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Morning Stephan,

        Since this is a little late, I am aware that some of what I have
included, and saved for a few days in "Drafts", is repetitious, but I
decided to send it anyway, because it is slightly different from Bob
Palmer's and might also be useful as I initially structured it.

        First of all, however, there is a great resource on the web that
will be of value to you at:

URL:
http://www.olympusmicro.com/primer/techniques/fluorescence/fluorotable1.html

        I can recommend one stain with which you can begin to learn not only
the instrument but also the methods of imaging.

        Acridine orange (AO) fluorescence can distinguish between
double-stranded and single-stranded nucleic acids in both preserved and
freshly killed (but permeabilized) cells.   [AO is also used to distinguish
between the living and the dead so it has the disadvantage of requiring
perhaps too much work.]  Excitation is at ~490nm and fluorescence occurs at
~530nm and ~630nm, a very appropriate beginning for a dual wavelength system
that has 488nm excitation and an interference filter in the 530-90 range to
separate the fluorescence channels, even if you must scan one channel at a
time to get decent spectral separation (depends on your dichroic and
bandpass filter combination).  The applications of AO can be found in the
journal literature for cell biology, microbiology and virology.  It has been
in use since the fluorescence microscope became accessible in the '50's and
'60's.  You should have no trouble finding references, even on the web, that
provide protocols for its use on cells in culture, etc.  I suggest AO,
because it is easily applied and relatively inexpensive, though for the work
you want to do, you should start with at least a relatively pure preparation
(the indicator of purity to a biologist, in a case like this, is that the
substance does NOT kill the cell - that is, can be used as a vital stain,
and doesn't combine with 'other' objects.  I have used old AO, which one can
find in most biology or microbiology department stores or in a pathology
department.  A detailed description of AO methodology can be found, at least
temporarily, at:

URL:  http://www.does.org/masterli/cy0703.htm

        Finally, you can find a wealth of information on fluorescence and
fluorescent materials at the site of Dr. Haugland's "Molecular Probes".
His catalog has become a manual/text for those who wish to use as well as
those who wish to learn fluorescence microscopy.

URL:  http://www.probes.com/

        I have some experience with a method that can demonstrate/prove? a
valuable comparison between the imaging (and resolving) powers of CLSM and
SEM at tissue levels of resolution rather than cellular.  While it is not as
easy as AO, it is straight forward and easily learned.  This method is
called "corrosion casting" and involves the perfusion of a vascular bed in a
tissue/organ with an unpolymerized Me-methacrylate mixture which is then
permitted to polymerize.  If one uses a proprietary, commercial methacrylate
preparation called "Mercox Blue", one has a preparation that will, after
removal of the surrounding tissue, cleaning, and critical point dying to
prevent collapse of the 3D vascular cast, a 3D network that is readily
suspended in immersion oil (1.47 n) and imaged with objectives from 20X to
100X (the Mercox Blue" is fluorescent using HeNe(R)(633nm) and a 660nm long
pass emission, though I have used an Argon laser (488nm) to collect a
green-yellow fluorescence signal).  The same network can be pre-viewed in a
SEM (or LVSEM/ESEM to reduce charging) to provide comparative SEM images
(stereo?).  NOTE that in the literature on SEM viewing of corrosion casts
there is a stack of publications that detail rigorously derived algorithms
by which vascular volume fractions can be calculated from tilted SEM pairs,
etc.  Needless to say, confocal microscopy provides an easier path to such
estimates than SEM.  The reference for this approach is:  Czymmek, K., et
al.(2000), Imaging and volumetric quantitation of vascular corrosion casts
with laser scanning confocal microscopy, Microscopy and Microanalysis,
6(Suppl. 2, Proceedings), pp562-563, ISSN: 1431-9276 MIMIF7, Springer.

URL:  http://www.sgi.com/features/2002/aug/dbi/dbi_pg2.html

        For the methodology, you may search PubMed for Hossler, F (the
expert caster) and/or Monson, F (the second of the two Fred's and the
baggage carrier of the pair).  The use of CLSM to image what amount to
anatomic objects is not usual but is very rewarding to one who is on a
confocal learning curve.

        Finally I cannot close without directing you to the following site
at which you can have relatively free access to the expertise of Dr. James
Pawley's writings on the subject of 3D microscopy.

URL:  http://www.cs.ubc.ca/spider/ladic/course/chptrpdf.htm

        On a second email, I will attach an example of a z-projection of an
image stack from a vascular cast from the wall of a pig urinary bladder that
I collected on an Olympus FV-300 CLSM (w/Argon(488nm), HeNe(G)(543nm) and
HeNe(R)(633nm) lasers).

Hope this helps,

Fred Monson

Frederick C. Monson, PhD
Center for Advanced Scientific Imaging
Mail Drop:  Geology
West Chester University
West Chester, PA, 19383
http://darwin.wcupa.edu/casi/
Phone/FAX:  610-738-0437



-----Original Message-----
From: Coetzee, Mr S. H Physics Science [mailto:[log in to unmask]]
Sent: Friday, April 04, 2003 12:26 AM
To: [log in to unmask]
Subject: Standard stains (New to CLSM)


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Dear All
I know this is a "how long is piece of string" question but I need help with
"general" stains.

We have a Zeiss LSM 5 pascal.  It has sadly not been in use for more than
two years due to a lack of interest.
I have spent most of my time establishing the EM lab.  Installing cupboards,
electricity connections, fume hood and all necessary ancillary equipment and
train the first users in ESEM.  I am now putting some energy towards
attracting users to the EMU including the CLSM.  Our mayor use will come
from Microbiology, Botany and hopefully one day some from the medical
profession.  I know it will take time and effort to coach the community as
well as train the users.  Before we can even dream of attempting this we
must "stock" some "general" stains to get people excited and going.
Specific stains for projects will come later.

Can you please send me a list of general stains and their protocols?  (We do
not have a UV laser.) One's with a long shelf life would be nice.

Thanks

Stephan H Coetzee
Department of Physics
EMU
Private Bag 0704
Gaborone,
Botswana

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