A new, fluorescence-based method for visualizing the pseudopupil and assessing optical acuity in the dark compound eyes of honeybees and other insects.

Recent interest in applying novel imaging techniques to infer optical resolution in compound eyes underscores the difficulty of obtaining direct measures of acuity. A widely used technique exploits the principal pseudopupil, a dark spot on the eye surface representing the ommatidial gaze direction and the number of detector units (ommatidia) viewing that gaze direction. However, dark-pigmented eyes, like those of honeybees, lack a visible pseudopupil. Attempts over almost a century to estimate optical acuity in this species are still debated. Here, we developed a method to visualize a stable, reliable pseudopupil by staining the photoreceptors with fluorescent dyes. We validated this method in several species and found it to outperform the dark pseudopupil for this purpose, even in pale eyes, allowing more precise location of the gaze centre. We then applied this method to estimate the sampling resolution in the frontal part of the eye of the honeybee forager. We found a broad frontal acute zone with interommatidial angles below 2° and a minimum interommatidial angle of 1.3°, a broader, sharper frontal acute zone than previously reported. Our study provides a new method to directly measure the sampling resolution in most compound eyes of living animals.