Nonlinear optical spectroscopy and two-photon excited fluorescence spectroscopy reveal the excited states of fluorophores embedded in a beetle's elytra.

Upon illumination by ultraviolet light, many animal species emit light through fluorescence processes arising from fluorophores embedded within their biological tissues. Fluorescence studies in living organisms are however relatively scarce and so far limited to the linear regime. Multiphoton excitation fluorescence analyses as well as nonlinear optical techniques offer unique possibilities to investigate the effects of the local environment on the excited states of fluorophores. Herein, these techniques are applied for the first time to study of the naturally controlled fluorescence in insects. The case of the male Hoplia coerulea beetle is investigated because the scales covering the beetle's elytra are known to possess an internal photonic structure with embedded fluorophores, which controls both the beetle's coloration and the fluorescence emission. An intense two-photon excitation fluorescence signal is observed, the intensity of which changes upon contact with water. A third-harmonic generation signal is also detected, the intensity of which depends on the light polarization state. The analysis of these nonlinear optical and fluorescent responses unveils the multi-excited states character of the fluorophore molecules embedded in the beetle's elytra. The role of form anisotropy in the photonic structure, which causes additional tailoring of the beetle's optical responses, is demonstrated by circularly polarized light and nonlinear optical measurements.

Optically ambidextrous circularly polarized reflection from the chiral cuticle of the scarab beetle Chrysina resplendens.

The evolution of structural colour mechanisms in biological systems has given rise to many interesting optical effects in animals and plants. The instance of the scarab beetle Chrysina resplendens is particularly distinctive. Its exoskeleton has a bright, golden appearance and reflects both right-handed and left-handed circularly polarized light concurrently. The chiral nanostructure responsible for these properties is a helicoid, in which birefringent dielectric planes are assembled with an incremental rotation. This study correlates details of the beetle's circularly polarized reflectance spectra directly with physical aspects of its structural morphology. Electron micrography is used to identify and measure the physical dimensions of the key constituent components. These include a chiral multilayer configuration comprising two chirped, left-handed helicoids that are separated by a birefringent retarder. A scattering matrix technique is used to simulate the system's optical behaviour in which the roles of each component of the morphological substructure are elucidated by calculation of the fields throughout its depth.

Circularly polarized reflection from the scarab beetle Chalcothea smaragdina: light scattering by a dual photonic structure.

Helicoidal architectures comprising various polysaccharides, such as chitin and cellulose, have been reported in biological systems. In some cases, these architectures exhibit stunning optical properties analogous to ordered cholesteric liquid crystal phases. In this work, we characterize the circularly polarized reflectance and optical scattering from the cuticle of the beetle Chalcothea smaragdina (Coleoptera: Scarabaeidae: Cetoniinae) using optical experiments, simulations and structural analysis. The selective reflection of left-handed circularly polarized light is attributed to a Bouligand-type helicoidal morphology within the beetle's exocuticle. Using electron microscopy to inform electromagnetic simulations of this anisotropic stratified medium, the inextricable connection between the colour appearance of C. smaragdina and the periodicity of its helicoidal rotation is shown. A close agreement between the model and the measured reflectance spectra is obtained. In addition, the elytral surface of C. smaragdina possesses a blazed diffraction grating-like surface structure, which affects the diffuse appearance of the beetle's reflected colour, and therefore potentially enhances crypsis among the dense foliage of its rainforest habitat.