some regions artefactual single-band patterns. In addition to removing the artifacts at the interfaces between antiparallel domains in both structures (PPLN and muscle), I-SHG also increases their visibility by up to a factor of 5. This demonstrates that I-SHG is a powerful technique to image biological samples at enhanced contrast while suppressing artifacts

Microwave-optical fiber lasers stabilized by frequency-shifted feedback

International audienceDual-frequency fiber lasers (DFFL) are shown to provide high purity beat notes for microwave signal generation and distribution. We study the stabilization of the beat from DFB DFFLs by using optical frequency-shifted feedback loop containing an electro-optic modulator (EOM). As a proof-of-principle of the method efficiency, a stabilized beat note with a phase noise level of -104 dBc/Hz at 1 kHz from a 1 GHz carrier, and of -90 dBc/Hz at 1 kHz from a 10 GHz carrier, is demonstrated when the EOM is driven by a synthesizer. Furthermore, the scheme is extended to a self-referenced scheme: a hybrid opto-electronic oscillator is obtained when the delayed DFFL beat note itself drives the EOM. Low-frequency phase noise is reduced by about 20 dB. Applications are discussed

Circular dichroism second-harmonic generation microscopy probes the polarity distribution of collagen fibrils

International audienceSecond-harmonic generation (SHG) microscopy is currently the preferred technique for visualizing collagen in intact tissues, but the usual implementations struggle to reveal collagen fibrils oriented out of the imaging plane. Recently, an advanced SHG modality, circular dichroism SHG (CD-SHG), has been proposed to specifically highlight out-of-plane fibrils. In this study, we present a theoretical analysis of CD-SHG signals that goes beyond the electric dipolar approximation to account for collagen chirality. We demonstrate that magnetic dipolar contributions are necessary to analyze CD-SHG images of human cornea sections and other collagen-rich samples. We show that the sign of CD-SHG signals does not reveal whether collagen fibrils point upwards or downwards as tentatively proposed previously. CD-SHG instead probes the polarity distribution of out-of-plane fibril assemblies at submicrometer scale, namely homogeneous polarity versus a mix of antiparallel fibrils. This makes CD-SHG a powerful tool for characterizing collagen organization in tissues, specifically the degree of disorder, which is affected during pathological remodeling. CD-SHG may thus serve to discriminate healthy and diseased collagen-rich tissues