Circular Oblique Lighting -COL (vs phase)

[Sansub2]

Hi,

I wanted try COL for some time after I read good information about it. My earlier half hearted try without proper understanding didn't yield good results so I just put it to "Todo" list. This long weekend gave me a chance to dig deeper. I carefully (?) read few articles online and started my trial and error. Most of the articles either didn't provide the patch detail or I don't understand it. This is my attempt to simply, share and learn along the way.

1. 10x objective - .25NA

Patch size:

Outer diameter: 32mm.
Inner block size: 6.9-6.8mm
Light window: 3mm.

After multiple trial and error, I noticed that the above comes close to getting the best hallow cone. Here are the sample images of COL and phase. I don't have see diatem test slide. Using zeeca seed.

Phase:
20220220_210722 by San Sub, on Flickr

20220220_210624 by San Sub, on Flickr

COL:
20220220_212130 by San Sub, on Flickr


20220220_212345 by San Sub, on Flickr

Sample COL video:


Here are the 2 versions of the patch. Not sure why you need the 2nd cone/light window. The first one works perfectly.


20220221_135726 by San Sub, on Flickr

On to 20x. Any guidance/tips are welcome.

[zzffnn]

Each scope model requires slightly different mask sizes. I eyeball it by looking at objective back focal plane (BFP); remove an eyepiece and see that the illumination ring takes up around 15-20% of the outside surface area.

I don't like the intense shadow effect (even though contrast will be enhanced), when the illumination ring gets too narrow, which tends to work better with the smallest protists that even 100x NA 1.32 won't resolve well.

If that illumination ring is too wide, then you would lose some contrast, which tends to work better with bigger and thicker protists.

I use a solid circle. The outside blocking comes from closing down the condenser iris to right around edge of objective perimeter (from viewing BFP).

I personally don't like effect produced by your type 2. Too much shadow to my eyes.

You may try increasing mask size by 2-3mm each time, with specific objective lens.

Concentric alignment is as important as mask size. As try to place mask as close to condenser iris as possible.

From your video, I think your mask size is slightly too big, but your eyes may be different than mine.

For below 20x NA 0.40, I prefer darkfield over COL. Just make the COL mask bigger to get true darkfield effect.

[Sansub2]

Zzffnn,

Thank you so much for the valuable response. You are right. I see a dark spot at the center. I will have to reduce it another.1 or .2mm to make it go away. It didn't appear in the slide test for some reason. Only shows in the live test.

As you mentioned, I am checking the illumination ring by using the phase telescope and using the iris to be just outside of the visibility from the objective ( by looking through the eye socket) and to make sure it is centered. Will try to get a picture for reference for others.

I do have dark field patches. Just trying the COL one step(objective) at a time .

BTW, did anyone try it with dark field condenser? I don't have one so no idea how it works. I take it, by closing it just enough to price COL using it? Only issue is, you have to do it every time.