Re; Nuclear volume, ploidy and Stain stochiometry

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Re; Nuclear volume, ploidy and Stain stochiometry

Alton D Floyd
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
Phone: 269.699.7182
Mobile: 574.215.0703
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Re: Re; Nuclear volume, ploidy and Stain stochiometry

Dale Callaham
It sounds like fluorescence of nuclei would be the way to go if the
mentioned factors could be adequately controlled.

I wasn't involved in the work but the professor I worked for in the
early 70's used the Feulgen staining and fluorescence of the Schiff's
reagent and a photometer to measure ploidy levels in root tissues; it
was a Leitz Ploem system with pinholes to isolate the nucleus to be
measured. I recall that there were preps of chicken RBCs (?) as a
calibration/standard. In root tips with rapid cell division I recall a
nice bimodal distribution was typical; blocking with colchicine gave a
switch to the higher peak. Older root cells often had multiples of the
2C level. It all seemed very reliable even back then - I recall them
letting everthing warm up and stabilize thouroughly.

Certainly instruments are more stable today, and LED light sources
should fix the excitation stability issue. Capturing a widefield image
can occur fairly quickly and so little bleaching should occur while an
image is captured. The pararosanilin-based Schiff's reagent is very
fluorescent (green excitation, Rhodamine set works nicely) and seems
very stable. Fluorescence should allow a much higher SNR relative to
abasorbance measurement. Antifade could be used to make it better.

A digital widefield image (with no saturation and a flat prep) should
allow multiple nuclei to be measured in one field using the ImageJ
features to integrate the pixel values in each delineated nucleus.

Dale



Alton D Floyd wrote:

> ---------------------- Information from the mail header -----------------------
> Sender:       ImageJ Interest Group <[hidden email]>
> Poster:       Alton D Floyd <[hidden email]>
> Subject:      Re; Nuclear volume, ploidy and Stain stochiometry
> -------------------------------------------------------------------------------
>
> 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
> Phone: 269.699.7182
> Mobile: 574.215.0703
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Re: Re; Nuclear volume, ploidy and Stain stochiometry

Hugo A. M. Torres
In reply to this post by Alton D Floyd
Dr. Floyd,

I appreciate your explanation on the stoichiometric problem.

Regarding this part:


> 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.

Should I take it then that in autoradiographic emulsions the silver
particles are uniform in size in  and do not change from manufaturer to
manufaturer, age, etc? If so can you recommend some reading on this? I
searched but found nothing.

> Al Floyd  
> 23126 South Shore Drive
> Edwardsburg, MI 49112
> Phone: 269.699.7182
> Mobile: 574.215.0703

--
Hugo Arruda de Moura Torres
==================================
Departamento de Biofísica
Universidade Federal de São Paulo
Rua Botucatu 862 7o. andar
CEP 04023-062 Vila Clementino
São Paulo - SP - Brasil
Tel:+55 (11) 5576 4530 r.220
Fax: 55 11 5571 5780
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Re: Re; Nuclear volume, ploidy and Stain stochiometry

Alton D Floyd
In reply to this post by Alton D Floyd
For autoradiographic grain counting, the type of emulsion, developer
used, and method of development all influences the grain size and
morphology.  When this technique was widely used, in the early 1960's,
most investigators used developers that produced tiny, round grains.  As
in any other type of quantitative measurement, the specimen preparation
and grain development must be highly controlled.  

When I was actively involved in this type of work, we found that we had
to create new calibrations each time we used a new batch of emulsion.
The calibration was created by making a series of slides, coating with
emulsion, and exposing for increasing lengths of time.  Then we took
reflectance measurements, and compared these to actual grain counts in
the measured areas.  With such a calibration, we found excellent
correlation between actual counts and reflectance measurements.
Obviously, the measurements were much faster, and permitted studies such
as localization of radioactive tagged materials in serial sections of
organs, such as rodent brains.

I don't remember all of the citations to these type of studies.  I will
check my old citation card file and see if I can come up with specific
citations that will give you a starting point for a citation search.

