FREQUENTLY ASKED QUESTIONS
Q. What is a PhosphorImager system?
A. A PhosphorImager system is a quantitative imaging device from
Molecular Dynamics that uses storage phosphor technology as a faster, more accurate
replacement for x-ray film in life science imaging applications. Models include the
PhosphorImager 400, PhosphorImager 425, PhosphorImager 445SI, PhosphorImager SI,
Typhoon, and Storm® series of imaging instrumentation from Molecular Dynamics.
Q. How does the PhosphorImager system work?
A. See: Johnston, R.J., Pickett, S.C. and D.L. Barker.
"Autoradiography using storage phosphor technology." Electrophoresis 11,
355-360. 1990. Reprints are available by request from Molecular Dynamics.
Q. How sensitive is the PhosphorImager system?
A. It's about ten times more sensitive than film exposed without fluors
or intensifying screens. For 14C, 2 dpm/mm2/hour will give a signal
that is at least twice background. This assumes that the 14C is not buried in
an absorbing or deflecting material such as a thick TLC silica matrix. This level of
sensitivity is easy to achieve using 14C-glucose spotted on Whatman filter
paper or nitrocellulose paper. Storage phosphor is not very sensitive to tritium-labeled
molecules in gels, since the gel matrix prevents the weak emissions from reaching the
screen. It's better to use a fluor, such as Amersham AmplifyTM, and a low temperature exposure to sensitive film for this application. However,
PhosphorImager systems work well for tritiated whole-body-autoradiography slices, because
tritium is right on the surface of the sample. Special tritium screens are required for
this application.
Q. How long should I expose my sample?
A. PhosphorImager systems are linear over a wide dynamic range so
exposure time doesn't matter as much as it does with x-ray film. Typically, an exposure of
one-tenth of the time you would expose the sample to film gives a useful image for
quantitative analysis. Longer exposures give the best-looking images.
Q. How do I know whether I've over-exposed my sample?
A. This is rare, but if your sample is hot you might suspect
over-exposure if any spot appears black when the upper limit of the gray/color adjust
range is set to the maximum (100,000 Counts). Check by drawing a line that goes through
the darkest part of the spot and creating an intensity profile for it. If the top of the
intensity profile is flat, you've over-exposed. Erase the screen for the maximum erasure
time and do a shorter exposure.
Q. Will the PhosphorImager system do westerns?
A. Yes. Unlike the old beta-imagers, storage phosphor screens are
sensitive to emissions from all laboratory radiochemicals, x-rays, and UV. The exception
is tritium, which because of its low energy, requires a special uncoated screen. The same
rule of thumb applies: multiply your film exposure time by one-tenth to estimate the best
exposure time for storage phosphor.
Q. Will the PhosphorImager system do TLC plates?
A. Yes. But take care not to contaminate your storage phosphor screen
with 14C. Make sure that your TLC plate is absolutely dry before starting your
exposure, and place a sheet of Mylar between the TLC plate and the storage phosphor
screen. Eight-by-ten inch sheets of .001 inch thick Mylar (polyethylene terephthalate) can
be purchased from Fralock Corporation in Canoga Park, California (tel. 818-709-1288).
Q. Will the PhosphorImager system do chemiluminescence?
A. No. Latent images are formed on storage phosphor screens during
exposure to ionizing radiation. Chemiluminescent reactions yield visible light, which
erases latent images from the screens. For nonradioactive imaging, Molecular Dynamics
recommends chemifluorescence. Chemifluorescent blots are read directly in FluorImager® and Storm imaging systems for maximum quantitative accuracy.
Q. Will the PhosphorImager system do wet gels?
A. Yes. General Purpose screens are not harmed by brief exposure to neutral aqueous
solutions, but for wet gel exposures, the gel should be blotted free of excess buffer and
wrapped in plastic wrap to prevent prolonged contact between the gel buffer and the
screen. Caution wet gels should not be used with Low Energy screens.
Wrapping samples in plastic film is not sufficient to prevent moisture damage to
LE screens. All samples must be absolutely dry.
Q. What is a PhosphorImager "Count"?
A. A PhosphorImager Count is an arbitrary unit of measure that describes
the intensity of photon emissions released from the storage phosphor screen during
scanning.
A. The linear dynamic range of PhophorImager systems begins at one
PhosphorImager Count. Given the sensitivity of the PhosphorImager system, if the average
pixel value for your band is below one PhosphorImager Count, there are insufficient
particle emissions from the band for statistically valid quantitation anyway.
Q. Can I expose my screen at -70C?
A. The screens and cassettes are not designed for prolonged low
temperature exposure and cannot be replaced under warranty due to damage from low
temperature exposures. Low temperature storage phosphor screen exposures do not improve
sensitivity. Freezing temperature exposures are sometimes used anyway to prevent bands
from diffusing in a wet gel, and some customers have done -20C exposures with no apparent
damage to the screen or cassette. If you go ahead with this procedure, avoid allowing
water to condense on the surface of the screen. Condensed water on the screen will blur
the image and reduce sensitivity. One way to prevent condensation is to place the whole
screen and cassette in a large Ziploc bag before placing it in the freezer. After
exposure, remove your gel quickly-avoid exposing the screen to both air and light-and seal
the Ziploc bag again as quickly as possible. It may be helpful to include some desiccant
in the bag with the screen and cassette. Put your gel back in the freezer before it melts.
Leave the screen and cassette in the bag while it warms up to room temperature, then scan.
Q. How long do the storage phosphor screens last?
A. The reactions involved with latent image formation and erasure are
completely reversible. With proper care, screens last indefinitely. Things that damage the
screen and limit their lifetime are compression lines from ball-point pens, acetic acid
burns, contamination with long-lived radioisotopes (most often caused by incomplete TLC
plate drying) and backing plates that are warped or bent from being dropped.
Q. How do I clean the storage phosphor screen?
A. Storage phosphor screens can be cleaned using "Kodak
Intensifying Screen Cleaner and Antistatic Solution." (Kodak catalog # 106 4930).
This item costs about $12.00 for 16 oz. and is distributed by VWR.
Q. Where can I get the copper foil required for double-label image analysis?
A. Goodfellow Corporation carries metals and materials for research applications. In the US, call Goodfellow Corporation in Berwyn, Pennsylvania. tel. 610-640-1612, fax. 610-993-8065. Elsewhere, contact their headquarters in Cambridge, England. tel +44 (0) 1223 568 068, fax. +44 (0) 1223 420 639.
Copyright ©2000. Molecular Dynamics.