good morning

Tim Fitzwater via methods%40net.bio.net (by tfitzwater from somalogic.com)
Wed Apr 16 09:21:08 EST 2008


Because an old Amersham Guide to Autoradiography contains the best explanation of pre-flashing that I've ever found, it seems simplest to generate a Reader's Digest version:

"Photographic emulsion consists of silver halide crystals suspended in a clear phase composed mainly of gelatin.  When a beta particle or gamma ray passes through such an emulsion the silver halide crystals respond, with each emission converting several silver ions to silver atoms."  

"These crystals constitute the grains of the film.  The silver bromide forms a regular lattice, however within each crystal there will be a number of faults.  When a photon of light enters the crystal, there is a high probability that it will give up its energy to one of the orbital electrons in the lattice.  When such an electron has acquired enough energy it will leave its orbit finally stopping at a fault.  Here, a silver ion accepts the electron to become an atom of metallic silver.  This silver constitutes the latent image."

"To produce a developable image each silver halide crystal requires several photons of visible light (approximately 5 in average emulsions), each of which produces an atom of metallic silver within the crystal."

"By comparison, when a beta particle or gamma ray passes through a photographic emulsion, energy is lost in a series of interactions with orbital electrons in the silver halide crystals.  This results in silver deposition at a number of faults, again forming a latent image."

"A single hit by a beta particle or gamma ray can produce hundreds of silver atoms from silver halide crystals in film emulsion, but a single photon of light from an intensifying screen produces only a single silver atom.  Although two or more silver atoms in a silver halide crystal are stable, a single silver atom is unstable and reverts to a silver ion very rapidly."

"This means that the probability of a second photon being captured before the First silver atom has reverted is greater for larger amount of radioactivity than for small amounts.  Hence small amounts of radioactivity are under-represented for both fluorography and intensifying screens."

"This problem can be completely overcome by a combination of pre-flashing the film and exposing at -70°C." -- Amersham

 

"Pre-exposure to an instantaneous flash of light overcomes the problem of low intensities of light producing disproportionately faint images.  It does so by providing many of the halide crystals with a stable pair of silver atoms.  Sensitivity is increased, allowing quantification of images because all photons contribute equally to the image on pre-flashed film."

"A linear response of the film to light produced from the radioactivity in the sample is obtained when the film has been pre-flashed sufficiently to increase absorbance of 0.1 to 0.2 (A540) absorbance units above that of unexposed developed film.  A further increase in the fog level above 0.2 reverses the deviation from linearity.  Thus a pre-flash of too much light causes over-representation of small amounts of radioactivity."

"Lowering the temperature to -70°C increases the stability of a single silver atom.  The reversion to a silver ion is slowed, increasing the time available to capture a second photon and thus produce a stable pair of silver atoms."

"Both pre-flashing and exposure at -70°C increase sensitivity on their own.  Only the combination of the two completely corrects the response of the film, giving a linear response to the amount of activity in the sample." --Amersham

 

Pre-flashing is essential if you want accurate densitometry data.  The 14C calibration strip (American Radiolabeled Chemicals, Inc. catalog # ARC-146) will allow you to determine the linear range of the film for each exposure and convert scan values to µCi.  This exposure strip can also be used to quantitate phosphorimager values as µCi rather than arbitrary units.  Densitometer and Phosphorimager can be calibrated for each exposure to directly convert to µCi values.

 

While not linear, temperature can be used to modulate exposure times when using a Lightning Plus intensifying screen.  If a -70°C exposure requires 1 hour, -20°C requires about 2 hours, 4°C requires about 3.5 hours and room temperature requires about 7 hours exposure.  This allows you to find an exposure that fits your schedule.  Note that if time in the freezer is too short the film won't have time to get cold.  The Lightning Plus intensifying screen has almost no effect at room temperature, so it does not need to be removed from the cassette.  A sheet of aluminum foil covering intensifying screens that are adhered to the cassette eliminates any effect by blocking screen response. 



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