Al Floyd
23126 South Shore Drive
Edwardsburg, MI 49112
Phone: 269.699.7182
Mobile: 574.215.0703
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Re: Re; Nuclear volume, ploidy and Stain stochiometry

Derek Ingram
First of all I would like to thank everyone for their input and interest
regarding my initial posting.  I should elaborate on my initial
question. Annually the company I work for evaluates ploidy via Propidium
Iodide and flow cytometry. Alternatively we would like to develop a
technique to determine ploidy in various strains of salmonids using
IMAGEJ and preferably unstained blood smears. I would be more then happy
to forward some journal articles to whomever is interested regarding
ploidy determination through means of nuclear measurement. It has been
done before. The reason I am trying to utilize IMAGEJ software is to
speed up the processing of samples. I have no problem measuring total
cell measurements with other software. The issue I have with IMAGEJ is
isolating the nucleus, I'm sure this should be a simple task, but due to
my inexperience with the software I am having some issues. Again I would
like to thank everyone who is responding.

Derek Ingram

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
Alton D Floyd
Sent: Wednesday, February 18, 2009 10:49 AM
To: [hidden email]
Subject: Re: Re; Nuclear volume, ploidy and Stain stochiometry

For autoradiographic grain counting, the type of emulsion, developer
used, and method of development all influences the grain size and
morphology.  When this technique was widely used, in the early 1960's,
most investigators used developers that produced tiny, round grains.  As
in any other type of quantitative measurement, the specimen preparation
and grain development must be highly controlled.  

When I was actively involved in this type of work, we found that we had
to create new calibrations each time we used a new batch of emulsion.
The calibration was created by making a series of slides, coating with
emulsion, and exposing for increasing lengths of time.  Then we took
reflectance measurements, and compared these to actual grain counts in
the measured areas.  With such a calibration, we found excellent
correlation between actual counts and reflectance measurements.
Obviously, the measurements were much faster, and permitted studies such
as localization of radioactive tagged materials in serial sections of
organs, such as rodent brains.

I don't remember all of the citations to these type of studies.  I will
check my old citation card file and see if I can come up with specific
citations that will give you a starting point for a citation search.

Al Floyd
23126 South Shore Drive
Edwardsburg, MI 49112
Phone: 269.699.7182
Mobile: 574.215.0703
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Re: Re; Nuclear volume, ploidy and Stain stochiometry

Hugo A. M. Torres
In reply to this post by Alton D Floyd
On Wed, 2009-02-18 at 13:48 -0500, Alton D Floyd wrote:

> For autoradiographic grain counting, the type of emulsion, developer
> used, and method of development all influences the grain size and
> morphology.  When this technique was widely used, in the early 1960's,
> most investigators used developers that produced tiny, round grains.  As
> in any other type of quantitative measurement, the specimen preparation
> and grain development must be highly controlled.  
>
> When I was actively involved in this type of work, we found that we had
> to create new calibrations each time we used a new batch of emulsion.
> The calibration was created by making a series of slides, coating with
> emulsion, and exposing for increasing lengths of time.  Then we took
> reflectance measurements, and compared these to actual grain counts in
> the measured areas.  With such a calibration, we found excellent
> correlation between actual counts and reflectance measurements.
> Obviously, the measurements were much faster, and permitted studies such
> as localization of radioactive tagged materials in serial sections of
> organs, such as rodent brains.
> I don't remember all of the citations to these type of studies.  I will
> check my old citation card file and see if I can come up with specific
> citations that will give you a starting point for a citation search.

Excellent! I am interest in how those control experiments should be
conducted in more detail, I could start by reading one of your papers.
Would you recommend one in particular?

Setting aside the problem of silver particle size in emulsion
radiography for a moment and back to the problem of DAB
semi-quantification, Dr. Al Floyd said previously:

> ... 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.

Dr Floyd, sir, after reading this statement I am really intrigued: an
article from the American Journal of Pathology (Open Access PMID:
11159179) claims that at least three times the linearity between DAB
absorbance or concentration staining and antigen concentration was
established by incorporating antigens and antibodies at different
concentration combinations in mounting media and therefore suitable for
densitometry (references number 32,34,43).

Unfortunately I don't have access to these works from my institution and
I can't ponder on their quality as of now.

I did find another work though, where a similar approach was taken and
it seemed pretty robust for me. Now, I am not particularly experienced,
so I would like if you could take a critical look (or anyone else in
this list, I gladly want to hear): PMID 8773563

Also mentioned:
 
> 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.

True, but by creating standards one would still be able to compare,
semi-quantitatively, relative amounts of antigen between experimental
and control groups, say, expression levels of neuropeptide P between
transgenic and naive mice groups.
--
Hugo Arruda de Moura Torres
==================================
Departamento de Biofísica
Universidade Federal de São Paulo
Rua Botucatu 862 7o. andar
CEP 04023-062 Vila Clementino
São Paulo - SP - Brasil
Tel:+55 (11) 5576 4530 r.220
Fax: 55 11 5571 5780