Q on intensifying screens --correct-correction of fallacies!

stebby at welchlink.welch.jhu.edu stebby at welchlink.welch.jhu.edu
Mon Jun 8 19:02:18 EST 1998



The ISP for Johns Hopkins is so sporadic that I have resorted to
dejanews for posting.  That said, I find dejanews cumbersome at best
for manipulating messages.  I'm not sure who said what so I'll just snip and
paste as I go with my apologies to the authors.  I assume we all agree
that the end result of getting the data straight is the most important
point, yes?

The following people are posting in the following clips...

> dspencer at is.dal.ca
> hwr2Wdn2gWtM at dawntreader.bio.ukans.edu
> PGegen at UKans.nolospamare.edu


The relevant papers I have in my files are the following:

Enhanced Autoradiographic Detection of 32P and 125I Using Intensifying
Screens and Hypersensitized Film.  1977.  R.A. Laskey and A.D. Mills.
FEBS Letters Vol. 82 #2, 314--316.

X-ray Intensifying Screens Greatl;y Enhance the Detection by
Autoradiography of the Radioactive Isotopes 32P and 125I..  1978.  R.
Swanstrom and P.R. Shank.  Analytical Biochemistry Vol. 86, 184--192.

The Use of Intensifying Screens or Organic Scintillators for
Visualizing Radioactive Molecules Resolved by Gel Electrophoresis.
1980.  R.A. Laskey in Methods in Enzymology Vol. 65, 363--371.



Now...into the muck!  :o)


> > The original question was simple:
> > Does one need one or two intensifying screens, and if two are used,
> > how should they be placed?   The answer is equally simple: one
> > screen works fine for most purposes. The placement is
> > sample:film:screen (active side against film).

> That is indeed the logical way to set things up but you are being
> very presumptious to assume that most or all readers realize
> that.

> > Greater sensitivity can be obtained with two screens, placed on
> > either side of the film with the active side of each screen in
> > direct contact with the film. This will work for film with emulsion
> > of one or two sides, although double-sided film will be somewhat
> > more sensitive. This is the only placement of two screens that has
> > ever been suggested, and its benefits were clearly documented in
> > the original paper describing the use of intensifying screens
> > for 32P samples.

> The original paper on use of intensifying screens in molecular
> biology is the Anal. Biochem paper from the late 70's (Swanson
> and Schenk ?) which I don't happen to have here at home. As I
> recall, their recommendation for two screens was for use with one
> of the iodine isotopes.



Ok, Ok, here's a good place to stop.  There is actually some confusion
on these points that I would like to point out.  In the first paper
mentioned above, Laskey and Mills make the following statement on page
315...

"Secondly enclosure of the film between two screens (sample:screen
A:Film:screen B) also increases efficiency for 32P, but causes loss of
resolution through scattering by the screen which lies between the film
and the sample.  Efficiency of detection of 125I is not significantly
increased by use of 2 screens, because a single screen absorbs most of
the emissions from this isotope."

Laskey repeats this arangement for sample, film, and screens in the
Meth. in Enzm. chapter on p. 367.  Here's the rub...Swanstrom and Shank
in their paper above show a diagram on p. 186 clearly depicting the
following...

-------------screen 1
-  -  -  -  -film
-- -- -- -- -test strip with isotope
-------------screen 2

Back when I was in grad school this set-up made more sense to me than
Laskey and Mills.  In my hands, this was slightly better than one
screen and definitely more fuzzy.  Since I was doing phospho-amino acid
analysis I did indeed ahve long (4--6 week exposures) and a fuzzy spot
is not allthat different than a less fuzzy spot.  Your mileage may
vary.  Which, applies to the notes below.  I am not mentally equipped
to argue the physics of what is or is not happening with any certainty.
I will add fuel to the fire, however, and report that I documented
shorter exposures of 35S labelled proteins using a fluor in the gel AND
an intensifying screen on the other side of the film.  Why I did this
initially is a lesson, perhaps, in ignorance.  None the less, it works,
and I'm willing to bet it has something to do with the white shiny
background reflecting the light flash vs. the black cardboard of a
Kodak exposure cassette.  Never did test white shiny paper
though...perhaps because I didn't want to write another chapter for the
thesis.  :o)

Regards,

Steve Dahl

> > Two screens are expected to yield a less-sharp image, for the
> > obvious reason that the beta particles will spread out from their
> > source as they travel through the first screen to the film. Those
> > which travel through the film will be spread more, and the light
> > they produce from the second screen will spread out, too. However,
> > we are not talking Ansel Adams here! If you're looking for
> > a few bands or spots, and your choice is between exposures of 1
> > week vs 2 weeks, or 1 month vs 3-4 months (because of reciprocity
> > failure), you aren't in a position to complain about image quality.

> Even assuming only a minimal 5-fold enhancement that a single
> screen setup should give, a 1 month autoradiography exposure
> with a screen would be the equivalent of more than 5 months
> without a screen. What on earth would you be trying to detect?
> Good lord.



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