Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Goran is right and the clever student is wrong.  I can't post
attachments to this list but I do have a diagram to illustrate
the point and you can find it in:
G.C. Cox, 1998.  Measurement in the confocal microscope.  In: Confocal
Microscopy, Methods and Protocols (ed. S. Paddock) .  Humana Press, Totawa,
NJ.  pp 357-371

The focus control on the microscope is NOT a ruler stuck into the
water, it is only measuring the APPARENT distance seen through the
microscope objective.  If you are using a dry lens and the sample
is is an RI of 1.5 then you will have to move the focus control
3µm (ie physical movement of the sample) to move the plane of focus
by 2µm.

                                                       Guy Cox

Quoting Stefan Gunnarsson <[log in to unmask]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi!
> We have been looking at rather thick sections of tissue, cut at 30-35
> microns, stained with propidium iodide, and mounted with DABCO mount
> (which I assume has RI around glycerol).
> With a 20x/0.7NA Imm lens with glycerol as immersion medium, the
> z-distance between top and bottom of the section (as measured by the
> fine focus device on the microscope) was the expected 30-35 microns.
> With a 40x/0.7NA dry lens, however, the same distance was only around
> 20 microns.
> The decrease in signal intensity away from the cover slip was slight in
> both cases, but a little bit more pronounced with the 40x lens.
> My immediate thought was that this was an example on RI mismatch, but
> the clever student objected, since the distance was actually measured
> with the step motor on the microscope. ("A stick in the lake will only
> seem to be bent, if actually measured with a meter the true distance to
> the surface will be revealed").
>
> Could someone please shed some light on this....
>
> tia,
> Stefan
> +++++++++++++++++++++++++++++++++++++++++++++++++++++++
> Dr Stefan Gunnarsson
> Evolutionsbiologiskt Centrum                    Evolutionary Biology Centre
> Enheten för biologisk strukturanalys          Microscopy and Imaging Unit
> Norbyvägen 18A
> SE75236 Uppsala, Sweden                 Tel &  Fax: +46 - 18471 2638
> +++++++++++++++++++++++++++++++++++++++++++++++++++++++
>


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