Phosphor imaging screens can be used as an alternative to film for recording and quantifying autoradiographic images (Johnston et al., 1990). They can detect radioiso- topes such as 32P, 125I, 14C, 35S, and 3H. There are several advantages of phosphor imaging over film: (1) linear dynamic ranges are 5 orders of magnitude, compared to ∼1.5 orders of magnitude for film (Fig. 6.3.2); (2) exposure times are 10 to 250 times faster than with film; (3) quantification is much easier and faster, and most imagers come with software to directly analyze data; (4) fluorography and gel drying are often unnecessary because of the sensitivity of phosphor imaging; and (5) phosphor screens can be reused indefinitely if handled carefully.
Phosphor imaging screens are composed of crystals of BaFBr:Eu+2. When the screen is exposed to ionizing radiation such as α, β, or γ radiation, or wavelengths of light shorter than 380 nm, the electrons from Eu+2 are excited and then trapped in an “F-center” of the BaFBr− complex; this results in the oxidation of Eu+2 to Eu+3, which forms the latent image on the screen. After exposure, the latent image is released by scanning the screen with a laser (633 nm). During scanning, Eu3+ reverts back to Eu+2, releasing a photon at 390 nm. The luminescence can then be collected and measured in relation to the position of the scanning laser beam. The result is a representation of the latent image on the storage phosphor imaging plates. The image can then be viewed on a video monitor and analyzed with the aid of appropriate software.
Some companies (e.g., Bio-Rad) offer different screens for use with different isotopes. They vary principally in the protective coating on the screen, which is optimized for low- or high-energy β particles or γ rays. No coating is typically used for weak β emitters such as tritium. More recently, screens have also been developed that measure chemilumines- cence. Such screens are particularly valuable for use with many nonradioactive labeling protocols.
The protocol below is for the PhosphorImager system from Molecular Dynamics; other phosphor imaging systems are available from Bio-Rad, Imaging Research, and National Diagnostics. 101 102 103 104 105 106 107 108 10-1 100 101 102 103 104 105 0 .05 1 1.5 2 2.5 3 Disintegrations/mm2
Phosphor imager signal Densitometric counts OD
Figure 6.3.2 32P dilution series quantified on Model GS-525 phosphor imager (squares), com-
pared to film (circles). Image courtesy of Bio-Rad, Hercules, Calif. Detection and
Quantitation of Radiolabeled Proteins in Gels and Blots
Materials
Gel or filter (e.g., from immunoblotting; UNIT 6.2)
PhosphorImager system (Molecular Dynamics) including: ImageEraser light box
Exposure cassette with phosphor screen Scanning software
1. Erase any latent image on the phosphor screen left by a previous user, or caused by background radiation, by exposure to visible light.
The PhosphorImager system comes with an extra-bright light box (ImageEraser) for this purpose. Standard laboratory light boxes may also be used.
2. Cover gel or filter with plastic wrap to protect the exposure cassette. Place wrapped gel or filter in the PhosphorImager cassette and close to begin exposure.
The gel does not have to be dried for this procedure. The phosphor screen is affixed to the lid of the cassette. Exposure times are typically one-tenth of the time required for film exposure.
3. After exposure, slide the screen face down into the PhosphorImager system. 4. Select the scanning area using the software supplied with the PhosphorImager and
start scanning.
The blue light emitted during scanning is collected to produce the latent image. 5. Analyze and quantitate the image using the software provided.
6. Erase the phosphor screen by exposing it to visible light as in step 1.
COMMENTARY
Background Information
The ability to detect radiolabeled proteins is critical to many studies in cell biology. A vari- ety of labeling methods are described through- out this manual, many of which are used to follow protein purification, protein processing, or the movement of proteins within the cell. More often than not, detection of radiolabeled proteins is coupled with the resolving power of SDS polyacrylamide gel electrophoresis (SDS-
PAGE; UNIT 6.1). Radiolabeled proteins sepa-
rated on gels can be used directly to obtain an autoradiographic image. Alternatively, pro- teins separated by SDS-PAGE are frequently
transferred to membranes (UNIT 6.2) and detected
using radiolabeled probes such as antibodies
and 125I-labeled protein A. The autoradio-
graphic image, whether generated on film or a phosphor screen, reflects the distribution of the radioactive proteins on the two-dimensional surface of the gel or filter. Molecular sizes of radiolabeled proteins, therefore, can be deter- mined by correlating their positions with mo- lecular markers. Also, the density of the band images can be used to determine the relative
quantities of the radiolabeled proteins in the sample.
