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TU Berlin

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


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:

Visualization of Synaptic Markers in the Optic Neuropils of Drosophila Using a New Constrained Deconvolution Method
Citation key Hiesinger2001
Author Hiesinger, P. and Scholz, M. and Meinertzhagen, I. and Fischbach, K.-F. and Obermayer, K.
Pages 277 – 288
Year 2001
DOI 10.1002/1096-9861(20000108)429:2
Journal Journal of Comparative Neurology
Volume 429
Number 2
Publisher Wiley
Abstract The fruitfly Drosophila melanogaster offers compelling genetic advantages for the analysis of its nervous system, but cell size precludes immunocytochemical analysis of wild-type structure and mutant phenotypes beyond the level of neuronal arborizations. For many antibodies, especially when immunoelectron microscopy is not feasible, it would therefore be desirable to extend the resolution limit of confocal microscopy as far as possible. Because high-resolution confocal microscopy suffers from considerable blurring, so-called deconvolution algorithms are needed to remove, at least partially, the blur introduced by the microscope and by the specimen itself. Here, we present the establishment and application of a new deconvolution method to visualize synaptic markers in Drosophila optic neuropils at the resolution limit of light. We ascertained all necessary parameters experimentally and verified them by deconvolving injected fluorescent microshperes in immunostained optic lobe tissue. The resulting deconvolution method was used to analyze colocalization between the synaptic vesicle marker neuronal synaptobrevin and synaptic and putative synaptic markers in photoreceptor terminals. We report differential localization of these near the resolution limit of light, which could not be distinguished without deconvolution.
Bibtex Type of Publication Selected:3dimages
Link to publication Link to original publication Download Bibtex entry

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