Methods

In landmark papers, Franceschini and Kirschfeld (Franceschini & Kirschfeld, 1971a) (Franceschini & Kirschfeld, 1971b) revolutionized the study of fly photoreceptor optics. Because the index of refraction of water or immersion oil approximate that of the cornea, the focusing function of these facet lenslets is eliminated when the eye is viewed with an immersion objective. Also, rhabdomeres carry light like a fiber optic (light pipe). Hence, this "optical neutralization of the cornea" technique allows viewing of rhabdomere tips when light is transmitted up (antidromically) through the eye. Because of the regularity of rhabdomeres in each ommatidium and the regularity of the arrangement of ommatidia, a low power air objective can be used to image the "deep pseudopupil," a virtual image of the rhabdomere tips, magnified deep (about 150 mm beneath the cornea) and superimposed from about 25 ommatidia (that number depending on the numerical aperture of the microscope objective).

(move beginning of paragraph in results section to here?)

(The following two paragraphs are in anticipation that the paper might include the ninaE driver, George's and Asmir's data)

Following another methodological advance (Stavenga et al, 1973), this laboratory soon developed methodology to make visual pigment measurements (Stark & Johnson, 1980) and fluorescence measurements (Stark et al, 1979) using the deep pseudopupil. In an extensive series of papers, (Stark et al, 1983) (Zinkl et al, 1990) visual pigment measurements were used to quantify photoreceptor demise in various mutants (and in flies after bright light treatment).

With the availability of the laser scanning confocal microscope, we extended our interest in fluorescence by studying rhabdomere autofluorescence, as well as fluorescence of labels such as green fluorescent protein using confocal microscopy of the deep pseudopupil and the optically neutralization of the cornea (Lee et al, 1996) (Stark & Thomas, 2004).

(Here is a plate made from GGA kd 43 days (left) [F1 from male eyGMRdriver x female GGA.E3], control eyGMR 42 days (middle) [eyGMRdriver;boss/+], GGA control 40 days (right) [GGA.E3/+]

Results

Because the deep pseudopupil and the optical neutralization of the cornea techniques are relatively straightforward, they have long served as a convenient entry point to diagnose the structural integrity of the retina. The deep pseudopupil of GGA knockdowns (driven by ey GMR) was in such disarray that there was no focal plane that showed any rhabdomere clearly. This was invariably the case at every time point assayed ranging from zero days (in the dark or in the light) out to 4 weeks. Focusing up and down, the eye color pigment seemed mottled and uneven.

(here is a selected depth series to demonstrate these points from GGA kd 43 days post-eclosion where 1 = distal, 6 = deeper than the deep pseudopupil)

Under optical neutralization of the cornea, rhabdomeres were present at all time points. However, rarely was a single rhabdomere imaged clearly. The number, size, and orientation of rhabdomeres that could be imaged per ommatidium was always variable. In contrast, rdgB (retinal degeneration B, even amid a wasteland of dead R1-6 cells and debris) and ora (outer rhabdomeres absent, lacking R1-6 rhabdomeres) gave clear images of R7 (and clearly lacking R1-6) (Harris et al, 1976).

Discussion

Why were the GGA knockdown's pseudopupils so disrupted? The diagnoses were straightforward when Harris et al (1976) utilized the pseudopupil techniques developed by (Franceschini & Kirschfeld (1971a; b): R1-6 was clear but R 7 was missing in sevenlses (sev); R7 was clear but R1-6 was missing in outer rhabdomeres absent (ora in what Washburn & O'Tousa, (1989) later showed to be a rhodopsin mutation) and in retinal degeneration (rdgB).

With hindsight, it is obvious that the optical neutralization of the cornea presaged the subsequent histological and ultrastructural findings of variable numbers, sizes, counts and configurations of rhabdomeres in the ommatidia. Since the deep pseudopupil is formed by the magnified, superimposed images of the rhabdomere tips collected from many ommatidia, it is reasonable to conclude that all the factors of rhabdomere disarray preclude a good deep pseudopupil image.

References

Franceschini N, Kirschfeld K (1971a) Etude optique in vivo des elements photorecepteurs dans l'oeil compose de Drosophila. Kybernetik 8: 1-13

Franceschini N, Kirschfeld K (1971b) Les phenomenes de pseudopupille dans l'oeil compose de Drosophila. Kybernetik 9: 159-182

Harris WA, Stark WS, Walker JA (1976) Genetic dissection of the photoreceptor system in the compound eye of Drosophila melanogaster. J Physiol (Lond) 256: 415-439

Lee RD, Thomas CF, Marietta RG, Stark WS (1996) Vitamin A, visual pigments and visual receptors in Drosophila. Micros Res Tech 35: 418-430

Stark WS, Johnson MA (1980) Microspectrophotometry of Drosophila visual pigments: Determinations of conversion efficiency in R1-6 receptors. J Comp Physiol A 140: 275-286

Stark WS, Stavenga DG, Kruizinga B (1979) Fly photoreceptor fluorescence is related to UV sensitivity. Nature (Lond) 280: 581-583

Stark WS, Thomas CF (2004) Microscopy of multiple visual receptor types in Drosophila. Mol Vis 10: 943-955

Stavenga DG, Zantema A, Kuiper JW (1973) Rhodopsin processes and the function of the pupil mechanism in flies. In Biochemistry and physiology of visual pigments, Langer H (ed). Berlin: Springer

Washburn T, O'Tousa JE (1989) Molecular defects in Drosophila rhodopsin mutants. J Biol Chem(264): 15464-15466

Zinkl G, Maier L, Studer K, Sapp R, Chen DM, Stark WS (1990) Microphotometric, ultrastructural and electrophysiological analyses of light dependent processes on visual receptors in white-eyed wild-type and norpA (no receptor potential) mutant Drosophila. Vis Neurosc 5: 429-439

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last updated 4/25/10