X-Ray film vs Phosphor imaging - Understanding quantification of mRNA (Oct/07/2007 )
I'm trying to learn the ins and outs of quantifying mRNA via Northern Blots and I don't understand the physics behind x-ray film and why it has a smaller dynamic range than phosphor imaging. I understand that phosphor imaging is linear because you can essentially measure every photon of light emitted (I think...), but why does x-ray imaging end up giving intensity in a sigmoidal curve? I would like to use horseradish peroxidase as a marker. I would have to run a chemiluminescent analysis, yes?
"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."
"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."
With preflashing and exposure at -70°C , you still only get 1 to 1 1/2 logs of linearity. 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.
Thank you, tfitzwater
Yes, Thank you very much!! Your explanation was clear and easy to understand.