Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>[snip]
>> interface, so these effects might not be as pronounced. Alternatively,
>> if you are using a high NA water immersion objective, make sure the
>> coverslip correction collar is correctly set. I would expect
>> significant aberration problems if this were not set optimally.
>
>As slight topic drift here but... In general do people bother to measure
>every coverslip with a micrometer before you use it or do people do
>something else, eg. measure a few from a box, or assume that #1.5 are
>170um?
>
>Ian
Just a short word or two on a long topic.
The exact coverslip thickness is much less important on oil-embedded
and immersed specimens. This is good.
But it sometimes leads people to think coverslip thickness is never
all that important to the quality of the image so you can have thin
ones when you want to look deep into thick specimens etc., and thick
ones to stand up to rough handling.
However, it is crucially important when you want to use high-NA dry
lenses or water lenses. This is because, while, the RI's of oil and
glass are very close, those of air or water and glass are not.
One more thing, knowing the thickness is good but you shouldn't
automatically assume that you just measure the thickness and set the
collar to the same number of microns. These collars are often
miscalibrated.
You know what you have it right when the image looks the same when
you go out-of-focus above as when you go out-of-focus below and, as a
practical matter, this can only really be done using a point object
such as a fluorescent bead or light coming through a small hole in a
metal film,
Cheers,
Jim P.
--
****************************************
Prof. James B. Pawley, Phone: 604-822-7801
3D Microscopy of Living Cells: Summer Course CELL: 778-869-2348
"If it isn't diffraction, it is statistics":Microscopist's complaint, Anon.
|
