Figure 4.

Correlation between intraretinal pathway and beam divergence. (a) Overlay of the cellular fluorescence (red) and the beam scattering (green) at two consecutive fiber positions, y₀ and y₁ (dotted lines). At both positions, the core of the fiber was placed in front of the cell axis of one of two adjacent Müller cells. The scattering light spot on the plastic membrane formed two distinct areas (indicated by the black circles) with minimal overlap. (b) The line profile of the scattered light intensity I_SL at position x₁ shows two peaks with nearly the same curve progression and maximum intensity. (c) A line profile of the cellular fluorescence intensity I_FL along the IPL (at position x₀) was used to localize the y position (and, indirectly, the z position) of the inner stem process of Müller cells in the retinal tissue (red). The intensity peaks 1–8 (red) each represent a distinct Müller cell process in the focal plane of the objective where the core of the fiber was also placed. In contrast, several fluorescence minima (1′–6′) represent interjacent retinal tissue compartments devoid of a Müller cell process. All other (intermediate) fluorescence intensities could not be clearly assigned to one of these two cases. The width w of the light spots at the plastic membrane (blue curve in panel c) indicates the beam divergence on its course through the retina; it was estimated as the y width of a two-dimensional Gauss fit of the transmitted light spot. Comparing the cellular fluorescence intensities (red) with the width values (blue) shows that w was low at the fluorescence peaks (1–8) and high at the fluorescence minima (1′–6′). (d) The width w of the well-defined data points (1–8, 1′–6′, red dots) was plotted against the corresponding fluorescence values of the line profile in panel c. The resulting correlation coefficient was r = −0.9. (e) Schemata of a straight Müller cell, the axis of which does not change in the y and z directions. The beam axis and the endfoot axis as well as the resulting spot center are at the same y position. (f) A “bended” Müller cell causes a displacement d between beam axis and spot center. (g) A displacement also occurs if the beam does not hit the Müller cell axis. Only oblique light rays pass the cell.