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Colourfulommatidiaofbutterflyeyes 1079 DPP L1 L4 D2 L5 Photomicroscope L2 D1 L3 * tr rh fc Fig. 1. The optical apparatus used to photograph the eye shine of butterfly eyes. The objective lens L1 has a large aperture. A light source is focused by lens L2 in its back focal plane, where the field diaphragm D1 is positioned. D1 is in the focal plane of lens L3, which is confocal with L1 because of a half-mirror placed at 45 ° with respect to the optical axes of L1 and L3. A more-or-less parallel beam, depending on the size of D1, enters L1 and is focused on the deep pseudopupil (DPP) in the centre of the butterfly’s eye. The telescope lens pair L1 and L4 images the DPP in the back focal plane of L4, where diaphragm D2 is positioned. The image of the corneal eye shine, projected by lens L5, confocal with L4, is photographed by a photomicroscope. The dotted lines are the back-focal planes of L1 and L5. Inset: incident light entering a butterfly ommatidium is focused by the facet lens and crystalline cone (fc) into the rhabdom (rh) and then propagates to the tapetal reflector (tr), where it is mirrored back into the rhabdom and out of the eye again, unless it is absorbed by visual pigments in the rhabdom or by screening pigments in the medium surrounding the rhabdom. The rhabdom organization of a pierid butterfly is indicated schematically: the distal part of the rhabdom consists of the rhabdomeres of photoreceptors R1–R4, the proximal part consists of the rhabdomeres of photoreceptors R5–R8 and the most basal part consists only of rhabdomere R9, which is indicated by an asterisk (see Qiu et al., 2002). The rhabdom is surrounded by photoreceptor screening pigment that absorbs light from the propagating light wave. of diaphragms D1 and D2 must be adjusted so that they are slightly wider than the image of the DPP. The number of ommatidia contributing to the eye shine depends directly on the aperture of objective L1. A large number can be captured with a Leitz LM32 0.60 objective, which combines a high numerical aperture with a long working distance. L2–L5 are 80, 100, 80 and 15 mm Spindler and Hoyer (Goettingen, Germany) lenses, respectively. The photomicroscope, with a Zeiss 3.2 0.07 objective, is equipped with a Kodak DC120 digital camera. The actual experimental apparatus used in the present study has two epi-illumination beams supplied by a 50 W halogen lamp and a 100 W mercury lamp. The halogen lamp provided the white light source in Figs 2 and 3, and the mercury lamp was used in Fig. 3 for applying monochromatic light at 670 or 550 nm (via Schott DAL interference filters, half-width approximately 15 nm). Although stray light and unwanted reflections are largely eliminated, some reflection on the lens surfaces of the microscope objective remains, and this is visible as a central ‘hot spot’. Its prominence can be diminished by reducing the bandwidth of the illumination beam, as was done in Fig. 2B. A long-pass filter, >550nm, was used in that case since the eye shine had no components in the shorter wavelength range. The eye shine photographs were made from dark-adapted eyes. Exposures were shorter than 1 s so that contamination by the pupil mechanism (Stavenga et al., 1977) was circumvented. The exposures lasted a few seconds with the 670nm illumination (Fig. 3), but this long-wavelength light did not activate the pupil. The apparatus resembles the ophthalmoscopes developed for the analysis of the visual fields of fly eyes by Franceschini (1975) and van Hateren (1984); the main difference is the added epi-illumination arm. Land and Osorio (1990) used an ophthalmoscope with a slightly different design to investigate the spatial properties of butterfly eyes (Land, 1984). Reflectance spectra Reflectance spectra (see Fig. 4) were measured with a conventional epi-illumination microscope (Leitz Ortholux) equipped with a Leitz NPL10, 0.22 objective. The goniometer with butterfly was positioned on the stage of the microscope. The eye shine due to illumination with a broadband, white (150 W Xe) light source was measured from a single facet by adjusting a diaphragm in front of an Oriel diode array spectrophotometer attached to the microscope.PDF Image | Reflections on colourful ommatidia of butterfly eyes
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