3D-Visualization of nerve fiber bundles by ultramicroscopy3D-Visualisierung von neuronalen Faserbündeln mittels Ultramikroskopie
Introduction
Ultramicroscopy is based on light sheet illumination, which originally was invented 100 years ago by Siedentopf and Zigmondy for their investigations of colloidal liquids [1]. We recently adapted this technique for the 3D-reconstruction of large microscopical specimens [2], [3] (Fig. 1).
In ultramicroscopy, only those parts of the specimen are illuminated which are in the focal plane of the objective thus avoiding the generation of stray-light and image blurring.
Optical sectioning is achieved by moving the specimen chamber vertically and stepwise through the light sheet. Using this approach, an excellent axial resolution can be obtained in the micrometer range [2].
Since ultramicroscopy requires specimens to be transparent, most specimens have to be cleared beforehand. This can be achieved by using a technique first applied by the German anatomist, Spalteholz, for studies in heart vascularization [4]. It relies on the incubation of a specimen in a medium, which has the same refractive index as proteins. As a result, light scattering is greatly reduced. If light absorption of the specimen is low, it will become transparent because no illumination light is deflected.
Ultramicroscopy is especially suitable for investigations of specimens labeled by fluorescence via immunohistological techniques or for green fluorescent protein (GFP) expressing transgenic mouse lines. Furthermore, it has already shown its applicability in investigations of GFP-labeled neurons in mouse brain, and in morphological studies of drosophila [2], [3]. Here, we present 3D-reconstructions of nerve fiber bundles in mouse embryos E12.5 obtained by ultramicroscopy and subsequent non-blind deconvolution.
Section snippets
Preparation of samples
Mouse embryos E12.5 from wild-type mice were killed by cervical dislocation. The according procedures were performed in accordance with the local animal committees.
Antibodies: Anti-neurofilament-160 (monoclonal, clone NN18, Sigma–Aldrich, Germany) was diluted 1:200 in blocking serum made up of four parts calf serum and one part dimethyl sulfoxide. Anti-neurofilament-160 is an antibody against an intermediate filament found specifically in neurons. The secondary fluorescent antibody (goat
Modeling of the light sheet
The optical sectioning quality greatly depends on the choice of an appropriate slit aperture of half-width d with respect to the focal length f of the cylinder lens and the specimen size (Fig. 1).
The cone angle of the asymptote to the beam profile at the beam waist w0 can be approximated by [7]Since angle θ approximately is d/f, the minimum beam wide w0 iswhere f is the focal length of the cylinder lens and λ the laser wavelength [7].
The squared 1/e2 intensity cut-offs w2(z)
Conclusion
Ultramicroscopy allows one to make 3D-reconstructions of immunostained nerve fiber bundles in mouse embryos. Artifacts are avoided because ultramicroscopy does not require the sample to be subjected to mechanical slicing. This makes ultramicroscopy a useful tool in embryology, especially for whole mount analysis. Immunostained nerve fibers can be localized precisely for all dimensions. In our experiments, the penetration depth of the antibodies was sufficient. For larger specimens XFP-labeling
Acknowledgements
The study was supported by the SFB 361 and the Hertie Foundation. We thank Dr. Saideh Saghafi for carefully reading this manuscript.
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