The determination of
A requires that flash intensities be expressed in photoisomerizations. An approach, applicable to monochromatic Ganzfeld stimulation in vivo,
15 is captured in the following expression:
\[{\Phi}\ {=}\ Q({\lambda}){\tau}({\lambda})\ \frac{\mathrm{S}_{\mathrm{pupil}}}{\mathrm{S}_{\mathrm{retina}}}\ a_{\mathrm{c,end-on}}({\lambda})\ {=}\ Q({\lambda})\ a_{\mathrm{c,\ cornea}}\ ({\lambda}).\]
In
equation 2 , Φ is the estimated average number of photoisomerizations produced by a flash of intensity
Q photons per square micrometer (measured at the cornea) and wavelength λ; τ(λ), the transmission of the ocular media distal to the outer segments;
S pupil, the area of the pupil;
S retina, the surface area of the retina at the photoreceptor layer; and
a c,end-on(λ) the end-on collecting area of a single photoreceptor at the retina. Collapsing all the factors in
equation 2multiplying the flash intensity
Q(λ) into a single parameter, one obtains a composite parameter,
a c,cornea(λ), which can be thought of as the effective collecting area of the photoreceptor at the cornea in a Ganzfeld. We previously estimated
a c,end-on(λ) and
a c,cornea(λ) for mouse rods and cones to analyze the sensitivity of components of the ERG,
15 17 but recently updated the estimates for rods by comparing the derived values with the measured rate of rhodopsin bleaching in the Ganzfeld.
23 For WT rods illuminated in vivo, the updated estimates are
a c,end-on = 0.87 μm
2,
a c,cornea = 0.11 μm
2, at the λ
max (498 nm) of mouse rhodopsin. To estimate the end-on collecting area of the
Nrl −/− photoreceptors, the simplest approach would be to assume that their collecting area scales relative to that of rods according to their OS volume ratio, which electron microscopy data presented herein show to be 1:4.3. However, there is a long history of investigations showing that all vertebrate photoreceptors guide or “funnel” light, and that funneling begins in the ellipsoid region of the inner segment, which is invariably larger in diameter than the outer segment, especially in cones.
24 25 The collecting area of turtle cone outer segments, for example, is increased ∼30-fold by light funneling in the inner segment.
26 Electron microscopy data presented herein show that
Nrl −/− photoreceptors, like WT cones, have ellipsoids wider in diameter than their OSs by twofold or more. Assuming these ellipsoids guide light, they should increase the end-on collecting area of
Nrl −/− photoreceptors by at least fourfold. Thus, the 4.3-fold smaller OS volume of
Nrl −/− photoreceptors relative to rods may be compensated by a roughly 4-fold contribution by light funneling, with the result that the effective end-on collecting area of
Nrl −/− photoreceptors may be about the same as that of WT rods:
a c,end-on = 0.87 μm
2,
a c,cornea = 0.11 μm
2 at the λ
max (360 nm) for
Nrl −/− photoreceptors. Although the light funneling factor cannot be considered precise, comparison of ERG results we present with suction electrode results presented elsewhere suggest that it does indeed contribute in the manner proposed.
27