[Histonet] Re: Reference to microtome micrometer thickness

Fri Nov 28 11:33:37 CST 2014

This has to do with section thickness. Would ne nice is did no0t have
several other topics un der the same heading.

Hi had a senior moment earlier so now will try to complete my sentences
before pressing the send key..
I am not sure that it is essential to know accurately the thickness of
sections, but the consistency of the sections in a ribbon.
The microtome setting is only an approximate guide as the  section
thickness depends on many factors including the tissue density and
homogeneity, temperature of the block and room, speed and angle of cutting,
time between individual sections, whether the block has been moistened with
ice water between sections and finally the skill and mood of the operator.
In limits between 10 and 1 microns the thinner the sections the greater the
chance of uneven thickness between sections. I think that Steedman's book
on section cutting explained that an individual section is cleaved from the
block, and like cheese slicing  with a regular knife (sorry I know that you
are all recovering from food overload) the thinner the slice the tendency
is for less consistency between individual slices.
If you cut a ribbon of sections then the consistency of individual sections
once the slide is stained should be fairly obvious and easy to measure
using image analysis of density and/or color. It is possible with
experience to
determine the difference between 4 and 7 micron sections but I do not
believe the difference of 1 micron between individual sections can be
easily determined.

There are a couple of ways to be very accurate but I am not sure the effort
is really worth it.
1.  Use a homogenous block of protein such that is radiolabeled,  embed and
section with your block of tissue and use a counter on sections to
determine how much activity and therefore thickness of individual sections.
2.  Use a block of protein stained with solvent resistant dye and measure
using image analysis. As you know the concentration of the dye you can
determine how much is in an individual section. Many Procion dyes (chloro s
triazines)  bind tenaciously to proteins and are resistant to most chemical
used in the histology lab.
3. Use labeled or stained micro-spheres of known diameter in the block and
determine how many profiles you have when focusing through a section.
4. Use built in markers such as red blood cell in small vessels, there
should be enough there to determine approximate thickness.

While this an interesting problem I would suggest that the easiest
approach, unless you have no social life,  is to rely on the skill of the
microtomist.
Barry

On Fri, Nov 28, 2014 at 3:47 AM, Vernon, Richard I. <
richard.vernon <@t> thermofisher.com> wrote:

> Hi Maxim,
>
> Does measuring section thickness on slide scanners get affected by the
> surface imperfections on the section?
>
> It has been indicated to me that surface imperfections certainly hinder
> scanning speed but what about the actual thickness measurements?
>
>
> Kind regards
>
> Richard Vernon
> Strategic Product Manager - Sectioning Products
> Anatomical Pathology Division
>
> Thermo Fisher Scientific
> Tudor Road, Manor Park, Runcorn, Cheshire, WA7 1TA, UK
> Tel:+44 (0) 1928 534122 | Mobile:+44 (0) 7825 119070
> richard.vernon <@t> thermofisher.com | http//:www.thermoscientific.com
>
>
>
>
>
> -----Original Message-----
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> Subject: Histonet Digest, Vol 132, Issue 29
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> Today's Topics:
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>    1. Re: Reference to microtome micrometer thickness (Barry Rittman)
>    2. Congo Red problems (Wheelock, Timothy R.)
>    3. Re: Congo Red problems (Carlos Defeo)
>    4. RE: Formalin smell in the last paraffin station in vip    6
>       tissue tek (Jamal)
>
>
> ----------------------------------------------------------------------
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> Message: 1
> Date: Wed, 26 Nov 2014 12:05:53 -0600
> From: Barry Rittman <barryrittman <@t> gmail.com>
> Subject: Re: [Histonet] Reference to microtome micrometer thickness
> Cc: "histonet <@t> lists.utsouthwestern.edu"
>         <histonet <@t> lists.utsouthwestern.edu>
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> It has been a while since I read articles regarding section thickness but
> I have some comments:
> 1.
>
> On Sat, Nov 22, 2014 at 8:37 PM, Maxim Peshkov <Maxim_71 <@t> mail.ru> wrote:
>
> > Reuel,
> > It is right, that there are many external reasons for a thickness of
> > paraffin sections.
> > It is possible to measure the final stained sections with mathematical
> > methods by slide scanner.
> > I hope that the slide-scanner can be easily to calculate thickness of
> > section by their soft.
> > Each individually sections will have own specific thickness.
> >
> > Sincerely,
> > Maxim Peshkov,
> > Russia,
> > Taganrog.
> >                        mailto:Maxim_71 <@t> mail.ru
> >
> >
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