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- D. Bucher, M. Scholz,
M. Stetter, K. Obermayer, and H.-J. Pflüger. Corrections Methods for
Three-dimensional Reconstructions from Confocal Images: I. Tissue Shrinking
and Axial Scaling.
.
J. Neurosci. Meth., 100:135-143, 2000.
(FTP Gzipped PDF, 456 kb)
We show here, using locust wholemount ganglia as an example, that
scaling artifacts in three- dimensional reconstructions from confocal
microscopic images due to refractive index mismatch in the light path and
tissue shrinking, can account for dramatic errors in measurements of
morphometric values. Refractive index mismatch leads to considerable
alteration of the axial dimension, and true dimensions must be restored by
rescaling the z-axis of the image stack. The appropriate scaling factor
depends on the refractive indices of the media in the light path and the
numerical aperture of the objective used and can be determined by numerical
simulations, as we show here. In addition, different histochemical procedures
were tested in regard to their effect on tissue dimensions. Reconstructions
of scans at different stages of these protocols show that shrinking can be
avoided prior to clearing when dehydrating ethanol series are carefully
applied. Fixation and mismatching buffer osmolarity have no effect. We
demonstrate procedures to reduce artifacts during mounting and clearing in
methyl salicylate, such that only isometric shrinkage occurs, which can
easily be corrected by rescaling the image dimensions. Glycerol-based
clearing agents produced severe anisometric and nonlinear shrinkage and we
could not find a way to overcome this.
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