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On Oct 7, 2011, at 10:02 PM, Milton Charlton system wrote:
> Has anyone compared confocal images obtained with #1 (0.13-0.17mm) and
> #1.5(0.16-0.19mm) cover slips using 40X(1.25NA) and 63X(1.32 NA) oil
> objectives or similar?
>
> Mounting media: Is there any consensus on the best mounting medium
> that
> combines high refractive index (near RI= 1.5) with preservation of
> fluorescence?
Sorry to contribute to this conversation kind of late, but a few
points –
Keep in mind that in practice there's *a lot* of variation in
thickness between commercially available coverslips, even within the
#1 and #1.5 range, respectively. A core director I know recently did a
microcaliper survey of a number of brands of #1 and #1.5 cover slips
and came up with descriptive statistics for them. I was shocked at how
far off the 0.17 mm mark most fell, with #1.5's generally being
notably thicker than the 0.17 average they are supposed to have.
The alternative is to buy coverslips specifically sold as 0.17 mm with
very low variance (Zeiss, and I believe other manufacturers, carry
these), which are expensive, or start picking through boxes of
coverslips with microcalipers. But if you have correction-critical
applications, such expense or effort may be called for.
This, of course, is assuming you're using an objective that is
optimized for a 0.17 mm cover slip.
As for mounting media, keep in mind that even where mounting media
matches glass, the further down below the coverslip the sample is, the
more the mounting media contributes to optical aberration. In other
words, optimal coverslip thickness is *really* optimal coverslip plus
mounting media thickness. Again, for fully optimized optics, try to
have the specimen actually in contact with the bottom of the coverslip.
Peter G. Werner
Program Assistant,
Merritt College Microscopy Program
[hidden email]
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