Read about the benefits of phosphor screens over x-ray film in high-sensitivity Western blot imaging.
Autoradiography for high-sensitivity Western blotting
Two methods of autoradiography are used in Western blotting:- X-ray film autoradiography
- Storage phosphor screen autoradiography (based on phosphor imaging luminescence)
Scientists have been using X-ray film autoradiography since radioactive isotopes were first used to tag biomolecules. Films remain detection option in today’s laboratories but new imaging techniques are quickly taking over. Autoradiography on photographic film converts emitted radiation into light. This conversion helps to increase the otherwise limited sensitivity of the radioisotope. However, this gain in sensitivity couples with decreased resolution and non-linear responses.
Alternatively, phosphor imaging offers a highly sensitive option for imaging western blots. The method deploys storage phosphor screens, which are up to 100-fold more sensitive than film for radioisotopes.
How does a phosphor screen work?
Each phosphor screen comprises a three-layer phosphor imaging plate. A photostimulable phosphor layer is sandwiched between a protective layer and a support film.
When exposed to high-energy radiation, photostimulable phosphor crystals excite and trap electrons. Placing the screen in an imager/laser scanner exposes it to visible light, releasing electrons to return to their ground state. An imager detects the emitting photons, producing a digital image that shows the location of radioisotopes.
In Western blot imaging, bands appear on the digital image wherever proteins labelled with radioisotopes are located.
As well as producing a visual representation of the blot, laser scanners (such as Typhoon) can precisely quantitate the radiolabelled proteins. This is because the amount of radioactivity is directly proportional to the amount of labelled protein.
Film vs phosphor screen
Storage phosphor screens overcome many of the issues associated with using film for radioactive detection:
1. Time
The exposure time for a film screen is significantly longer than for a phosphor screen. Exposure time for X-ray film is typically between 24 and 72 h. Phosphor imaging can be performed in 3 h or less due to increased detector efficiency.
2. Sensitivity
The high sensitivity of phosphor imaging is 10 to 100-fold that of film screens. This large range is dependent on the isotope used and is the result of high-density excitable particles on the phosphor imaging storage plate.
3. Dynamic range
The dynamic range for phosphor imaging (five orders of magnitude) is broader than for film (two orders of magnitude). A wider dynamic range is beneficial in imaging as it enables capturing of a wider range of signal intensities in the same exposure.
4. Logistics
Phosphor imaging does not require development chemicals or a dark room, which are both necessary for film. Storage phosphor screens also work on a variety of media including the gels themselves, membranes, microarray plates, and tissue slices.
5. Reusability
Storage phosphor screens are reusable as they can be erased following each use. In comparison, film creates a permanent record. Combined with the lack of chemicals required, phosphor imaging helps to reduce waste in the lab.