papers available

[Klaus Obermayer]

Dear Connectionists,

attached please find abstracts and preprint locations of two manuscripts
with results in the neuronformatics area (confocal microscopy).

Comments are welcome!

Cheers

Klaus

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Prof. Dr. Klaus Obermayer         phone:  49-30-314-73442
FR2-1, NI, Informatik                     49-30-314-73120
Technische Universitaet Berlin    fax:    49-30-314-73121
Franklinstrasse 28/29             e-mail: oby at cs.tu-berlin.de
10587 Berlin, Germany             http://ni.cs.tu-berlin.de/

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Visualization of synaptic markers in the optic neuropils of Drosophila
using a new constrained deconvolution method.

P. R. Hiesinger^1, M. Scholz^2, I. A. Meinertzhagen^3, K.-F. Fischbach^1,
and K. Obermayer^2.

^1 Institut fuer Biologie III, Universitaet Freiburg, Freiburg, Germany
^2 Fakultaet fuer Elektrotechnik und Informatik, TU Berlin, Berlin,
   Germany
^3 Neuroscience Institute, Dalhousie University, Halifax, Canada


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.

in: Journal of Comparative Neurology 429, 277ff.

available at: http://ni.cs.tu-berlin.de/publications/


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

D. Bucher^1, M. Scholz^2, M. Stetter^2, K. Obermayer^2, and
H.-J. Pflueger^1

^1 Institut fuer Biologie, FU Berlin, Berlin, Germany
^2 Fakultaet fuer Elektrotechnik und Informatik, TU Berlin, Berlin,
   Germany


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.

in: Journal of Neuroscience Methods 100, 135ff

available at: http://ni.cs.tu-berlin.de/publications/