title: Nonlinear microscopy of chitin and chitinous structures: a case study of two cave-dwelling insects.
abstract: We performed a study of the nonlinear optical properties of chemically purified chitin and insect cuticle using two-photon excited autofluorescence (TPEF) and second-harmonic generation (SHG) microscopy. Excitation spectrum, fluorescence time, polarization sensitivity, and bleaching speed were measured. We have found that the maximum autofluorescence signal requires an excitation wavelength below 850 nm. At longer wavelengths, we were able to penetrate more than 150-um deep into the sample through the chitinous structures. The excitation power was kept below 10 mW (at the sample) in order to diminish bleaching. The SHG from the purified chitin was confirmed by spectral- and time-resolved measurements. Two cave-dwelling, depigmented, insect species were analyzed and three-dimensional images of the cuticular structures were obtained.
authors.affiliation: University of Belgrade, Institute of Physics, Pregrevica 118, 11080 Zemun, Belgrade, Serbia.
authors.forename: Mihailo D
authors.initials: MD
authors.lastname: Rabasović
authors.affiliation: University of Belgrade, Institute of Physics, Pregrevica 118, 11080 Zemun, Belgrade, Serbia.
authors.forename: Dejan V
authors.initials: DV
authors.lastname: Pantelić
authors.affiliation: University of Belgrade, Institute of Physics, Pregrevica 118, 11080 Zemun, Belgrade, Serbia.
authors.forename: Branislav M
authors.initials: BM
authors.lastname: Jelenković
authors.affiliation: University of Belgrade-Faculty of Biology, Institute of Zoology, Studentski Trg 16, 11000 Belgrade, Serbia.
authors.forename: Srecko B
authors.initials: SB
authors.lastname: Ćurčić
authors.affiliation: University of Belgrade, Institute of Physics, Pregrevica 118, 11080 Zemun, Belgrade, Serbia.
authors.forename: Maja S
authors.initials: MS
authors.lastname: Rabasović
authors.affiliation: University of Belgrade-Faculty of Biology, Institute of Zoology, Studentski Trg 16, 11000 Belgrade, Serbia.
authors.forename: Maja D
authors.initials: MD
authors.lastname: Vrbica
authors.affiliation: University of Belgrade, Institute of Physics, Pregrevica 118, 11080 Zemun, Belgrade, Serbia.
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Lazovic
authors.affiliation: University of Belgrade-Faculty of Biology, Institute of Zoology, Studentski Trg 16, 11000 Belgrade, Serbia.
authors.forename: Božidar P M
authors.initials: BP
authors.lastname: Ćurčić
authors.affiliation: University of Belgrade, Institute of Physics, Pregrevica 118, 11080 Zemun, Belgrade, Serbia.
authors.forename: Aleksandar J
authors.initials: AJ
authors.lastname: Krmpot
mesh_terms: D000818:Animals; D001181:Arthropods / Q000737:chemistry; D059552:Caves; D002686:Chitin / Q000737:chemistry*; D001517:Coleoptera / Q000737:chemistry*; D004867:Equipment Design; D021621:Imaging, Three-Dimensional / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D017711:Nonlinear Dynamics
chemical_list: D002686:Chitin