Posted by
Julien Cau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslips-tp6865241p6870643.html
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Hi there,
A main advantage of the SIM technique is that you need few if any
modifications in your sample preparation (but you need strong signal).
SIM, as any confocal/widefield, needs microscopy best practices.
That's said, then of course, it is more sensitive (as STED is), to
coverslip thickness errors as it needs the pattern to be well projected
for instance. But the coverslips are not any different from coverslips
you would use with confocal and listers should not believe SIM needs
fancy preparation of the sample. It just need care and the right
coverslips, as any nice confocal image deserves as well.
My understanding so far, and I might be wrong, is that the right
coverslip thickness is very important with good dry/water objectives
(and we noticed the difference) as well as oil objectives. Even if you
use a mounting medium with the right refractive index, you never really
have no RI mismatch in your preparation. And our experience with these
high precision coverslips shows, when using oil objectives, improved
light collection and image quality in sample like drosophila embryos,
even if we used the right refractive index mounting medium. So my
personal conclusion is it is important in any case anyway and not using
right coverslips (which cost a few more cents per item) is of no
advantage. If it doesn't help, it doesn't harm.
To answer another question, we usually recommand liquid vectashield or
hardening Prolong Gold as mounting media for oil lenses. Six years ago,
we requested users to bring their mounting media and measured their RI
with an Abbe refractometer. Among a dozen of immersion media tested
(including home-made mowiol), liquid vectashield (and glycerol) had the
right refraction index (hardening vectashield had a lower RI, although
it is hard to measure has you have to leave it harden in the
refractometer and I don't know whether the protocol is really correct or
not). Then, later on, when released, we tested Prolong Gold and
basically reproduced the RI over time ( a week!) chart the manufacturer
claimed. The advantage of Prolong Gold is it hardens (no idea whether it
modifies the geometry of the sample when hardening). Of course, there
might be other mounting media we did not test, or our measures might be
not precise enough, but we quite like them. We find both vectashield and
Prolong Gold are doing ok to protect sample from severe photobleaching.
Of note, at the time we tested several mounting solutions, from all
commercial media specs sheets provided by users, the only one to mention
the refractive index was vectashield. Again, this might be different
now, but in a "right refractive index mounting medium chase" I would
suggest the following criterion : does the specs sheets indicate the RI?
Then most of them usually include antifading reagents.
Hope it helped
Julien
--
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*/Julien Cau, PhD./*
/Montpellier RIO Imaging Facility manager/Responsable technique MRI/
Montpellier RIO Imaging
Montpellier BIOCAMPUS, UMS3426
Arnaud de Villeneuve Campus Imaging Facility
Institut de Génétique Humaine-CNRS
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