Figure 2.

Pathway of light through the vital retina. (a) Position of a single mode fiber (left) in front of the retinal surface (right) recorded by transmission microscopy. (b) The divergent light beam emanating from the core of the glass fiber was visualized by inserting the fiber into an agarose gel that caused scattering of the light (λ = 532 nm, green) in all directions and thus allowed light detection orthogonal to the beam. (c) Slice preparation of a living retina, monitored by using a 63×/0.95 water-immersion objective of an upright confocal microscope. The vital dye, Mitotracker orange (red), predominantly stained the Müller cells, the photoreceptor segments, and the plastic membrane (M). The positioning of the fiber core (dotted line at position y₀) between two Müller cell processes caused light scattering in both plexiform layers (IPL, inner plexiform layer; OPL, outer plexiform layer), as demonstrated by the x-line profiles (at position x₀) of the cellular fluorescence (I_FL, red) and the scattered light intensity (I_SL, green) along the IPL (d). The scattered light spots on the membrane (c and f) visualize the light that was transmitted by the retina. Their yellow appearance results from a merge of strong red fluorescence (vital dye sucked up by the membrane) and the green laser light scattering. (e) The x-line profile of I_SL along the membrane (at position x₁ in panel c) showed a rather wide-spread intensity distribution with a low maximum intensity. (f) In a position where the fiber and the Müller cell process are aligned to each other (along the dotted line at position y₀), the scattered beam pattern changed. The almost complete lack of light scattering in the Müller cell processes is represented by a reduced I_SL in the IPL (g) and OPL. This was accompanied by a narrowing light spot on the membrane with an increased maximum intensity (h).