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

Zhiqian Liu wrote:

> I am currently measuring leaf cuticle thickness using the vertical section
> images acquired from CLSM (either using reflection mode or after staining
> with a lipophilic fluorescent dye). The problem I am having is that the
> thickness values I get this way is far greater than those obtained with TEM
> for the same plant species and growth stage. Does anyone have experience
> with similar situations?

Dear Dr. Liu--

There are at least 3 possible sources for distortions in thickness:
dehydration, differences in index of refraction, and recording outside
the dynamic range of the instrument.

Dehydration will shrink tissue.  If your TEM sections were dehydrated
and the confocal sections were not, that might be the source of the
difference.

If you were not using an oil objective (or if you were, but weren't
using the correct immersion oil), datasets will be foreshortened in the
z-axis.  (It's like looking into an aquarium and having things appear
closer than they are.)  However, this will make the thickness measured
on the confocal appear LESS than it really is--not more.

Recording outside the dynamic range of the instrument (as well as using
too large a pinhole, spherical aberration, etc.) will allow objects that
are out of the plane of focus to appear to be in focus.  Cut the gain
and/or laser power back so that all pixels fall within the range of
1-254.  (The issues of spherical aberration and of choosing the best
pinhole size are worth considering but would take a while to discuss.)

One question: just how different are these measurements?

Good luck!

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                       office:  (612) 626 0145
Assoc Prof, Dept Neuroscience                     lab:  (612) 624 2991
University of Minnesota                 Preferred FAX:  (612) 624 8118
6-145 Jackson Hall, 321 Church St. SE        Dept FAX:  (612) 626 5009
Minneapolis, MN  55455              e-mail:  [log in to unmask]