Digital camera images and What can you see

[WSchick at aol.com]

In a message dated 3/13/01 1:39:22 AM Pacific Standard Time, jjmirujo at unav.es 
writes:

> And remember than most systems work with visible light, so do not expect to
> find any difference  your eye do not see...
> 
> 
On both scanners and digital cameras, you CAN find significant differences 
that your eye cannot see.  Our eyes are optimized for color, not B&W, so the 
general level of grayscale differences our eyes can see is about 30-40 
grayscales, or a little over 1 OD. 

Film such as a Polaroid or X-Ray is linear to about 80 grayscales, maybe you 
can calibrate to 100  which give you 1.8 to 2.0 OD.  And of course, your eye 
can still see only 30-40 grayscales on the film.  Cameras and scanners begin 
at 8bit imaging (256 grayscales) and some are 12 bit or even 16 bits.

This is why digital scanning for visible samples is useful in getting more 
information.  Or digital cameras for fluorescence, visible and even 
chemiluminescence if you get a cooled camera.

Just try the BWG controls on a digital image, and you will see faint bands 
that would not be noticed on film by eye.  The BWG compresses the black and 
white to any range, so you can see say 0-40 grayscales of a 0-256 grayscale 
image.  Without changing quantitation.  The original pixel by pixel value is 
what the software compares for quantification, not what an eye may see or not 
see.

Special curve algorithms are available for non-linear densitometry--these 
work fine for a digital image. And the internal standards in every gel can be 
measured and used to quantify PCR products, or unknown proteins, etc.

SCANNERS

You can use a $99 office scanner and get good MW reports.  But for precision, 
I'd recommend a professional $2000 transmission scanner from UMAX or Epson. 
These are only useful for visible stains--they don't have capability for 
fluorescence.

SOFTWARE

NIH Image is free, but if you have lots of users, an easy to use commercial 
software is a better long term choice.  General imaging software is 
$1800-$5000 from BioRad, AlphaInnotech, MediaCybernetics, but you should talk 
to reps or users before buying. The user interfaces differ greatly depending 
on the software writer.

CCD Camera Systems for fluors and visible stains

The least costly hood mounted models, including analysis software are about 
$4000.
Flexible general gel documentation should run around $10,000.  Top megapixel 
systems that also do chemiluminescence list aroung $40,000.  If I did a lot 
of isotope work, I'd consider the chemilumnescence imagers.

Walt Schick
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