title: Efficient second-harmonic imaging of collagen in histological slides using Bessel beam excitation.
abstract: Second-harmonic generation (SHG) is the most specific label-free indicator of collagen accumulation in widespread pathologies such as fibrosis, and SHG-based measurements hold important potential for biomedical analyses. However, efficient collagen SHG scoring in histological slides is hampered by the limited depth-of-field of usual nonlinear microscopes relying on focused Gaussian beam excitation. In this work we analyze theoretically and experimentally the use of Bessel beam excitation to address this issue. Focused Bessel beams can provide an axially extended excitation volume for nonlinear microscopy while preserving lateral resolution. We show that shaping the focal volume has consequences on signal level and scattering directionality in the case of coherent signals (such as SHG) which significantly differ from the case of incoherent signals (two-photon excited fluorescence, 2PEF). We demonstrate extended-depth SHG-2PEF imaging of fibrotic mouse kidney histological slides. Finally, we show that Bessel beam excitation combined with spatial filtering of the harmonic light in wave vector space can be used to probe collagen accumulation more efficiently than the usual Gaussian excitation scheme. These results open the way to SHG-based histological diagnoses.
authors.affiliation: Laboratory for optics and biosciences, Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128 Palaiseau cedex, France.
authors.forename: Nelly
authors.initials: N
authors.lastname: Vuillemin
authors.affiliation: Laboratory for optics and biosciences, Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128 Palaiseau cedex, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Mahou
authors.affiliation: Laboratory for optics and biosciences, Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128 Palaiseau cedex, France.|Laboratory of interdisciplinary physics, Université Joseph Fourier, CNRS, 38402 St Martin d'Hères, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Débarre
authors.affiliation: Laboratory of condensed matter physics, Ecole polytechnique, CNRS, Université Paris-Saclay, 91128 Palaiseau cedex, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Gacoin
authors.affiliation: Paris-Cardiovascular Research Centre, INSERM, European Georges Pompidou Hospital, 75015 Paris, France.
authors.forename: Pierre-Louis
authors.initials: PL
authors.lastname: Tharaux
authors.affiliation: Laboratory for optics and biosciences, Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128 Palaiseau cedex, France.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Schanne-Klein
authors.affiliation: Laboratory for optics and biosciences, Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128 Palaiseau cedex, France.
authors.forename: Willy
authors.initials: W
authors.lastname: Supatto
authors.affiliation: Laboratory for optics and biosciences, Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128 Palaiseau cedex, France.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Beaurepaire
mesh_terms: D000818:Animals; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D004195:Disease Models, Animal; D005355:Fibrosis / Q000000981:diagnostic imaging* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton; D017785:Photons
chemical_list: D003094:Collagen