RE: Slide "Plains"

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From:"Gary W. Gill" <> (by way of histonet)
To:histonet <>
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"Fuzzy or hazy in some places AND crisp in other" does indeed point to too
much mounting medium IF you see a gradient of microscopic sharpness across
the slide, from one end to another.  That happens especially in cytology
(Pap smears)that can be so thick in some areas that it's impossible to fill
in the surrounding area with mounting medium. The problem can also occur
with thin cyto and histo preps when the technician applies viscous mountant
medium initially at one end of the slide and applies the cover glass by
lowering it from one side to the other, rather than front to back.  In the
former fashion, the cover glass creates a wedge-like layer of mountant, so
that it is thicker at one end and progressively thinner towards the other.
The artifact is more apparent when slides are freshly mounted than after
several days or weeks, when the mounting medium layer becomes more uniform
in thickness as it dries.  The artifact is also more apparent under a 40x
objective than it is under a 4x or 10x objective.  The latter two are
insensitive to cover glass thickness.

Therefore, try these tests to rule in/rule out mounting medium thickness as
the responsible factor:

*	Hold the preparation at eye level and carefully look at the
thickness of
mounting medium.  Is it thicker towards one end of the long edge than it is
the opposite end?  If yes, the mounting medium is suspect.

*	Look at the problematical areas under 10x and 40x.  If the haziness is
apparent under 40x but not 10x, excessively thick mounting medium is the

*	If the image quality improves under 40x when you close the substage
condenser aperture diaphragm slightly, the mounting medium is it.


*	Use No. 1 thickness cover glasses.

*	Apply as little mounting medium as is consistent with a permanent mount
after it dries.

*	Keep the microscope clean.

*	Use Kohler illumination  When a microscope is adjusted for Kohler
illumination, the substage condenser aperture diaphragm controls the working
numerical aperture of the objective.  Closing the diaphragm somewhat reduces
the working numerical aperture (which is different than the theoretical
maximum NA engraved on the objective [e.g. 0.65 for 40x planachromats]),
thereby reducing the sensitivity of the objective to cover glass and
mounting medium thickness. This is a very practical tip and is the working
microscopist's image salvage tool to deal with real life variation in
mounting medium and cover glass thickness.  Generally, the substage
condenser aperture diaphragm should be open more widely for higher NA
objectives than for lower ones.

The KEY to this troubleshooting advice is your having noted that the
sharpness varies across the slide.  If you said everything were hazy, then
cover glass and mountant thickness could be the culprit.  However, there are
other factors that can create the same appearance.  The haziness associated
with overly thick MM and CG thickness is due to spherical aberration, while
that associated with other causes is due to glare and flare (e.g., dirty
lens surfaces, wide open field and substage condenser diaphragms, pale
staining).  The latter two are scattered light that does not contribute to
image formation.  The visible outcomes of spherical aberration and
glare/flare are indistinguishable.  Systematic troubleshooting can pinpoint
the cause(s).

Gary Gill

> -----Original Message-----
> From: []
> Sent: February 25, 1999 7:20 PM
> To:
> Subject: Slide "Plains"
> Hello Histonetters, I was hoping you could help me a couple problems.My
> pathologist was complaining that the stained H & E sections in
> our daily work
> was somewhat on different "plains" and needed constant focusing
> while under
> the scope. I sat down with her and went over some slides, these
> sections were
> somewhat "fuzzy or hazy" in some places and crisp in other, which makes me
> think possibly it's the mounting medium...we've been using this medium for
> sometime (resinous) and unfortunately we're on contract so my options are
> limited (for purchasing that is).

> Kari Zajic HT,MLT
> Lead Histotech
> Florida

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