Elsevier

Medical Laser Application

Volume 23, Issue 4, November 2008, Pages 209-215
Medical Laser Application

3D-Visualization of nerve fiber bundles by ultramicroscopy3D-Visualisierung von neuronalen Faserbündeln mittels Ultramikroskopie

https://doi.org/10.1016/j.mla.2008.06.001Get rights and content

Abstract

Ultramicroscopy has recently been shown to be an outstanding tool for the 3D-visualization of large microscopical structures with μm resolution. In ultramicroscopy, mechanical slicing is replaced by optical sectioning which eliminates drawbacks such as mechanical distortion and misalignment. Simulations of the illumination light sheet show that the optical sectioning quality of an ultramicroscope greatly depends on the choice of an appropriate slit aperture, thereby the distance between specimen chamber and cylinder lens has to be considered. We labeled nerve fibers in mouse embryos by binding primary antibodies to neurofilament-160 in combination with fluorescent Alexa 488 secondary antibodies. After non-blind deconvolution, 3D-reconstructions of even fine nerve fiber branches were possible. These results demonstrate that ultramicroscopy is a valuable tool for developmental studies in embryology.

Zusammenfassung

Wie jüngst gezeigt wurde, ist die Ultramikroskopie hervorragend geeignet, auch große mikroskopische Strukturen dreidimensional in μm-Auflösung darzustellen. Da bei dieser Technik die Anfertigung mechanischer Schnitte überflüssig ist, lassen sich mechanische Verzerrungen und Ausrichtungsprobleme vollständig vermeiden. Wie Simulationen des Beleuchtungsstrahlengangs zeigen, ist die Wahl einer geeigneten Schlitzblende für die Bildqualität entscheidend. Hierbei muss der Abstand zwischen Probenkammer und Zylinderlinse berücksichtigt werden. Mittels eines Primärantikörpers gegen Neurofilament 160, kombiniert mit Alexa 488 als fluoreszierendem Sekundärantikörper, wurden neuronale Faserbündel von Mausembryonen markiert. Durch Anwendung eines mathematischen Dekonvolutionsverfahrens lassen sich auch feinste Verzweigungen von Nervenfasern in ihrem dreidimensionalen Verlauf sichtbar machen. Die Ultramikroskopie kann daher ein wertvolles Hilfsmittel bei embryologischen Entwicklungsstudien sein.

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]θλπw0.Since angle θ approximately is d/f, the minimum beam wide w0 isw0=λfπd,where 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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