Critical Parameters
The sensitivity of the detection device and the strength of the radioactive signal are the two most important parameters for autoradiogra- phy. Sensitivity can be enhanced by treating samples with fluors or by using intensifying screens (Table 6.3.2). Because phosphor imag- ing is 10 to 250 times more sensitive than film (Johnston et al., 1990), this technology makes it possible to monitor radioactive samples that would previously have gone undetected with film.
A second important parameter is the range over which the measurement device is linear. Film requires preflashing in order for the inten- sity of the image to be linear with respect to the amount of radioactivity, particularly for weakly radioactive samples (Laskey and Mills, 1975, 1977). Phosphor imaging offers a much wider linear range of measurement (5 orders of mag- nitude compared to 1.5 for film; Johnston et al.,
1990). This makes it possible to accurately Electrophoresis
and
quantitate very weak or very strong radioactive samples.
Troubleshooting
Cracking is one of the most common prob- lems encountered when drying gels. This often occurs if the gel is removed from the dryer before it has adequately dried or if drying tem- peratures are too high. To overcome this prob- lem, drying times should be extended and the performance of the vacuum pump and heater unit should be checked. For many gels, particu- larly for those with high percentages of poly- acrylamide or >1.5 mm thick, cracking can be reduced by using an alternative fixing solution containing glycerol (3% glycerol/10% glacial acetic acid/20% methanol; see Support Proto- col 1).
Among the biggest problems encountered in autoradiography are images that are either too weak or too intense. Such problems can be solved by varying the exposure time. Estimat- ing initial exposure time is difficult, since the amount of radioactivity in the sample is often unknown. A Geiger counter can offer some guidance with certain isotopes. For highly ex- posed film, the length of time in developer can be reduced to produce a lighter image. It is particularly important to remember that if ac- curate quantification of the film image is de- sired, film must be preflashed so that there is a linear relationship between the amount of ra- dioactivity in the sample and the image inten- sity.
Artifacts, such as black spots and stripes, can be avoided during developing by making sure that no moisture comes in contact with the film
and that films exposed at −70°C are brought to
room temperature before developing. Also, it
must be noted that β particles from weak iso-
topes such as 3H cannot penetrate plastic wrap,
and plastic wraps can attenuate signals from 35S
and 14C up to two-fold.
Anticipated Results
The protocols described here should yield a film image of a gel that can be quantified, stored, and photographed for publication.
Time Considerations
Fixing a gel will require ∼45 min. Drying
will take an additional 2 hr for a gel 1 mm in
thickness. Incorporation of a fluor will add ∼45
min to the processing time.
For gels >1.5 mm thick or with >15% acry- lamide, an additional 30 min will be required
for fixing and ∼30 additional minutes will be
required for drying.
The length of exposure for films in autora- diography can range from a few minutes to a few weeks, depending on the strength of the radioactivity in the sample. Most exposures last from several hours to a few days. Exposure time can be reduced more than 10-fold with a phos- phor imager.
Literature Cited
Chamberlain, J.P. 1979. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal. Biochem. 98:132-135.
Johnston, R.F., Pickett, S.C., and Barker, D.L. 1990. Autoradiography using storage phosphor tech- nology. Electrophoresis 11:355-360.
Laskey, R.A. 1980. The use of intensifying screens or organic scintillators for visualizing radioac- tive molecules resolved by gel electrophoresis. Methods Enzymol. 65:363-371.
Laskey, R.A. and Mills, A.D. 1975. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56:335-341. Laskey, R.A. and Mills, A.D. 1977. Enhanced auto-
radiographic detection of 32P and 125I using in- tensifying screens and hypersensitized film. FEBS Lett. 82:314-316.
Contributed by Daniel Voytas and Ning Ke Iowa State University
Ames, Iowa
Table 6.3.2 Film Choice and Exposure Temperature for Autoradiography
Isotope Enhancement method Film Exposure
temperature
3H Fluorography Double-coated −70°C
35S, 14C, 32P None Single-coated Room temperature
35S, 14C, 32P Fluorography Double-coated −70°C
32P, 125I CaWO
4 intensifying screens Double-coated −70°C
Detection and Quantitation of Radiolabeled Proteins in Gels and Blots