Fluorescence autoradiography

The inclusion of a fluorescent dye into thin-layer plates can be used to detect substances that quench its fluorescence and so result in dark zones when the chromatogram is examined under ultraviolet radiation. Autoradiography can also be used in thin-layer chromatography and electrophoresis when samples are radio-labelled. 

Fluorescein-labelled deoxyribonucleotide triphosphates may be used in place of those labelled with 32P. Once the DNA sequences are separated by electrophoresis, the resulting DNA bands fluoresce and are analysed by a flu-orogram imager, which produces a picture of the fluorescent bands similar to the autoradiography produced when using 32P-labelled nucleotides. 

After drying the sheet with a cold fan P-labeled nucleotides are identified by autoradiography or a Phospholmager. Unlabeled substances are identified by fluorescence quenching (see below). 

Chemiluminescent labeling systems have been developed, based on the incorporation of fluorescein-11-dUTP into a DNA probe. An anti-fluorescein antibody covalently bound to the enzyme HRP is then bound to the incorporated fluorescein label. HRP catalyses the breakdown of luminol and the chemiluminescent signal is detected by autoradiography with X-ray film or by fluorescence scanning instrumentation. Chemiluminescence is more sensitive than enzyme-based color detection systems. Furthermore the labeled gene probes are stable and give results quickly (160). 

Analysis is best carried out by a fluorescence activated cell sorter (see 10.7.5) but, if the cells are pulse labelled with [3H]-thymidine immediately before harvesting the proportion of cells in S-phase in the various fractions can be estimated by autoradiography (see 12.3). The problem with this procedure is that the machines can become contaminated with radioactivity and the tritium may interfere with subsequent enzyme assays. Labelling of a sample after fractionation is a poor alternative, but prior pulse labelling with bromodeoxyuridine allows S-phase cells to be detected using a fluorescent antibody 12.7.5. 

Another approach is whole-manuscript neutron activation analysis (NAA) in a nuclear reactor, followed by gamma-ray analysis and autoradiography (4). Gamma-ray analysis permits identification of the elements contained in the manuscript, and autoradiographic analysis indicates the specific locations of these elements. A similar approach applied to art objects of historical interest is energy-dispersive X-ray fluorescence (XRF) analysis, which permits nondestructive semiquantitative elemental analysis (5). 

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