Digital camera images and What can you see

J. Martinez-Irujo jjmirujo at unav.es
Tue Mar 13 14:47:36 EST 2001


WSchick at aol.com wrote:

> 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.
>

I agree that these devices are able to precisely measure the
optical density of the film. In our lab we calibrate the
scanner using a commercial photographic step tablet (density
range approximately 0.05 to 3). By eye and putting the film
on a white paper (reflectance mode )  you can see
differences in the 0 –1 O.D. range. If you  put the film
towards the light source (transmittance mode) you can see
differences up to 1.7 O.D. This density corresponds to an
overexposed film (by eye). By using the scanner it is
possible to extend this range and see differences above and
below this level that may not be evident to the eye (in this
case I prefer to put another film to get  the right
exposition). Within the 0.05-1.7 O.D. range, however,
significant differences must be evident both before (by eye)
and after image analysis, unless very small differences are
expected (30-40 grayscales correspond to 2.5-3% difference
in intensity).

While image analysis will provide an accurate measure of
density in the film, usually this is not the critical factor
in quantitation. If you run an internal standard, for
example several dilutions of a radioactive sample, and plot
O.D. vs radiactivity, you usually will find a linear
relation in a range. Below or above this range the
proportion does not apply. Moreover, in many cases the
extrapolated line does not cross the x=0, y=0 origin, so
doubling the amount of sample does not result in doubling
the measured O.D. There may be many explanations for this
behaviour and this is the reason that internal standards
must be present. In fact, is the user, not the software, who
by chosing a variety of user-selectable parameters that
affect the quantitation (background subtraction, band
intensity, band contour...) takes the responsibility of the
result.

> 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.

I agree. Non-linear dose-response curves can be easily
managed by non-linear regression using commercial
user-friendly software (Grafit being my favourite, no
affiliation, etc.)

--
Juan J. Martinez Irujo
Depart. de Bioquimica
Universidad de Navarra

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