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Micro ion beam: New software module for quantification of protein stainings

10.05.2006

The evaluation procedures of radiobiological experiments performed on the microbeam were extended and refined so that the radiation effect can now be made visible and quantified for each individual cell. Within the scope of an EU project, the module serves to investigate the so-called bystander effect which characterizes the occurrence of a radiation effect in non-irradiated cells when neighbouring cells are irradiated.

In biology, processes in the cell such as the production or decomposition of a protein or the spatial aggregation of proteins to so-called foci, can be made visible with the aid of colours. The procedure called immunohistochemistry makes use of antibodies which specifically ligate to specific proteins (primary antibody) and of a second type of antibodies which carries a fluorescence colour and on its part ligates to the primary antibody.

Within the scope of the “Interstander” EU project, the apoptosis (programmed cell death) of the cells investigated is detected with the aid of immunohistochemistry. As the spatial arrangement of the cells at the moment of irradiation is important for investigation of the bystander effect, the cells must remain on the irradiation dish for evaluation. We have, therefore, implemented analysis routines for the images taken with microscope and CCD camera which allow the fluorescence intensity of the cells to be quantified and correlated with the positions of irradiated cells.

Here, the following aspects are important:

  • Standardized preparation of the cells to guarantee comparability
  • Transformation of the cell coordinates exact to the micrometer (Figures 1a and1b)
  • Effective cell recognition with a DNA staining (Figures 2a, 2b and 2c)
  • Post-processing of the images, in particular compensation of inhomogeneous illumination
  • Automatic processing of large data quantities
(1a)
(1b)

Figure 1: Transformation of the cell coordinates.
1a. Result of the cell recognition before the irradiation cycle.
1b. Assignment of the transformed irradiation positions after evaluation.

(2a)
(2b)
(2c)

Figure 2: Extraction of p53 intensities from the image files:
2a. Cells with blue fluorescent cell nuclei. This serves to determine the cell positions.
2b. Immunohistology of protein p53 (here: red fluorescence, untreated original image).
2c. False colour representation of the immunohistochemical colouring for the recognized cell nuclei. Cell nuclei which could be unequivocally assigned to an irradiation position are marked:
o = unirradiated (75 %), x = irradiated (25 %).

The tests so far performed show a significant activation of the protein p53 for the irradiated cells compared to unirradiated cells on the same dish (Figure 3). The comparison with completely irradiated and completely unirradiated carriers is not yet meaningful, as the variations between different cell dishes are of the same order of magnitude as the intensity of the effect to be determined. Consequently, our experiments do not yet allow a clear statement regarding the intensity of the bystander effect to be made. This is why further technical improvements and additional measurements are required. Limitations of the method are the generally high variability of biological parameters and the fact that influences of manual cell preparation (pipetting, washing processes, cleanliness of the cover slips "prior history" of the cells, etc.) cannot be controlled completely.

Figure 3: Histogram of p53 staining
Normalized distributions of the brightness integrated over the cell nucleus. Irradiated (25 %) and unirradiated (75 %) cells show significantly different distributions, which are overlapping.