What is wrong with this DIC/fluorescence picture (image)?

I have a great interference image and I have a great fluorescence image
on a Leica TCS-SP system. However I cannot get these two great images at
the same time. Can confocal fluorescence and confocal interference be
done simultaneously? Are these compatible optical technologies or are
they incompatible?

The optical path of interference contrast (DIC) consists of a polarizer,
Wollastrom prism, objective lens, sample, condensor, Wollastrom prism,
analyzer and detector. When these components are properly configured the
microscope will yield excellent images using either conventional
fluorescence microscopy or confocal microcopy.

However these interference optical components will attenuate the
fluorescent excitation and emission light. Therefore in order to obtain
fluorescence simultaneously with interference it has been recommended
that the polarizer be removed to increase the fluorescence transmission.
This however decreases the quality of the interference signal greatly.

If the polarizer is not removed then the fluorescence signal attenuation
is huge necessating high PMT's and averaging and usually bad images.
.
What is the solution to this problem? Is the only solution to use very
brightly stained samples so signal attenuation is not a problem? Should
the samples be measured separately with each excitation system and then
accept the slight frame shift that will naturally occur?

Are interference and fluorescence really compatible on a confocal
microscope?

Bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
MD 72
National Health and Environmental Effects Research Laboratory
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: [log in to unmask]