Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: The fluorescence of the induced fluorescent pseudopupil originates from the rhabdomeres. (a) Diagram of a single ommatidium of a dipteran compound eye. Dotted lines denote the plane where optical cross sections of the eye in (b–d) were taken. From top to bottom: corneal facet (C), pseudocone (PC) and distal tip of the rhabdomeres (Rh). (b,c) Eristalis tenax compound eye after application of Lucifer Yellow and scanned with a confocal microscope, with a 63× glycerol objective. Scale bar 100 µm. The fluorescence observed at the surface of the eye (b) originates from the fluorescent rhabdomere tips as no other cells or parts of the photoreceptors are fluorescent when we focus below the cornea to the plane of the rhabdom tips (c) . (d) Magnified view from a single ommatidium, showing the distinctive trapezoidal shape formed by the distal tips of 7 adjacent rhabdomeres, typical of dipteran flies. Scale bar 5 µm. (e) Maximum intensity projection of a z-stack of the left eye of a female Eristalis tenax. When the dye (in this case Neurobiotin 488) had been left in the head for more than 3 h we experienced glowing in the entire eye. Image acquired with a Leica SP8 DLS confocal microscope and a 2.5× air objective lens (see also Supplementary Information video , part 2).

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Fluorescence, Microscopy

Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: 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 “ ). 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.

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Staining, In Vivo

Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: Dark and induced fluorescent pseudopupils. (a) Section of an insect apposition compound eye illustrating formation of the principal pseudopupil when illuminated by incident co-axial light ( continuous black lines ); modified after . To the experimenter, looking through the lens of the microscope, the surface of the eye ( diagram below ) will appear pale in the majority of the facets where incident light is scattered from secondary pigment cells ( black dashed lines ). However, in ommatidia aligned with the viewing axis, light is absorbed by rhabdoms and primary pigment cells, making them appear dark ( diagram below ). C corneal facet, Cc crystalline cone, Rh rhabdom. (b) Section of an apposition compound eye illustrating formation of the fluorescent pseudopupil. In this case the compound eye has been injected with a fluorescent dye that has been taken up by the rhabdomeres ( green shade in the diagram, e.g. Lucifer Yellow or Neurobiotin 488, see “ ” section and Fig. ). Short wavelength incident light ( blue shade ) will excite the fluorophore in the rhabdomeres so that only those ommatidia aligned with the optical axis of the microscope will re-emit longer wavelengthlight ( green line ). These re-emitted light rays will cause the facet lenses through which they pass to be bright compared to the surrounding facet lenses which will be dark ( diagram below ). (c) Images taken from the eye of a dragonfly, Aeshna cyanea , whose induced fluorescent pseudopupil was obtained using Lucifer Yellow (see “ ). Scale bar: 500 µm. Images from Supplementary Information video 1, part 1. A. cyanea has a frontal acute zone that is very broad ( black arrows ) as seen by the dark pseudopupil occupying a large area of the frontal part of the eye when the animal is perpendicular to the axis of the objective, i.e. at 0° longitude. However, the induced fluorescent pseudopupil ( blue arrow ) is easily identified and because it is confined to a smaller region of the eye, its centre is easy to pinpoint.

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Modification, Microscopy, Injection

Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: The dimensions of the induced fluorescent pseudopupil depends on the numerical aperture (NA) of the objective lens. (a) Heatmap of the induced fluorescent pseudopupil in a honeybee forager eye stained with Lucifer Yellow (see “ ) and acquired with a Nikon SMZ18 microscope fitted with a P2-EFLGFP-B filter cube, a 1 × SHR PlanApo objective with two different numerical apertures (NA), 0.15 ( top ) and 0.075 ( bottom ). (b) Relative pixel values of the rectangular area in (a) showing that the two pseudopupils have the same centre despite subtending a broader area when the NA is higher ( orange line ).

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Staining, Microscopy

Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: A induced fluorescent pseudopupil in the dark-pigmented compound eye of the honeybee, Apis mellifera . (a) The pigmented compound eye of a naive honeybee has no visible pseudopupil due to the presence of pigments around the retinula cells . The red arrow shows the direction of rotation in (b–d) . (b–d) Zoomed-in view of the left eye of a honeybee forager, Apis mellifera (area of the eye as in the red inset in (a) ), acquired using a Nikon SMZ18. The eye was stained with Lucifer Yellow as described in the Methods and the bright induced fluorescent pseudopupil is thus visible ( red arrow ) and shifts its location in the eye as the head of the honeybee is turned (c, d) . (b) The head is in its frontal view and rotated around its vertical axis at 15° and 30° in (c, d) , respectively. Scale bar: 500 µm.

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Staining

Journal: Scientific Reports

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

doi: 10.1038/s41598-021-00407-2

Figure Lengend Snippet: The induced fluorescent pseudopupil is a good tool for estimating the sampling resolution of a compound eye. (a) Projections of the interommatidial angle, Δ φ , (average of horizontal and vertical components) and (b) facet diameter (values in µm) of a single forager honeybee, Apis mellifera . The highest resolution is found in the frontal part of the eye where there is an averaged Δ φ of 1.3°. Note that the location of this minimum value does not coincide with the location in the eye having the largest facet diameter. The boundary of the visual field ( dashed lines ) is reconstructed from . Dotted lines show 0° azimuth. F frontal, D dorsal, L lateral.

Article Snippet: A few small crystals of a fluorescent dye (we used either Neurobiotin 488 (BioNordika Sweden AB) or Lucifer Yellow (Sigma-Aldrich Sweden AB)) were then applied on the open head capsule before the head capsule was resealed by replacing the removed section of cuticle and waxing it in place to avoid desiccation.

Techniques: Sampling