Justin,

Your question seems pretty good - I wouldn't consider it basic. I'm
replying off-line because I'm only a software person - I've never
actually operated a microscope.

I know that one effect that we have to be concerned about is distortion
of intensity measurements from lower layers caused by optical density in
upper layers. If you have an object in an upper layer that absorbs a
given wavelength, it reduces the intensity measured from lower layers at
the same wavelength. The reason is that the light has to pass through
upper layers to reach the detector.

Your measurements, made at several wavelengths, seem to indicate that
this is not an obvious problem in your case. However, I wonder if such
an effect might still cause an apparent bias?

-- Dan

--
Dan Brown
Bruxton Corporation, Seattle
Phone: (206) 782-8862
Web: http://www.bruxton.com