E-600

Immunofluorescence images were captured on a Zeiss Axiophot epifluorescence microscope equipped with an ORCA camera (Hamamatsu). Immunoperoxidase images were obtained on a Nikon E600 microscope equipped with Spot RT Slider camera. Image files were pseudocolored and enhanced globally for brightness and contrast using Photoshop 6.0 (Adobe Systems, San Jose, CA). Final figures were composed in Photoshop, Canvas X (Deneba Software, Miami, FL) or PowerPoint (Microsoft Corporation, Redmond, WA).
To reconstruct the dendritic profiles of individual melanopsin-expressing ganglion cells, we analyzed in detail a quadrant of retina that had been optimally stained by the immunoperoxidase method (see Figs. 2A and 2E). We assembled a comprehensive high-resolution library of digital photomicrographs in which every melanopsin immunoreactive element was in sharp focus in at least one image. With the optic axis positioned at one margin of the retinal sample, we used a 20× objective lens (numerical aperture: 0.75) to capture in a series of approximately 30 images incremented in focal depth by 1 µm and ranging from the optic fiber layer to the outermost immunopositive dendrites. The optic axis was then offset laterally and the process repeated. Altogether, we captured 17 such “z-stacks”, each covering an area of 435 × 580 µm. Each stack slightly overlapped those adjacent to it so that individual processes could be followed continuously from one stack to the next. These stacks were assembled in Photoshop, with one image per layer. Individual somadendritic profiles were traced in a process similar to that used to make a camera lucida drawing. Using the Photoshop pencil tool, we traced each cell’s profile on a separate, overlying transparent layer, displaying or hiding underlying layers as needed to visualize each process as it coursed in depth. Each cell’s profile was drawn in a unique color on a separate layer, so that cells could be displayed individually or as a mosaic.
We used the same three-dimensional digital photomontage for several other purposes. We assessed the basic stratification pattern of every immunolabeled cell in the retinal piece (many more than it was possible to fully trace) to develop the map of cell types shown in Fig. 3C. We traced the outlines of each labeled cell body and the minimal convex polygon enclosing the dendritic field of each traced somadendritic profile. These outlines were exported to ImageJ (http://rsbweb.nih.gov/ij/) which we used to measure the areas of somatic and dendritic profiles. Soma and dendritic field sizes were expressed as equivalent diameters, that is, the diameter of a circle of equal area. To estimate the level of stratification of M2 dendrites within the IPL, we identified, at many retinal locations, the plane of best focus for the innermost and outermost processes of the M2 dendritic plexus. These depths were then expressed as a percentage of the full thickness of the IPL, with 100% depth corresponding to the ganglion cell layer, taken as the depth of best focus of ganglion cell somata, and 0% corresponding to the outer border of the IPL, inferred from the plane of best focus of the outermost dendrites of the M1 plexus.
To generate the images in Fig. 2A–C, we manipulated the z-stacks to correct for the imperfect flatness of the retina. Using the outer margin of the M1 plexus and the inner margin of the M2 plexus as benchmarks, we generated a contour map encoding the displacement in the z-dimension of every point in the retinal piece relative to a reference region of excellent flatness. We then used this contour map to correct the distortion by selecting the relevant region within the image and transposing it, in every image of the stack, either up or down the stack by the appropriate number of images. These corrected images were used only to illustrate the inner and outer melanopsin plexuses as they would appear in a perfectly flat piece of tissue (Fig. 2A–C). All reconstructions of dendritic profiles and analyses of dendritic stratification were done on the uncorrected z-stacks, with the depth of any process being inferred by reference to the local position within the stack of the inner and outer plexuses. Dendritic profiles shown in Fig 4 were subjected to Sholl analysis to compare their dendritic arborizations quantitatively. We used the ImageJ Sholl analysis plug-in courtesy of the Ghosh Lab (http://www-biology.ucsd.edu/labs/ghosh/software/) with the following settings: starting radius, 10 µm; ending radius, 400 µm; radius step size, 20 µm; radius span, 3 µm; and span type, median.