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In reply to Guy's comment in regard to using the hair sample for testing
the confocal microscope I should have mentioned that the hair can just
as readily be mounted in water (although physically glycerol is easier
to use as a mountant). Guy is right, spherical aberration is an
important consideration and so if you do have a water immersion lens
with correcting collar then water mounting would be better.

The good thing about the hair sample is that it has a low level of
fluorescence, is very stable from one day to the next and has fine
structural detail within the hair. The same sample can be used over an
extended period to check the sensitivity and resolution of the
instrument. Yes, the hair will act as a lens, but then so will all other
cell samples - and they are very difficult to reproduce even the next
day, never mind the next month.

Hair is not a perfect sample, but it does have some advantages over
using live cells to test the instrument.

Regards, Alan Hibbs.

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Australia


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Guy Cox
Sent: Tuesday, 3 June 2003 12:46 AM
To: [log in to unmask]
Subject: Re: Confocal service issue

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I can only agree with Alan about the difficulty in testing confocal
performance but I must disagree with his choice of sample!

The major testing difficulty is that few of us can compare different
confocal heads with identical optics, and as soon as we use different
optics their differences are likely to swamp the differences between
systems.  Just changing between standard and phase-contrast versions
of the same lens makes 20-40% difference in the final image intensity.

If we are working at the high  end of the market and are mostly geared
towards living cells we can use high-NA correction collar water
immersion coverslip lenses since most makers only have one offering in
that category and that can be part of our overall system performance
assessment.  But in other areas we might well find that the choice of
lens is at least as relevant as the choice of confocal head.

But I do feel that we should use imaging conditions that are as
aberration-free as possible which is why I think that a hair in
glycerol is not an ideal sample.  A hair is a cylindrical lens and
few of us use glycerol immersion routinely so there will also be SA.

In the past I've used the opposite strategy - samples which are
consistent in staining but which fade very fast.  The merit figure
is then the best signal/noise ratio that can be obtained from the
sample, period. We don't have to worry about such virtually impossible
tasks as matching laser intensities at the specimen.  Operator
skills do become important in this case, though.

                                                   Guy

--
Associate Professor Guy Cox
Electron Microscope Unit, F09
University of Sydney NSW 2006
+61 2 9351 3176

Until 25th July:
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