Dear Mark,

I cannot comment whether it is worth to upgrade your particular system, but DIC
in combination with confocal microscopy sure is great, in particular if you
want to do live cell imaging. If the microscope is set up efficiently, it will
offer you lots of additional information without increasing the light load on
the cells. I used to work on a Leica NT confocal that was set up this way: The
laser is used simultaneously for excitation of the fluorochrom (in the "normal"
confocal mode) and as transmission light source for DIC. Since the laser emits
polarized light anyway, you don't need a polarizer in front of the specimen
that would steal your photons from the excitation. The Analyzer is coming
behind the specimen and after that a cheap PMT (cheap compared to the
"confocal" PMTs: Since you're working with transmitted light you can get away
with comparatively low sensitivity.) So you're doing nothing to the cells that
you wouldn't do anyway for fluorescence microscopy, but you get the extra DIC
image with possibly additional information. Compared to Phase contrast, you do
not loose photons to the ring in the objectives. The theoretical drawback of
the setup described is that you have a prism in the light path of the confocal
light. However, with my specimens I didn't notice a loss in the resolution.

I put a couple of Quicktime Movies on the Web, showing live Drosophila embryos
and the distribution of Polycomb-GFP fusions in some tissues. The site is
http://www.life.uiuc.edu/belmont/dietzel/droso.html
In one of the movies I included the DIC-image side by side with the
GFP-Fluorescence. To get there, follow the link "Distribution of Polycomb
during blastoderm mitosis". When you look at these images, be reminded that the
embryo is about 200 to 300 microns thick. It also has a whole lot of optically
active ingredients like fat droplets. Whereas this does not influence the
resolution of the fluorescencent image, for the DIC the light has to travel
through all of that. With cell monolayers you should be able to achive much
better resolutions.


Steffen


--- "Dr. Mark W. Tengowski" <[log in to unmask]> wrote:
> Greetings again,
>
> I am thinking about adding DIC optics to my current confocal system (Nikon
> Diaphot 200, BioRad 1024 scanhead, Kr/Ar laser, transmitted light
> detector).  Before getting quotes, I have a few questions to ask the list
> in hopes that someone has been successful in this type of endeavor.
>
> Is it worth upgrading the Nikon base, since objectives are mostly
> discontinued?
>
> While it seems important for one of my more animated user, is adding DIC
> optics really a cost-effective upgrade?  The TLD currently sees limited use.
>
> If anyone has DIC merged with confocal pictures, please send me a jpg or
> reference.  I can tweak the TLD images to look more like phase, but the
> detail of DIC is definitely lacking.
>
> Respectfully,
> -Mark
>
> Mark W. Tengowski, DVM, MS, PhD
> University of Wisconsin Medical School
> W. M. Keck Neural Imaging Laboratory
> Department of Obstetrics & Gynecology
>

===
Dr. Steffen Dietzel
Dept. of Cell and Structural Biology
University of Illinois at Urbana Champaign
601 S. Goodwin Ave.
Urbana, IL 61801
Phone: +1/217/333-8372  Fax: +1/217/244-1648
e-mail: [log in to unmask] Web:  http://www.life.uiuc.edu/belmont/dietzel
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