Capture of a moving filament. The images shown here were selected from a time-lapse movie acquired at 4 s intervals. The white arrowheads mark the leading and trailing ends of the moving filament. The filament moved rapidly into the gap, pausing intermittently, and then stopped shortly after the start of the flow of the permeabilization solution. Proximal is right, and distal is left. Scale bar, 6 μm. See also QuickTime movie 1 (available at www.jneurosci.org as supplemental material).

Quantitative analysis of captured and splayed filaments. A, Histogram of the normalized fluorescence intensities for 169 unpaired filaments from splayed axonal cytoskeletons. B, Histogram of normalized fluorescence intensities for 44 paired filaments from splayed axonal cytoskeletons. The curves represent Gaussian fits of the binned data. C, Histogram of normalized fluorescence intensities for 17 captured filaments. The mean normalized fluorescence intensity of the captured filaments is not significantly different from the unpaired filaments (p > 0.05; unpaired t test), but it is significantly different from the paired filaments (p < 0.05; unpaired t test). The insets in A-C show examples of the NFL immunofluorescence for unpaired, paired, and captured filaments, respectively. The white arrowheads label the unpaired filaments in A and the paired filament in B. Scale bar, 5 μm.

Electron microscopy of a captured filament using semithin sectioning. A, GFP fluorescence of a permeabilized axon before fixation, showing a captured filament in a gap. This filament was moving anterogradely at the time of capture. Proximal is left, and distal is right. B, Phase-contrast image of the same axon. The blebs along the axons are a consequence of the saponin treatment. Scale bar, 8μm. C, Schematic line drawing of the axon and neurofilaments. The filament was traced from a montage generated from the digitized electron micrographs. Scale bar, 2 μm. The entire axon was contained within one semithin (blue/green) section that was the first to come off the block (i.e., the section immediately adjacent to the glass coverslip). The neurofilament is represented by the black line. This line was thickened to make the location of the filament visible; therefore, it is not to scale. The axon membrane was extracted for semithin sectioning (see below); therefore, the outline of the axon was drawn artificially based on the phase-contrast image and is represented by the gray lines. The shaded region represents a large piece of cellular debris, also visible in the phase-contrast image, which facilitated alignment of the fluorescence image with the electron micrographs. The arrows in A-C mark the ends of the filament. One end of the filament extended further in the electron micrographs than in the image of the GFP fluorescence, indicating that this neurofilament had not incorporated GFP-NFM along its entire length. D, Electron micrograph of the boxed region shown in C. The axon membrane is not present because we treated with 0.5% Triton X-100 after glutaraldehyde fixation to permit unambiguous detection of the neurofilament along its entire length. Note the continuity of the captured filament here, comparing to the discontinuity in ultrathin section in Figure 3. Scale bar, 100 nm.

Electron microscopy of a captured filament using ultrathin sectioning. A, GFP fluorescence of a permeabilized axon before fixation, showing acaptured filament in a gap. This filament was moving retrogra delyat the time of capture. Proximal is left, and distalis right. B, Phase-contrast image of the same axon. The blebs along the axon area consequence of the saponin treatment. Scale bar, 8 μm. C, Schematic line drawing of the axon and neurofilaments. The filaments and axon were traced from a montage generated from the digitized electron micrographs. Scale bar, 2 μm. The entire axon was contained within three ultrathin (silver/gold) sections. The section shown here was the first to come off the block (i.e., the section immediately adjacent to the glass coverslip). The neurofilaments are represented by the black lines, and the axon outline is represented by the gray lines. The black lines have been thickened to make the location of the filaments visible; therefore, their thickness is not to scale. The shaded regions represent cellular debris, also visible in the phase-contrast image. The varicosities and cellular debris facilitated alignment of the fluorescence image with the electron micrographs. The arrows in A-C mark the ends of the filament. D-F, Electron micrographs of the three boxed regions shown in C. Note the two segments of the captured filament marked by white arrowheads shown in D, the absence of neurofilaments in the gap shown in E, and presence of several filaments in the region flanking the gap shown in F. The membrane is mostly intact, which indicates that saponin perforates the membrane without stripping it away. Note that microtubules are absent from these images because we destabilized axonal microtubules by including 5 mM calcium in the permeabilization solution to permit unambiguous detection of the captured neurofilament. Scale bar, 150 nm.