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

Stefan, I think you are right it is a RI mismatch. If we dive in to your
pond an bring a camera in a waterproof housing then the distance scale on
the camera lens will not show the right distance to your object because the
camera has a non-immersion lens. As a result you get spherical aberration
and crazy size measurements. You can also say that if you see a bent stick
then the microscope will also see a bent stick.

Göran Ocklind :-)

-----Ursprungligt meddelande-----
Från: Confocal Microscopy List [mailto:[log in to unmask]] För
Stefan Gunnarsson
Skickat: den 2 december 2003 10:41
Till: [log in to unmask]
Ämne: RI mismatch?

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
+++++++++++++++++++++++++++++++++++++++++++++++++++++++