Re; Nuclear volume, ploidy and Stain stochiometry

Posted by Alton D Floyd on Feb 18, 2009; 3:41pm
URL: http://imagej.273.s1.nabble.com/Re-Nuclear-volume-ploidy-and-Stain-stochiometry-tp3693629.html

Derek Ingram recently asked about using nuclear volume measurements to
determine ploidy levels.  Setting aside the volume determination for the
moment, the real question is, can one accurately determine ploidy level
based on nuclear area as projected in an image?  This subject was
extensively researched in the 1960's, and one should look up papers by
Swartz related to such studies in rodent liver.  These older studies
demonstrated that only in the case of mammalian liver could nuclear
diameter (in thick sections) be demonstrated to have a consistent
relationship to nuclear ploidy, as determined by Feulgen absorption
photometry.  Remember that all of this absorption photometry (called
microspectrophotometry at the time) was done using end window
photomultipliers, with small spot sizes on the optical axis of the
device, or with scanning stages or Nipkow disc type scanners.  Anyone
contemplating doing Feulgen ploidy determinations should become familiar
with the extensive literature of the 1950's and 1960's.  With careful
work, CV's of approximately 1% (as good as flow cytometry) can be
achieved with careful work.  Remember that the flow cytometer was
invented specifically to speed up ploidy determinations.  However, by the
time the instrument was sufficiently developed, diagnosticians had lost
interest in ploidy (even though the literature was clear that this was a
powerful diagnostic technique).  The flow cytometer simply languished
until the advent of monoclonal antibodies.

With respect to fluorescence, this technique was introduced into
microspectrophotometry as a way to avoid the necessity for scanning
systems to integrate signal.  One of the authors who contributed greatly
to this effort (1960's) was Frank.  The many problems inherent in
fluorescence made this a problematic technique.  Issues are stochiometry
of dye binding, fading of dues, evenness of illumination, energy
transfer, and stability of light sources.  Many of these issues are still
very real, yet are generally ignored by modern workers.  The issue of
even illumination is a major problem in excitation of fluorescence.

In absorption photometry (brightfield), field correction is necessary.
Assuming a Feulgen stained specimen, only nuclei should be stained.  Any
area of the slide that does not contain stained or unstained specimen
should have the exact same value, it integrated nuclear measurements are
to be meaningful.  Microscope optics simply cannot do this, without a
correction for field illumination.  The routines often coded into cameras
or capture software are not very good at this.  Seldom do they provide an
constant value for "background".  In a transmitted light system, every
"white" area of the specimen must have an identical value for integrated
stained specimen components to be useful.  In the case of stained nuclei,
one way to check validity of data is to measure the same object (nucleus)
at many different locations in the field of view.  The obtained value
should be the same, assuming identical illumination intensity, exposure,
etc.

The issue of immunohistochemical staining raises a number of problems
that are ignored in current literature, and in fact, there are many
papers in the literature in the recent past that are simply misleading
because the technique used to generate the data are based on false
assumptions.  First, there are no current methods in use that can control
the multiple steps of amplification that are inherent in immunostaining.
Without this type of control, any derived data is meaningless.  Even
worse, the current practice in the U. S. is to employ peroxidase as a
readout enzyme system, with DAB as the chromogen.  DAB cannot be used for
absorption photometry, as it does not meet the Beer-Lambert criteria for
a phometric material.  It is not a true absorbing material, as it is
actually a particulate (the reason it is an effective stain for electron
microscopy).  As the concentration of DAB in a stained specimen
increases, more and more light is scattered outside the capture cone of
the objective, and therefore any measured signal is very nonlinear.  It
might be assumed that one could create a calibration chart, much as was
done with photographic grains for counting of autoradiographs.  This does
not work for DAB, as the particle size changes from manufacturer to
manufacturer and with age of the DAB solution.  Whether this is a real
change of particle size, or is due to aggregation of particles has yet to
be determined.

I hope this clarifies some of the recent issues raised regarding ploidy
and stochiometry.

Al Floyd  
23126 South Shore Drive
Edwardsburg, MI 49112
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