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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A095
The high specificity of molecular in situ techniques in combination with new
3D techniques in fluorescence microscopy opens new possibilities for
investigating structural and functional molecular and cell biology. Since 3D
volume images are difficult to evaluate quantitatively by visual methods,
computer assisted methods are needed. We have developed a two step method for
studying the spatial relationships between fluorescent foci. First we find
fluorescent foci in 3D images by locating local intensity maxima. This method
in combination with 3D neighborhood mean filtering can be used to detect
fluorescent foci also in noisy and low contrast images. The method only takes
the nearest neighborhood into consideration when detecting foci and is
therefor independent of variations in fluorescence intensity due to uneven
illumination and light attenuation. After detecting the foci we create a
distance map by applying a 3D distance transform to the detected foci of one
wavelength channel. The map gives direct information about the shortest
distance between foci of the wavelength channel where the distance map was
created and foci in other wave length channels. This two step method is
currently being used for evaluation of the spatial relationships between
newly synthesized DNA and replication complexes. Cells are double stained
for BrdU, a thymidine analogue, and PCNA, an auxiliary protein of DNA
polymerase d. Foci for BrdU and PCNA co-localize at sites of DNA replication
when cells are fixed directly after exposure to BrdU. This result will be
compared with cells incubated with fresh medium and cultured for 2 hours
after the BrdU incorporation, a so called pulse-chase experiment. By varying
the pulse-chaise time, this method could be used to measure the speed of
movement for DNA replication units during S-phase.
DETECTION OF FLUORESCENT FOCI AND EVALUATION OF SPATIAL RELATIONSHIPS IN
3D-FLUORESCENCE MICROSCOPY IMAGES OF MAMMALIAN CELLS
Linnman C *, Bengtsson E *, Ekholm-Jensen S **, Zetterberg A **
* Centre for Image Analysis, Uppsala University, Uppsala, **Dept.
of Oncology-Pathology, Div. of Tumorcytology, Karolinska Institute,
Stockholm, Sweden