Phosphoimagers vs Instantimager

[Tracy Aquilla]

In Article <372o52$1no at charm.magnus.acs.ohio-state.edu>,
rsaldanh at magnus.acs.ohio-state.edu (Roland J Saldanha) wrote:
>>Very interesting. Where did you get this information (do you trust sales
>>reps)? It is my understanding that phosphorimagers are relatively accurate
>>for quantitation; at the very least, it's much better than time-tested X-ray
>>film autoradiography (see BioTechniques 16(2); 290-294; Carcinogenesis
>>13(8): 1475-1479; Electrophoresis 11: 355-360). The dynamic linear range of
>>the instrument I have used is reported to be approximately 65,000:1
>>(compared to X-ray film, which is about 300:1). I have checked the screens I
>>use for a linear response, and find them to be at least as good as film
>>(within the range that can be used for comparison), and several-fold more
>>sensitive. I don't know anything about the Instaimager. Care to elaborate on
>>this technology?
>>
>>Tracy Aquilla, Ph.D.
>>Molecular Physiology and Biophysics
>>University of Vermont
>>aquilla at salus.med.uvm.edu
>
>The most serious problem with the quantitation is the plate to plate variation
>and within plate variation for phosphorimagers.  In a test of Uniformity done
>by placing slice of the same activity 14C methacrylate slice over the 35x43 cm
>area Molecular Dynamics Technical Note #55 says "MD phosphorimagers have less
>than 10%(!!!mine) difference between the highest and lowest signals of all
>slices."  Clearly MD itself confesses to plate variation within a plate.  For
>any application requiring serious quantitation this is disastrous variability.
>

Does anyone know whether MD manufactures the screens sold by Bio-Rad for the
Bio-Rad phos-imagers? Any references pertaining to the Bio-Rad screens?

>These screens also exhibit reciprocity failure and thus very low signals could
>be lost.
>
>The linearity over five logs does not guarantee linearity in a single exposure
>- in the article you cite in Electrophoresis 11 355-360 the authors expose the
>plates for 10min, 4.5 hour and 17 hours.  This was undoubtedly done to avoid
>saturating the plate for high activity samples when detecting the low activity
>sample.  Clearly, this demonstrates a difficulty in measuring samples of
>greatly differing activity in a non cumbersome way (multiple exposure).
>
>Another problem also discribed in MD Technical note#55 is laser bleed.  Again
>to quote them " Bleed is caused by stray laser hiting high intensity signals on
>the storage phosphor screen around the pixel being excited.  Bleed generates a 
>real signal & will interfere with accurate quantitation.  This is especially a 
>problem when weak bands are within the bleed area of intense bands."

Again, does this apply to all imagers, or just Molecular Dynamics?

>
>Lastly, the time dependant decay of the signal can cause problems if 
>appropriate corrective measures are not taken (described in some of the 
>articles you cite or references within).
>
>A Technical note from Ambis a company producing competing technology claims 
>greater than 23% variation from run to run, greater than 25% variation when 
>same sample grid was placed in different orientations and a 4% plate to plate 
>variation for an MD phosphorimager.  You make your own judgement.
>
>The competing technology (companies such as Betagen, Ambis and Packard 
>Instantimager)  use some form of multi wire proportional counting & colimation 
>of the signal.  MWPC  consists of a 3-D array of wire grids with a suitable 
>electrical potential surrounded by a suitable gas.  Decay of particles are 
>sensed by the wires through secondary ionisations of the gas.  The advantages 
>are they offer, real time imaging, true counting (data corresponds to original 
>ionization events, not conversion from ionization to electron hole pairs to 
>light to digital data), no reciprocity failure, truely large dynamic range.
>
>The downside of these instruments is the poorish resolution (0.5 mm for the 
>Instantimager the current best machine in my opinion by contrast the MD machine
>is about 0.3 mm for 14C), small sample window 20 x 24 cm which will not 
>accomadate sequencing gels.
>
>Roland Saldanha

Thanks for this info.


Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aquilla at salus.med.uvm.edu