336 J. E. Rash et al.: Double-Replica FRIL Analysis of Gap Junctions fractured single replicas (procedure described below) revealed that neuronal processes often had thin astrocyte ‘‘ﬁngers’’ interposed, in this case with a small A:A gap junction labeled for both Cx26 and Cx30 (Fig. 2c; 12 nm gold = Cx26, 20 nm gold = Cx30). The nominal LM limits of resolution in the x, y and z axes are indicated in stereo- scopic images by the inscribed three-dimensional box, which corresponds to a single ‘‘voxel’’ (volume pixel) at the reso- lution limit of confocal LM (0.2 9 0.2 9 0.4 lm in the x, y and z axes, respectively). Moreover, in the red wavelength (which had been used to visualize the margins of the neurons in Fig. 2a), the limit of resolution is *0.4 lm, or several times the width of the space occupied by the astrocyte ﬁngers (Fig. 2c, crossing red arrows). If this conﬁguration had been Fig. 2 Comparison of limits of resolution of light microscopy (a) with ultrastructural resolution (b, c). a Neurons double-stained for Cx43 (green ﬂuorescence) and the neuronal marker MAP-2 (red ﬂuores- cence) but without visualization of intervening glial cells. Without companion bright-ﬁeld or differential interference optics to reveal other cell types, it is not possible to assign Cx43 unambiguously to the visualized cells, regardless of apparent close proximity. This deﬁciency is implicit in representative thin-section TEM images. b Modiﬁed from Peters et al. (1991). The limits of resolution in the blue and red wavelengths are indicated by superimposed red and blue discs, each of which overlaps cell margins of all three cell types, as well as multiple cytoplasmic membranes. c Two neuronal dendritic processes (red overlays), with a gap junction linking two thin intervening astrocyte processes (blue overlays). The astrocyte gap junction (shown at higher magniﬁcation in the inset) is double-labeled for Cx26 (12 nm gold) and Cx30 (20 nm gold). The limits of resolution in the x, y and z axes are indicated by the inscribed three-dimensional box, which corresponds to a single voxel (volume pixel) at the limit of resolution of confocal LM. If this region had been visualized by LM, with neurons stained red, astrocytes and oligodendrocytes not stained and connexins visualized using green ﬂuorescence (as in a), Cx26 and Cx30 would have appeared 123 to be localized to the decussating, small-diameter neuronal processes. Crossing red arrows indicate the limit of LM resolution in the red wavelength, suggesting that these two neuronal processes would have been in direct contact, with no room for intervening astrocyte processes. Barred Circle = gold bead on top of replica, as ‘‘noise’’ (Rash and Yasumura 1999). d, e ‘‘Serial sections in which gold–silver labeling for Cx32 (straight open arrows) was identiﬁed on the cytoplasmic surface of a peroxidase-labeled TH dendrite and in an apposed glial process (asterisks) that separates two TH-positive dendrites from one another’’ (Alvarez-Maubecin et al. 2000). However, we note that Cx32 is an oligodendrocyte connexin and is not found in astrocytes, nor has it been detected in ultrastructurally deﬁned neuronal gap junctions, so we consider these images to represent background ‘‘noise’’ on two nonserial sections, each showing an astrocyte process between two different sets of TH neurons. Calibration bars 0.2 lm. f Comparison FRIL image of two neuronal gap junctions (red overlays) in adult rat retina that were immunogold-labeled for Cx36 (13- and three 20-nm gold beads). Unlabeled glutamate receptor postsynaptic density (yellow overlay). Modiﬁed from Rash et al. (2001). Calibration bars 0.1 lm, unless otherwise indicated