Figure 3

Induced fluorescent and dark pseudopupil overlap. (a) Head of the bee Anthophora sp. These bees have clear eyes and its dark pseudopupil is evident as a black spot on the eye surface. Image acquired with a Nikon SMZ18 equipped with a bright field filter cube (P2-EFLBF) and a ¼ wave plate. The eye was stained with Neurobiotin 488 (see “Methods”). Scale bar: 500 µm. (b) Zoomed-in view of the area marked by the white rectangle in (a) showing more clearly the dark pseudopupil in the left eye. It is common to see multiple pseudopupils resulting from the incident light from the environment around the eye. (c) Zoomed-in view of the area marked by the white rectangle in (a), after using a P2-EFLGFP-B filter cube—the induced fluorescent pseudopupil was visible in the same sample (in vivo). (d) Co-visualization of the dark (b) and induced fluorescent pseudopupil (c) showing a clear overlap of the two. (b–d) Scale bar: 200 µm. (e) Relative pixel values for the induced fluorescent and dark pseudopupils in the area marked in (d) by a white rectangle. Note that the centres of the two (i.e. max relative pixel value) coincide, with the induced fluorescent pseudopupil having the centre much more easily identifiable compared to the dark pseudopupil.