>Thickness of an optical section is actually a confusing term. Some people
>use it for the stepsize in z and some use it for the optical resolution in
>z. To get correct answers, you may want to clarify what you are referring
>to. (Although, personally I wouldn't know for the MRC600 in either case)

This is absolutely right.  But whether you use an MRC600 or any other
model of confocal microscope will make no difference (so long as it has
a variable pinhole).  The things that determine the optical thickness
(ie what depth of the sample is actually imaged in a single slice) are
1) the opening of the pinhole and
2) the numerical aperture of the lens (it depends on 1/NA^2)
The mechanical step thickness is determined by the stepper motor (or
other focussing device) and the fine-focus mechanism.  On a BioRad
MRC600 the stepper motor does 100 steps per rotation, and if you look at
the scale on your fine-focus knob you will see how many microns it does
per turn.  With a Zeiss Axiophot one step is 0.18µm and with a Nikon
one step is 0.1µm.

A simpler method of measuring the optical thickness is to do an XZ
section through a mirror surface (the mirror coating should be on the
underside of a number 1.5 coverslip - get your SEM lab to sputter-coat
some coverslips for you).  Take a vertical line through this using the
'length' command in MPL (the profile you get from the COMOS menu isn't
acurate enough) and read off the Full Width Half Maximum (FWHM).  This
is the distance from halfway between peak and background on the upper
side to halfway between peak and background on the lower side.  This
gives the figure you want - it should be around 500nm with an oil-immersion
lens of NA 1.4 and the pinhole closed all the way down.

                                                        Guy Cox

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