Suitability of epi-illumination for exhibition slide

pwnell · Post by **pwnell** » Wed Jan 09, 2013 11:13 pm

Charles mentioned a while ago to me that trying to use the epi-illuminator in the microscope to illuminate an exhibition slide such as this hand of flowers would be futile. So I tried it to see just how bad perpendicular lighting would be.

First the oblique lighting using my trusted Ikea lamps.

Then trying only the epi-illuminator (through the objective), single photo.

I do not even see the background as black anymore. Everything is completely washed out. And I have a weird concentric halo circling the butterfly scales - any idea what that is about?

curt0909 · Post by **curt0909** » Thu Jan 10, 2013 1:21 am

There are two problems with using epi bright field for this subject. 1. the glass slide and coverslip is reflecting some light back up through the objective. 2. the subject is not completely reflective which reduces contrast. Epi dark field would give better results. If that is unavailable try crossed polarization. This will give you a black background and reduce the reflection caused by the glass slide and coverslip. Hope this helps.

pwnell · Post by **pwnell** » Thu Jan 10, 2013 10:57 am

How do one perform epi-dark field? I use the fluorescence arm to perform epi-illumination, and I have therefore no condenser with a dark field stop. Oh by the way, this slide has a black background - the glass slide is not visible, it is only the cover slide.

Pau · Post by **Pau** » Thu Jan 10, 2013 11:24 am

You need an epi darkfield condenser with a stop ring and BD type objectives that have an external illumination path and of course a nosepiece to mount them because they have a wider diameter.

curt0909 · Post by **curt0909** » Thu Jan 10, 2013 11:44 am

I wasn't aware the slide had a black covering. The cover glass would be enough to cause quite a bit of reflection on its own.

Like Pau said, epi darkfield requires a BD(bright/dark) objective and an illuminator with BF/DF slider. I've never seen a dark field stop ring for epi illumination though. The kind I'm familiar with (Reichert and Nikon) is a special mirror. The bright field position is a 50/50 prism at 45 degree angle. The dark field position is a mirror at 45 degrees with a hole in the center that runs vertically.

Charles Krebs · Post by **Charles Krebs** » Thu Jan 10, 2013 7:19 pm

With a coverslipped slide, epi-brightfield is is like standing squarely in front of a window and trying to take a flash photograph of something just on the other side of the window (with a direct flash close to the lens). It is actually worse than that, because the light is coming through the objective itself, and is reflected straight back into it. In Epi-darkfield the light travels through a "cylinder" around the objective optics, and then a circular reflector at the tip directs the light onto the subject. You need a vertical illuminator that has a darkfield setting built in, or uses a "darkfield cube" to direct the light into the cylinder surrounding the objective optics while preventing light from entering the objective optics as would be done for epi-brightfield.

Epi-darkfield:
http://www.olympusmicro.com/primer/tech ... flect.html

pwnell · Post by **pwnell** » Thu Jan 10, 2013 8:12 pm

Thank you all for the valuable information.