Correction methods for three-dimensional reconstructions from confocal images: I. Tissue shrinking and axial scaling

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.