Color Imaging with Multimodal Three-Photon Microscopy

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Microscopie non linéaire multicolore de grands volumes de tissu cérébral

Multiphoton microscopy has transformed neurobiology since the 1990s by enabling 3D imaging of thick tissues at subcellular resolution. However the depths provided by multiphoton microscopy are limited to a few hundreds of micrometers inside scattering tissues such as the brain. In the recent years, several strategies have emerged to overcome this depth limitation and to access larger volumes of tissue. Although these novel approaches are transforming brain imaging, they currently lack efficient multicolor and multicontrast modalities. This work aims at developing large-scale and deep-tissue multiphoton imaging modalities with augmented contrast capabilities. In a first chapter, we present the challenges of high-content large-volume brain imaging, with a particular emphasis on powerful multicolor labeling strategies which have so far been restricted to limited scales. We then introduce chromatic serial multiphoton (Chrom-SMP) microscopy, a method which combines automated histology with multicolor two-photon excitation through wavelength-mixing to access multiple nonlinear contrasts across large volumes, from several mm3 to whole brains, with submicron resolution and intrinsic channel registration. In a third chapter, we explore the potential of this novel approach to open novel experimental paradigms in neurobiological studies. In particular, we demonstrate multicolor volumetric histology of several mm3 of Brainbow-labeled tissues with preserved diffraction-limited resolution and illustrate the strengths of this method through color-based tridimensional analysis of astrocyte morphology, interactions and lineage in the mouse cerebral cortex. We further illustrate the potential of the method through multiplexed whole-brain mapping of axonal projections labeled with distinct tracers. Finally, we develop multimodal three-photon microscopy as a method to access larger depths in live settings.La microscopie non linéaire a transformé le domaine de la neurobiologie depuis les années 1990, en permettant d'acquérir des images tridimensionnelles de tissus épais avec une résolution subcellulaire. Cependant, les profondeurs d'imagerie accessibles sont limitées à quelques centaines de micromètres dans des tissus diffusants tels que le tissu cérébral. Au cours des dernières années, plusieurs stratégies ont été développées pour dépasser cette limitation de profondeur et accéder à de plus grands volumes de tissu. Ces avancées récentes ont jusqu'à présent été limitées en terme de modes de contrastes accessibles, et ont souvent été réduites à des approches monochromes. Ce travail de thèse vise à développer des techniques d'imagerie non linéaires de grands volumes et de grande profondeur dotées de diverses possibilités de contrastes, indispensables pour l'étude de tissus complexes tels que le tissu cérébral. Dans un premier chapitre, nous présentons les difficultés associées à l'imagerie de grand volume de tissu cérébral, avec une emphase particulière sur les puissantes stratégies de marquages génétiques dont l'usage à jusqu'à présent été limité à des faibles étendues. Ensuite, nous introduisons la microscopie Chrom-SMP (chromatic serial multiphoton), une méthode développée au cours de cette thèse et consistant à combiner l’excitation deux-photon multicouleurs par mélange de fréquences avec une technique d'histologie automatisée (i.e découpe sériée) pour accéder à plusieurs contrastes non linéaires à travers de grands volumes de tissus ex vivo, allant de plusieurs mm3 à des cerveaux entiers, avec une résolution micrométrique et un coalignement intrinsèque des canaux spectraux. Dans un troisième chapitre, nous explorons le potentiel de cette nouvelle approche pour la neurobiologie. En particulier, nous démontrons l'histologie multicouleur de plusieurs mm3 de tissu "Brainbow" avec une résolution constante dans l’ensemble du volume imagé. Nous illustrons le potentiel de notre approche à travers l'analyse de la morphologie, des interactions et du lignage des astrocytes du cortex cérébral de souris. Nous explorons également l’apport du Chrom-SMP pour le suivi multiplexé de projections neuronales marquées par des traceurs de couleurs distinctes sur de grandes distances. Enfin, nous présentons dans un quatrième chapitre le développement de la microscopie à trois photons multimodale, approche permettant d’augmenter la profondeur d’imagerie sur tissus vivants

Probing neural codes with two-photon holographic optogenetics

Optogenetics ushered in a revolution in how neuroscientists interrogate brain function. Because of technical limitations, the majority of optogenetic studies have used low spatial resolution activation schemes that limit the types of perturbations that can be made. However, neural activity manipulations at finer spatial scales are likely to be important to more fully understand neural computation. Spatially precise multiphoton holographic optogenetics promises to address this challenge and opens up many new classes of experiments that were not previously possible. More specifically, by offering the ability to recreate extremely specific neural activity patterns in both space and time in functionally defined ensembles of neurons, multiphoton holographic optogenetics could allow neuroscientists to reveal fundamental aspects of the neural codes for sensation, cognition and behavior that have been beyond reach. This Review summarizes recent advances in multiphoton holographic optogenetics that substantially expand its capabilities, highlights outstanding technical challenges and provides an overview of the classes of experiments it can execute to test and validate key theoretical models of brain function. Multiphoton holographic optogenetics could substantially accelerate the pace of neuroscience discovery by helping to close the loop between experimental and theoretical neuroscience, leading to fundamental new insights into nervous system function and disorder

