Dear Heather,

I learned my confocaling on that very instrument shortly after it was
installed.  Given how heavily used it was then, I'm sure there must be a few
experienced users who could help you out.

Fluorescently doped paper was the standard setup sample back then.  I
preferred to use freshly cut linen bond thesis paper which fluoresces reliably
without the stain.  Stick a piece to a slide with superglue, then douse it in
immersion oil or use it dry.  I have since become a fan of fluorescent beads.
Molecular Probes has some good alignment standards which I helped them
develop.  They excite with all common laser lines and clearly show loss of
colocalization when the system is misaligned.  For quantitation of
fluorescence signal, I prefer FCSC Quantum series of fluorescent beads, which
are what flow cytometrists use (hence Flow Cytometry Standards Corp.).  They
come in a range of intensities corresponding to a few thousand FITC molecules
right up into the millions.  I have ordered but not yet tried their PE
intensity series.

If you are the only person using the instrument, pick a pinhole setting and
don't change it.  If not, you will have trouble recreating scanning parameters
from a previous day's work.  PMT and black level dials have numbers on them,
so you could set them the same, then tweak the pinhole slider until a known
sample looks about the same.  This will be close, but not really rigorous
enough to call quantitation.  Another useful trick would be to view the image
in false color so that the level of saturation and black level cutoff is
clearly visible.

By the way, what dye are you using, and what does it stain for?

Good luck,

David Carter
oQQQQQ@


In a message dated 6/16/98 21:25:09, [log in to unmask] wrote:

<<As a new-comer to the wonderful world of confocal microscopy, I am
amazed at its potential but at the same time I often find
myself way out to lunch :)

If you haven't already guessed, I'am a novice user, of a Biorad MRC
600 confocal microscope to be exact.  As part of my masters research
I am using the microscope to observe fluorescently stained yeast
cells.  The dye I am using stains viable cells a diffuse green
(cytoplasm) with red intravacuolar structures.  I would like to
measure (quantitate) the fluorescence intensity of both colours
independently (I realize there will likely be some overlap - if
anyone has any suggestions for dealing with this situation your
advice would be greatly appreciated).  I have already been advised
that I will require some non-fading fluorescent standards (red &
green) that I can use to set up the microscope before testing my yeast
samples.  I have been told that some plastics (some kinds used to make
cheap toys) can work very well.  I don't know how true this is or
what plastics would be good.  I would really appreciate any
suggestions anyone might have on appropriate standards that I could
use.

Thanks in advance for any advice offered

Heather Heggart
M.E.Sc. candidate
University of Western Ontario, Canada
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