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Section images (2040×2048) in the z-dimension (z-spacing, 0.5 µm) were collected, ensuring that segments of both dendrites, spanning multiple confocal planes, were fully captured.
We collected several optical section images (1024×1024) in the z-dimension (z-spacing, 1 µm), ensuring that each 293 cell, spanning multiple confocal planes, was fully captured.
We collected a variable number of optical section images in the z-dimension (z-spacing, 0.5 µm) ensuring that segments of dendrites, spanning multiple confocal planes, were fully captured.
Several optical section images (2040×2048) in the z-dimension (z-spacing, 0.5 µm) were captured, ensuring that segments of the dendritic tract, spanning multiple confocal planes, were fully captured.
The figures provide images derived from the projection of a series of different optical sections spanning multiple confocal planes, in order to visualize the entire dendrite, but the spine density evaluation was calculated by collecting only the spines emerging from the proximal and distal dendrite in the central image of the series.
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Spinning disk confocal microscopes (SDCMs) instead use a parallelized scanning approach with multiple confocal beams that simultaneously illuminates the sample.
These methods include using multiple confocal pinhole apertures, lenses, and focusing mirrors.
Micrographs were generated with several merged confocal planes or vertical sections of confocal stacks using Olympus Fluoview FV1000 software (version1.3b).
The average distance between the confocal planes throughout the study was in the range of 1.7 2 µm, as 6 9 confocal planes were recorded for all the points measured.
Images in the same confocal planes were scanned sequentially to avoid fluorescent channel emission cross-talk.
Moreover, ChAT bands are useful to align the different sections on different confocal planes ensuring the dendrites are reconstructed properly.
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