The method of slit scanning involves the illumination by a ribbon shaped laser beam of elongated chromosomes stained with deoxyribonucleic acid (DNA) specific dyes. The chromosomes are aligned in the direction of the flow canal perpendicular to the laser beam. The morphology of the chromosomes is analysed through the digital time dependent registration of the fluorescence signal as the chromosome passes through the narrow laser beam.
The optical system was improved using a new detector lens system. This allowed an improvement of the resolution from 2.7 um to 1.0 um.
For the analysis of chromosome profiles computer programmes were developed to recognise dips in the profiles which indicate the place of the centromere. A pulse shape analyser was build which detects dips with an amplitude of 8% of the plateau value and a width of 1 um. A shape parameter named pulse dip index (PDI) was defined as the ratio of the integrated signal from the beginning of the pulse until the first dip, relative to the integrated signal of the complete profile. The PDI is similar to the centromeric index of chromosomes.
The composition of aggregates in mixtures of fluorescent particles of different sizes was evaluated by PDI analysis. In our experiments the PDI was determined within 30 us from the onset of the pulse profile and particles with a specified morphology of interest were selected for online registration of their profiles as digitized pulse shapes. In a cell sorter system, the PDI can be used as a parameter for sorting.
In general, particles with dips in their fluorescence profiles have elongated shapes inducing them to align in the direction of flow. This process facilitates the slit scanning analysis of particles such as sperm, cell aggregates and chromosomes. Using pulse shape analysis, objects with equal fluorescence intensities but of different shape can be distinguished by the PDI criterion. In this way peaks with overlapping fluorescence intensity distributions can be separated. Our results show that the PDI analysis can be used as an alternative method to increase the resolution of a measurement when dual beam equipment for 2 parameter fluorescence analysis is not available.
The main requirement for a successful application to small chromosomes is that they are isolated according to procedures developed to produce elongated chromosomes.
The length of the chromosomes was increased by incubating the unfixed chromosome suspension in a 37 C waterbath under constant movement. This procedure resulted in an appropriate length of the chromosomes without noticeable deterioration of chromosome quality. This was assessed by microscopic and slit scanning analysis of chromosome morphology, and by flow cytometric analysis of the chromosome fluorescence. The length of the larger chromosomes from human cells could in this way be in this way be increased up to 20 um. The chromosomes were kept on melting ice until flow cytometric sorting. About 10 minutes before sorting paraformaldehyde was added to a final concentration of 0.025% (w/v). This was found to be necessary for preservation of the morphology during the process of sorting and slide preparation.
Propidium iodide was chosen for isolation and staining because its intercalating action results in long chromosomes and it shows no base specificity.
It is generally accepted that only a fraction of the primary changes caused by a dose of ionising radiation results in effects observable as asymmetric chromosomal exchanges. In flow karyotyping analysis, on the other hand, it is to be expected that both asymmetrical and symmetrical translocations are detected. It has been suggested that damage in chromosomes, normally not expressed as breaks, might be exposed by the isolation of the metaphase chromosomes from the protective environment of the cell. Changes in the degree of condensation of the chromatin induced by radiation treatment, moreover, might affect the interaction of the inc alating fluorochrome propidium iodide with the DNA. This could result in a change in fluorescence intensity. These different factors could offer an explanation for the relatively large number of radiation induced events deduced from changes in the flow karyotypes.
The developments described make it now possible to apply the automated detection of aberrations to derive dose effect relationships for radiation exposure of human cells and finally to assess exposure of persons who received radiation (eg as a consequence of working conditions or treatments).