A call to Circular Oblique Lighting

[75RR]

Plan 40x/0.65, Brightfield, Oblique and COL, single images in Photoshop

This is a call to all fans of diatoms to consider Circular Oblique Lighting (COL) when choosing an illumination technique to view and photograph them.

http://www.microscopy-uk.org.uk/mag/ind ... pjcol.html

Navicula lyra Kemp 8 Form

[gekko]

I am not sure: are you making a case for COL? In terms of resolution, I think COL can resolve as well as brightfield provided the outer zone of the objective's aperture is illuminated (think of an undersized darkfield patch stop). My apologies if I am misreading your post; it is rather cryptic to me.

[75RR]

I am not sure: are you making a case for COL?

Yes. I think it works very well with diatoms
The 3 images (left to right - brightfield, oblique and COL) speak for themselves I think.
Personally I find the COL image jumps out and grabs one. Is it just me?
I have left it to the link to explain how to achieve it.

[zzffnn]

The even shadowing effect of COL does have its appeals, especially for highly symmetrical subjects like that diatom. Sometimes it produces too much glare and not enough contrast though (especially at low magnification), while offset oblique may cast too much and uneven shadow (especially at high magnification).

I have been wondering, what if one adds and slides a gradient grey filter to normal COL, for example under condenser. That would reduce glare, add contrast and a slight oblique effect. And no, I am not talking about oblique+gradient (as in UGF/GUF), I am talking about COL+gradient.

David Walker told me that how narrow (or wide) the COL ring is also matters (in addition to ring diameter). He seems to prefer slightly too narrow than slightly too wide, but overall he like offset oblique better for some unknown reason.

[gekko]

75RR, I agree that the third (COL) image is the "nicest", but to be fair, I think the first two are much too contrasty which detracts from their appearance. But despite that, I do think the COL image has more appeal, at least in your example above.

[75RR]

I agree that the third (COL) image is the "nicest", but to be fair, I think the first two are much too contrasty which detracts from their appearance.

Decided to have another go. This time I oiled the condenser to the slide.
Plan 40x/0.65, Brightfield, Oblique and COL, single image Photoshop

[zzffnn]

I did the experiments myself. COL+gradient grey or COL+Pol does reduce glare, but contrast enhancement is not really there (they both darken all parts of the image indiscriminately, but I want dark parts to be darker and light parts to be lighter).

My favorite is the middle ground between offset oblique and COL. In other words, COL offset slightly, so that the dark center circle touches on one side of the light ring. I tested this with a Frustulia diatom, which was resolved to dots at NA 1.23 and the balance among brightness/3D effect/contrast was pretty good. The key is not to over-do the offset and carefully use condenser-centering screws to fine tune shadow effects.

With slight offset, I did not have to use polarizer or gradient. I think a better internal-reflecting COL condenser, like Leitz Heine or Reichert Polyphos, should work much better, than my Abbe condenser with mask stops. It is kind of like cardioid darkfield condenser vs Abbé with darkfield mask/stops. Have to save up for a Leitz Heine.........

Here is an article by Yannis, who resolved Frustulia to dots using COL, a Leitz Heine condenser and a 60x NA 0.85 objective and green light: http://www.microscopy-uk.org.uk/mag/art ... atoms.html

[JimT]

To my eyes COL is less sharp down the center axis and better along the edges. Wouldn't stacking be able to do the same?

BTW, all three are images I would be proud of

JimT

[75RR]

Thanks zzffnn and JimT

Here is an article by Yannis, who resolved Frustulia to dots using COL, a Leitz Heine condenser and a 60x NA 0.85 objective and green light: http://www.microscopy-uk.org.uk/mag/art ... atoms.html

Interesting article - might try and see how close I can get to his results.
Surprised he sets his Optovar at 2.0, I find 1.6 is about the max I am comfortable with.
I have a couple of "unknown provenance" green filters, will try them as well.

[75RR]

Plan 40x/0.65 and Ph3 annulus, single image converted to black and white in Photoshop

Kemp's 8 Test Form

Gyrosigma balticum and Navicula lyra

Stauroneis phoenicenteron and Nitzschia sigma

Surirella gemma and Pleurosigma angulatum

Frustulia rhomboides and Amphipleura pellucida

[zzffnn]

Very good results with most diatoms.

Resolved diatoms are consistent with objective NA. Though not exceeding expection, like how Yannis did it (granted, Yannis "cheated" by using green light and a special reflecting condenser with a variable lighted annulus - Leitz Heine - which gives a significant advantage).

I think your 40x NA 0.65 may be able to do slightly better with Nitzschia, if you offset your PH3 annulus slightly to enhance contrast (reduce flare).

It may also due to your COL mask size not being optimal - your PH3 annulus may be too small or too big (I am sure with your skills, if you did carefully check objective back focal plane, you would have achieved optimal positioning).

What did your objective back focal plane look like with PH3 COL and Nitzschia? Usually, the closer the mask size to objective edge NA, the better (with reasonable range, if light output is enough).

With my rig, changing mask sizes/positions makes significant difference for balance between resolution and contrast. I have lots of mask sizes to choose from though, which makes it more precise than using phase annuli.

[75RR]

View through the Optovar's Bertrand lens - Plan 40x/0.65, Ph3 annulus, focused on a diatom.

[zzffnn]

It looks like lighted annulus falls within objective's edge NA, not touching or going beyond, right? Maybe offseting a bigger annulus would get your even better resolution/contrast, some DIY annulus may be needed though.