Unbiased Imaging of Dendritic Spines

Using an approach that precludes sampling bias of spines, dendritic segments were selected with a systematic-random design42 (link)43 (link)44 (link). Dendritic segments, 20–25 μm in length, on secondary and higher order branches and at 50 and 100 μm from the soma, were imaged on the Zeiss LSM 510 confocal microscope (Zeiss) using a 100×/1.4 N.A. Plan-Apochromat objective with a digital zoom of 3.5 and an Ar/Kr laser at an excitation wavelength of 488 nm. All confocal stacks were acquired at 512 × 512 pixel resolution with a z-step of 0.1 μm and approximately 1 μm above and below the identified dendritic segment, a pinhole setting of 1 Airy unit and optimal settings for gain and offset. On average 3 z-stacks were imaged per apical and basal tree and 5 neurons per animal. In order for a dendritic segment to be optimally imaged it had to satisfy the following criteria: (1) the entire segment had to fall within a depth of 50 μm; (2) dendritic segments had to be either parallel or at acute angles to the coronal surface of the section; and (3) segments did not overlap other segments that would obscure visualization of spines42 (link)43 (link)44 (link). To improve voxel resolution and reduce optical aberration along the Z-axis, the acquired images were deconvolved using an interactive blind deconvolution algorithm (AutoDeblur version 8.0.2; MediaCybernetics).