Microscopies multiharmoniques pour l'imagerie structurale de tissus intacts [Second- and third-harmonic generation microscopies for the structural imaging of intact tissues]

Abstract : Depuis son introduction en 1990, la microscopie de fluorescence excitée à deux photons (Fluo-2P) s'est peu à peu imposée comme une méthode incontournable d'imagerie de tissus intacts à l'échelle sub-cellulaire. En effet, la caractéristique la plus remarquable de la microscopie multiphotonique est de maintenir une résolution tridimensionnelle micrométrique lors de l'observation en profondeur d'un milieu optiquement diffusant. Combinée aux technologies de protéines-fusion (type GFP), cette approche est aujourd'hui utilisée dans de nombreux domaines, notamment en neurophysiologie. Un autre attrait de ce type d'imagerie réside dans l'utilisation possible d'autres phénomènes optiques non linéaires (c'est-à-dire impliquant l'interaction simultanée de plusieurs photons avec une molécule observée) comme source de contraste. Ainsi, les microscopies par génération de second harmonique (GSH) et par génération de troisième harmonique (GTH) permettent également d'observer des milieux complexes et fournissent des informations complémentaires par rapport à l'imagerie de fluorescence. Certaines structures cellulaires ou tissulaires fournissent, en effet, ce type de réponse optique sans nécessiter de marquage exogène. La microscopie GSH permet, par exemple, de détecter le collagène fibrillaire et la microscopie GTH permet d'observer sans marquage le développement embryonnaire de petits organismes.

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy.
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Delphine Débarre, Ana-Maria Pena, Willy Supatto, Thierry Boulesteix, Mathias Strupler, et al.. Microscopies multiharmoniques pour l'imagerie structurale de tissus intacts [Second- and third-harmonic generation microscopies for the structural imaging of intact tissues]. médecine/sciences, EDP Sciences, 2006, 22 (10), pp.845. ⟨10.1051/medsci/20062210845⟩. ⟨hal-00827919⟩

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