Background

The Drosophila compound eye is comprised of approximately 750 ommatidia that are packed into a remarkably ordered three-dimensional ommatidial array covered with transparent cuticles secreted by cone cells [1]. This layer scatters light, which makes it challenging to capture high-contrast eye images using a stereomicroscope equipped with a standard illumination system. Analysis of such images by a trained researcher or an automated image analysis system can only give an approximation with the possibility of misinterpretation [2,3]. Scanning electron microscopy (SEM) is a popular technique for high-resolution imaging of the eye, but it requires imaging skills and resources [4,5]. Furthermore, SEM images provide no information about pigmentation alteration in the adult fly eye. Nail polish imprint is another high-contrast imaging technique to image the molds of the fly eye, but this technique requires hand skill and does not provide pigmentation details [6,7].

To fix this problem, we tested different light sources, including a bar light, a gooseneck light, a diffused/straight backlight, and a ring light to illuminate the eye under a stereomicroscope. Images of bar- or gooseneck light–illuminated eyes had considerable glare with shaded regions (Figure 1A). The dome light alignment, which provided even and diffused light, generated less glare with slightly improved image contrast (Figure 1B). The transmitted light source provided some structural details, but the contrast was poor (Figure 1C). The ring illuminator, fitted around the objective lens of the stereomicroscope, provided a cylinder of light, illuminating the eye from the top. It generated an image with no unwanted shades but an intense glare on the ommatidia (Figure 1D). Also, the ring illumination images provided better contrast but lacked details of the ommatidial structure. We removed the ring illuminator from the objective lens assembly and positioned it closer to the fly on the stereobinocular stage. The glare disappeared when the light source and fly were at the same plane, and a high-contrast ommatidial structure appeared (Figure 1E). However, the central part of the eye lost its contrast because of less illumination than in the peripheral regions of the curved eye surface. This issue was resolved by lifting the ring illuminator approximately 2° upward to the eye position. This improved optical alignment—called low-angle ring illumination stereomicroscopy (LARIS)—did not generate any glare and equally illuminated all parts of the eye, thereby providing a high-contrast image of the entire eye (Figure 1F). In this approach, the position of the ring light source is only slightly above the sample in the LARIS alignment so that the camera receives only the scattered light from the curved surface of the eye. This makes the top surface of each ommatidium slightly brighter than its basal areas, which creates high contrast between neighboring ommatidia and allows distinct visualization of each ommatidium (Figure 1F).

The LARIS method provides better contrast of ommatidial structure, orientation, and pigmentation status than what is obtained by conventional imaging methods of the Drosophila compound eye. A comparative analysis of different approaches used in eye imaging of Drosophila is given in Table 1. Here, we provide a detailed protocol for the LARIS method to image compound eyes of adult flies or other insects.

Figure 1. Microphotographs of an adult Drosophila compound eye under a simple stereomicroscope with different illumination methods: (A) bar light, (B) dome light, (C) back light, (D) ring light, (E) low-angle ring illumination stereomicroscopy (LARIS) (0°), and (F) LARIS (2°), as mentioned above each panel. All panels are the same sample imaged sequentially at the same magnification without remounting. Scale bar, 100 μm.

Table 1. Comparison of LARIS with other imaging methods for the adult Drosophila compound eye