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

Citation
D. Bucher et al., Correction methods for three-dimensional reconstructions from confocal images: I. tissue shrinking and axial scaling, J NEUROSC M, 100(1-2), 2000, pp. 135-143
Citations number
29
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Neurosciences & Behavoir
Journal title
JOURNAL OF NEUROSCIENCE METHODS
ISSN journal
0165-0270 → ACNP
Volume
100
Issue
1-2
Year of publication
2000
Pages
135 - 143
Database
ISI
SICI code
0165-0270(20000731)100:1-2<135:CMFTRF>2.0.ZU;2-Z
Abstract
We show here, using locust wholemount ganglia as an example, that scaling a rtifacts in three-dimensional reconstructions from confocal microscopic ima ges due to refractive index mismatch in the light path and tissue shrinking , can account for dramatic errors in measurements of morphometric values. R efractive index mismatch leads to considerable alteration of the axial dime nsion, and true dimensions must be restored by rescaling the Z-axis of the image stack. The appropriate scaling factor depends on the refractive indic es of the media in the light path and the numerical aperture of the objecti ve used and can be determined by numerical simulations, as we show here. In addition, different histochemical procedures were tested in regard to thei r effect on tissue dimensions. Reconstructions of scans at different stages of these protocols show that shrinking can be avoided prior to clearing wh en dehydrating ethanol series are carefully applied. Fixation and mismatchi ng buffer osmolarity have no effect. We demonstrate procedures to reduce ar tifacts during mounting and clearing in methyl salicylate, such that only i sometric shrinkage occurs, which can easily be corrected by rescaling the i mage dimensions. Glycerol-based clearing agents produced severe anisometric and nonlinear shrinkage and we could not find a way to overcome this. (C) 2000 Elsevier Science B.V. All rights reserved.