Radiation & Fluorescence Imaging
Radiation and fluorescence imagers that can be purchased from companies like Bio-Rad, Molecular Dynamics, Fuji and Canberra Packard are generally obtained for two reasons: a) faster and more efficient band detection as compared with x-ray film; b) the greater dynamic range of these instruments compared with film. These imaging devices are reported to cope with a range of 6 OD units or even more. This makes it possible in principle, to quantify very weak and very strong bands in the same experimental run.
These instruments produce suitable images for analysis using image analysis programmes. While many instruments such as those listed in these Web pages are purchased with such software, there is frequently a real need for further processing specific to electrophoretic separations (both 2D and 1D). Phoretix software tackles all the problem areas including background subtraction and band edge detection with a graphical display of software or user decisions and their consequences. [see also WWW links]
Storage Phosphor Imaging
x-ray film emulsion detects beta and gamma radiation inefficiently (95% of beta particles
emitted from 32P - labelled material in gel or TLC media go unrecorded). Good quality
x-ray film has a response range of up to 3 OD units. This will permit differences in
labelling up to 1 part in 300 to be recorded. For quantitation, however, the useful linear
range of film is far less, perhaps one-third of the response range.
Fluorescence or luminescence labelling is increasingly being used as an alternative to radioisotopic labelling. The image is acquired and digitised either by a low-light camera (sample illuminated with a UV trans-illuminator UVP, RAYTEK) or using a photomultiplier combined with a laser used to excite the fluorescence emission.
Bioluminescence & Chemiluminescence
Some fluors (for example fluorescein), chemiluminescent and bioluminescent samples provide much weaker images that require scanning imagers analogous to the storage phosphor imaging systems described above. Molecular Dynamics has the PhosphoImagerŪ FluorImagerŪ and StormŪ instruments, in which samples are excited with a laser and scanned with a photomultiplier. This excitation wavelength is appropriate for a limited number of fluors, including fluorescein, and the instruments are suitable for moderately bright fluorescence emission. Chemifluorescence is useful for the detection of both proteins and nucleic acids on blots and other solid media. Chemiluminescence can be captured with CCD cameras with a high dynamic range e.g. 16 bits (1 - 65,535 counts). Applied Biosystems Perkin.-Elmer now offer Tropix Wester-Star Blotting kit. This uses the chemiluminescent substrate 1,2-dioxetane which offers high sensitivity protein detection via alkaline phosphatase-labelled antibody conjugate at exposure times of 1s to 30 min. The substrate produces a chemiluminescent signal with enhanced kinetics. This allows multiple exposures over many hours, unlike peroxidase - luminol systems.
How it works
Exposure of a storage phosphor screen (Kodak, Fuji) to ionising radiation produces a latent image. This is read out with a red light laser which scans the image plate inducing a blue phosphorescence from the stored image. The light is collected by a photomultiplier to form a digitised spatial image.