Project information
Automated micro-axial tomography of tumor-correlated FISH pattern

Recently a 2pi-tilting device for fluorescence microscopes has been
developed at the Institute of Applied Physics, University of Heidelberg
(Bradl J et al. 1994, 1996a, 1996b, 1996c), which enables to rotate
observed biological specimens (especially suitable for
fluorescence-labeled chromosomes and chromatin regions in 3D conserved
cell nuclei) up to the full rotation angle (2pi). This can be performed by
fixation of the cell nuclei into a quartz glass capillary or on a glass
fiber mounted perpendicularly to the optical axis by means of a mounting
adapter for the microscope stage. This technique is also called
micro-axial tomography. Due to the rotation, the best resolution of the
given microscope (usually the lateral resolution) can be obtained in any
direction of observation (Rinke et al. 1996). Therefore, the 3D point
localization and distance measurements of fluorescence labeling sites are
much more precise than with a standard microscope setup (Bradl et
al. 1996b).

Meanwhile, a so called high-resolution cytometry (HRCM) technique has been
developed at the Faculty of Informatics and Institute of Biophysics in
Brno (Kozubek M et al. 1999) which enables fully automated 2-D and 3-D
acquisition and analysis of a large number of cells (comparable to flow-
or laser scanning cytometry) using computer-driven fluorescence microscopy
and a computer-driven CCD camera with the precision and resolution
comparable to confocal laser-scanning microscopy.

The aim of the project is to combine these two novel techniques by adding
an optimized 2pi-tilting device to the HRCM instrument. This will enable
detailed high-precision automatic 3D acquisition and analysis of large
number of fluorescence-labeled cell nuclei which can not be performed
using any other currently available instrument. The instrument will be
used for the analysis of the 3D topology of selected chromosome regions
stained using fluorescence in situ hybridization technique (FISH). Several
gene loci located in close vicinity will be stained with different dyes in
order to determine their 3D conformation.