Speaker: Josef Neumüller (Zentrum für Anatomie und Zellbiologie)
ET is a powerful tool for visualization of the highly dynamic structures of organelles in living cells. Although investigation using transmission electron microscopy requires fixed and embedded material which is stable in high vacuum and under a high voltage electron beam, the modern preparation method of high pressure fixation (HPF) allows obtaining snapshots from the arrangement of organelles in living cells in relation to a particular experimental condition.
In order to obtain appropriate 3D data, tilt series from semithin sections (200-300 nm), cut in parallel to the plane of the monolayer of cell cultures, are performed using a Tecnai-20 200KV transmission electron microscope (FEI, Eindhoven, The Netherlands) equipped with an eucentric goniometer. In addition a rotation holder (Gatan, Inc., Pleasanton, USA) is used for orientation of rod-like structures parallel to the tilt axis and also for dual axis acquisition. Series of tilted images (range: -70° to +70°) are acquired with a tilt increment of 1°. After holder calibration, dislocations in x, y and z axis are corrected by the Explore 3D acquisition software (FEI). The volume of the semithin sections is reconstructed by the back projection method into serial slices using the software package Inspect 3D (FEI). This software implicates also an alignment tool using cross correlation which is prerequisite for an appropriate reconstruction. Dual axis reconstruction requires acquisition of perpendicular orientated tilt series. It is performed using the Matlab software platform and an advanced version of the "Tomo Toolbox", kindly provided by Dr. Jürgen Plitzko, Dept. of Molecular Structural Biology (Head: Prof. Dr. Wolfgang Baumeister), Max Planck Institute of Biochemistry in Martinsried near Munich.
3D models are performed by tracing the structures of interest in every slice with colored contours that are merged in the Z axis by the help of the Amira 3.0 software (Mercury Computer Systems, Merignac Cedex, France). Models, generated in this way can be rotated in the space and presented as movie.
The aim of this presentation is to introduce interesting applications from cell biology and to discuss problems and limitations in 3D visualization using the commercial software as described above.