CSC Visualization Group

Electron Microscopy Tomography (EMT) of DNA-Depleted Human (HeLa) Cell-Line Chromosome

The information shown here was presented at International Conference on 3D Image Processing in Microscopy, April 25-28, 1994, Munich, Germany (/1/).

Peter Engelhardt,
Department of Virology, University of Helsinki, Helsinki, Finland
Juha Ruokolainen,
CSC, Espoo, Finland

Metaphase chromosome preparations from Human (HeLa) cell-lines, were collected and fixed according to conventional cytogenetical methods modified for electron microscopy (EM). Before processing to EM the preparate was digested with DNase which removed all DNA detectable by the DNA-specific DAPI fluorescence. Electron microscope picture series taken at angles of +-60 degrees with 3 degree increments is shown below. The pictures shown here have been aligned (see e.g./2/) as regards to rotations, translations, and scaling using colloidal (15nm diameter) gold particles as reference points. After the alignment the reconstruction was made using the weighted back projection-method. The EMT was fulfilled with a EMT-program KIT developed by EMT-group (Ulf Skoglund et al.) at Dept. of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.

One picture of the series zoomed here.

A movie of the reconstruction viewpoint changing with 3 degree increments around tilt axis is shown below.

Click here.
The size of the movie is 506 Kb

Also a 'hardcopy', i.e. a physical 3D-model of a isosurface of the reconstruction was manufactured at SINTEF Norway, after the data had been processed for holes etc. (/3/) by André Dolenc at Institute of Industrial Automation, Helsinki University of Technology . The manufactured model is shown here spinning on top of a record player.

Click here.
The size of the movie is 575 Kb

References

1) P.Engelhardt#, J. Ruokolainen%, A.Dolenc$, L.-G. Överstedt@,
         H. Mehlin@, and U. Skoglund@
   3D-Reconstruction by Electron Microscope Tomography (EMT)
   of Whole-Mounted DNA-Depleted Metaphase Chromosomes Show
   Scaffolding Macro Coils, 30-nm Fibers, and 30-nm Particles
   Abstract for International Conference on 3D Image Processing
   in Microscopy, April 25-28 , 1994, Munich, Germany.

 # Department of Virology, PO Box 21,
    00014  Univ. of Helsinki, Finland
% Centre for Scientific Computing (CSC),
     PO Box 40, SF-2101, Espoo, Finland
@ Dept. Of Molecular Genetics, PO Box 60400,
  Karolinska Institute (KI), S-10401 Stockholm, Sweden
$ Institute of Industrial Automation,
  Helsinki University of Technology

2) Joachim Frank (Editor)
   Electron Tomography
   Plenum Press, 1992
   See Chapter 8 by Michael C. Lawrence

3) André Dolenc, Reidar Hovtun,
   Peter Engelhardt, Juha Ruokolainen
   Manufacturing Chromosomes And Viruses:
     A New Application Area For RPT?
   EARP Newsletter, April 1994