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TU Berlin

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Confocal Microscopy: Semi-Automatic Segmentation, Tracing and Analysis of 3D Images

Lupe

In this project we developed algorithms for the computer assisted segmentation and 3D-reconstruction of neurons from confocal microscope image stacks. We investigated methods for the correction of scaling artifacts due to refractive index mismatch and tissue shrinking. We also developed blind deconvolution techniques in order to comensate for the strongly anisotropic point-spread functions measured in the stained preparations. Deconvolution techniques were validated in preparations of optic neuropils where the resolution of the confocal microscope scans could be sufficiently enhanced in order to study colocalization between synaptic vesicle markers near the resolution limit of light. We evaluated techniques based on the wavelet transform for increasing the signal-to-noise ratio of the confocal images, and we developed semi-automatic algorithms for segmentation, tracing and reconstruction of connected tubular structures. 3D-reconstruction techniques were evaluated on 3D scans of neurons from Maduca Sexta.

Acknowledgement: Research was funded by BMBF, DFG, and the Technische Universität Berlin.

Selected Publications:

Corrections Methods for Three-dimensional Reconstructions from Confocal Images: I. Tissue Shrinking and Axial Scaling
Citation key Bucher2000
Author Bucher, D. and Scholz, M. and Stetter, M. and Obermayer, K. and Pflüger, H.-J.
Pages 135 – 143
Year 2000
DOI 10.1016/S0165-0270(00)00245-4
Journal Journal of Neuroscience Methods
Volume 100
Number 1-2
Publisher Elsevier
Abstract 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.
Bibtex Type of Publication Selected:3dimages
Link to publication Link to original publication Download Bibtex entry

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