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Large volume multicolor nonlinear microscopy of neural tissues

Abstract : 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.
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  • HAL Id : tel-02299759, version 2


Lamiae Abdeladim. Large volume multicolor nonlinear microscopy of neural tissues. Optics [physics.optics]. Université Paris Saclay (COmUE), 2018. English. ⟨NNT : 2018SACLX070⟩. ⟨tel-02299759v2⟩



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