Phosphorimager

Abstract

Phosphorimaging is a form of solid‐state liquid scintillation where radioactive material can be both localised and quantified. Similar to traditional autoradiographic techniques (in that it relies on high‐energy particle decay), phosphorimaging can recognise radiolabelled deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein targets in a variety of sample preparations, such as gels, blots, homogenised tissues, cell populations, arrays and tissue slices. Although more expensive than traditional X‐ray film technology, phosphorimaging is more sensitive, develops images more rapidly and has a greater dynamic range than X‐ray film, which has made it a popular choice for several applications in molecular biology. Current applications include, but are not limited to, in situ hybridisation, ligand‐binding pharmacology, as well as Southern, northern and western blotting.

Key Concepts:

  • Phosphorimaging allows for both localisation and quantification of targets.

  • Potential targets include radiolabelled DNA, RNA, proteins and posttranslational protein modifications.

  • Phosphorimaging can be performed on media including gels, homogenised samples, cell populations, arrays and tissue slices.

  • Phosphorimaging identifies and quantifies its target through the measurement of light emitted from excited electrons returning to their ground state. These electrons are stimulated to higher energy levels by radioactivity emitted from the target and light is released as the electrons return to their ground state.

  • Although phosphorimaging is more expensive and has lower resolution than traditional X‐ray film detection and quantification methods, phosphorimaging affords greater sensitivity, faster image development, reusable detection plates and enhanced linear dynamic range.

Keywords: autoradiography; image plate; radioactivity

Figure 1.

The physics of phosphorimaging. Each phosphorimaging plate is coated with photostimulable crystals (BaFBr:Eu2+). When the plate is exposed to a sample (e.g. tissue section) containing a radiolabelled species (i.e. DNA/RNA/protein), the radiation emitted from the species in question (green line) excites an Eu2+ electron from a corresponding crystal, forming Eu3+ (red crystals). The electron is trapped within the bromine vacancies in the crystals until exposure to visible light at a specific wavelength (red line). This exposure releases the trapped electrons from the bromine vacancies, allowing Eu3+ to return to its Eu2+ ground state, causing photons to be released at a different wavelength (yellow circles). This permits accurate quantitation and localisation of the radiolabelled species within the sample. Following imaging, the plate can be erased via exposure to visible light, which returns the excited electrons to the ground state and enables reuse of the plate.

Figure 2.

Dynamic range of the phosphorimager versus X‐ray film. The phosphorimager maintains a direct relationship between signal intensity and radioactivity through an extremely wide dynamic range. Reproduced from Mori K and Hamaoka T (1994) Protein, Nucleic Acid and Enzyme (38)11.

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References

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Further Reading

Alwine JC, Kemp DJ and Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl‐paper and hybridization with DNA probes. Proceedings of the National Academy of Sciences of the USA 74: 5350–5354.

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Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

Neumaier JF (1998) Quantitative in situ hybridization detection of 5‐HT receptor mRNA in rat brain using storage phosphor imaging. American Biotechnology Laboratory 16: 61–62. International Scientific Communications, Shelton, CT.

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Van Kirk, C, Feinberg, LA, Robertson, DJ, Freeman, WM, and Vrana, KE(Sep 2010) Phosphorimager. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002973.pub2]