Fast in vivo multiphoton light-sheet microscopy with optimal pulse frequency

International audienceImproving the imaging speed of multiphoton microscopy is an active research field. Among recent strategies, light-sheet illumination holds distinctive advantages for achieving fast imaging in vivo. However, photoperturbation in multiphoton light-sheet microscopy remains poorly investigated. We show here that the heart beat rate of zebrafish embryos is a sensitive probe of linear and nonlinear photoperturbations. By analyzing its behavior with respect to laser power, pulse frequency and wavelength, we derive guidelines to find the best balance between signal and photoperturbation. We then demonstrate one order-of-magnitude signal enhancement over previous implementations by optimizing the laser pulse frequency. These results open new opportunities for fast live tissue imaging

Fast in vivo multiphoton microscopy using optimized light-sheet illumination

International audienceLight-sheet fluorescence microscopy is a method of choice for multiscale live imaging. Indeed, its orthogonal geometry results in high acquisition speed, large field-of-view and low photodamage. Its combination with multiphoton excited fluorescence improves its imaging depth in biological tissues. However, it appears femtosecond laser sources commonly used in multiphoton microscopy at an 80 MHz repetition rate may not be optimized to take full advantage of light-sheet illumination during live imaging. Hence, we investigated the nature of induced photodamage in multiphoton light-sheet microscopy and the influence of laser parameters on the signal-to-photodamage ratio. To this end, we used zebrafish embryonic heart beat rate and fluorophore photobleaching as sensitive reporters of photoperturbations. We characterized linear and nonlinear disruptions depending on laser parameters such as laser mean power, pulse frequency or wavelength, and determine their order and relative impact. We found an optimal pulse frequency of ~10 MHz for imaging mCherry labeled beating hearts at 1030 nm excitation wavelength. Thus, we achieved high-speed imaging without inducing additional linear heating or reaching nonlinear photodamage compared to previous implementation. We reach an order-ofmagnitude enhancement in two-photon excited fluorescence signal by optimizing the laser pulse frequency while maintaining low both the laser average power and its peak irradiance. It is possible to reach even larger enhancement of 3photon excited fluorescence using such laser parameters. More generally, using low laser pulse frequency in multiphoton light-sheet microscopy results in a drastic improvement in signal level without compromising live sample, which opens new opportunities for fast in vivo imaging

Balancing signal and photoperturbation in multiphoton light-sheet microscopy by optimizing laser pulse frequency

Improving the imaging speed of multiphoton microscopy is an active research field. Among recent strategies, light-sheet illumination holds distinctive advantages for achieving fast imaging in vivo. However, photoperturbation in multiphoton light-sheet microscopy remains poorly investigated. We show here that the heart beat rate of zebrafish embryos is a sensitive probe of linear and nonlinear photoperturbations. By analyzing its behavior with respect to laser power, pulse frequency and wavelength, we derive guidelines to balance signal and photoperturbation. We then demonstrate one order-of-magnitude signal enhancement over previous implementations by optimizing the laser pulse frequency. These results open new opportunities for fast live tissue imaging

Three-photon microscopy with a monolithic all-fiber laser format laser emitting at 1650 nm

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High-repetition-rate ultrafast source emitting concomitantly in the 1.3 and 1.7 μm SWIR water-transparency bands for dual-color 3-photon microscopy

International audienceWe present a novel ultrafast laser design providing simultaneous excitation in the two water-transparency windows at 1.3 and 1.7 µm, and we demonstrate its potentiality for efficient 3-photon microscopy of nervous tissues

Cortical astrocytes develop in a plastic manner at both clonal and cellular levels

International audienceAstrocytes play essential roles in the neural tissue where they form a continuous network, while displaying important local heterogeneity. Here, we performed multiclonal lineage tracing using combinatorial genetic markers together with a new large volume color imaging approach to study astrocyte development in the mouse cortex. We show that cortical astrocyte clones intermix with their neighbors and display extensive variability in terms of spatial organization, number and subtypes of cells generated. Clones develop through 3D spatial dispersion, while at the individual level astrocytes acquire progressively their complex morphology. Furthermore, we find that the astroglial network is supplied both before and after birth by ventricular progenitors that scatter in the neocortex and can give rise to protoplasmic as well as pial astrocyte subtypes. Altogether, these data suggest a model in which astrocyte precursors colonize the neocortex perinatally in a non-ordered manner, with local environment likely determining astrocyte clonal expansion and final morphotype

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

International audienceProtoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines