Oblique vs COL +Pol +offset retarder vs UGF vs phase and others

[zzffnn]

gekko,

I filmed the test diatoms because I usually only shoot videos. Also, I wanted to push my rig to its light sensitivity limit. Videos need more continuous light and demand balance between brightness, contrast and resolution.

My camera (Olympus E-PM2) does videos at 1080p and 20Mbps, which seems decent considering its age.

Those two videos' resolution and contrast was optimized for Navicula lyra, by the way.

Edit: I apologize, as my test diatom slide is partially damaged / shifted by heat (from my previous halogen light). Klua Kemp sent me a perfect slide, but I messed it up

The combo of COL+POL+retarder gave significantly better 3D effect than UGF and it resolved Navicula to dots in video (whereas UGF only resolved it to lines in video), at least to my eyes.

But the combo filter takes more time and patience to set up optimally. And for different test diatoms, this combo performs very differently - Navicula lyra and Pleurosigma angulatum looked best at the diaphragm/filter stop setting of the COL+POL video, but not other diatoms for example Frustulia rhomboides looks worse.

In comparison, UGF seems to flatten out some thick diatoms like Navicula lyra and favors thin diatoms like Frustulia rhomboides.

[gekko]

zzffnn, many thanks for your elaboration. I must admit that this seems too complex for my simple old brain. I will post the results I got with details of what I did (which I suspect will not be useful in addressing the problem you pose) and then I will do it again hopefully more correctly after I get your feedback.
By the way, your camera is superb (I use an E-P1, much older than yours).

[gekko]

zzffnn, this is my first attempt (basically a proof-of-concept). I can repeat it using a different diatom or method, depending on what you think would be most useful. In order to make the comparison as fair as possible, I wanted simply to post a crop from the center of the full-resolution image without any editing or downsizing. I hope this is somewhat useful, but as I said, I would be glad to repeat the comparison, taking your feedback into account.

Diatom: I used Pleurosigma angulatum from Kemp's 8-form test plate. This has a distance between striae of about 0.53 µm.

Microscope: I used my 40x phase contrast objective (Nikon, CF N Plan 40/0.7 DL, PH3) on the Nikon Optiphot microscope with 1.25x magnification in the head, and a CF PL2.5x projection lens. I used a green interference filter over the field lens as otherwise the chromatic aberration fringes would have been very prominent. The condenser was Nikon Phase Contrast 1.25 condenser (Abbe type), except that for the DIC test I used the Nikon DIC achromatic-aplanatic condenser. Both condensers were used dry and the condenser aperture size was just shy of the objective aperture. For COL, I used the Ph4 setting of the phase condenser. For Phase + Oblique, I blocked the right side of the light by holding an opaque object below the condenser. PH + POL + FW Ret = phase contrast with cross-polarized light and a full-wave retarder.

Camera: I set my camera (Olympus E-P1, resolution: 4032x3024 pix.) to save the files as jpeg, monochrome; I set the following camera parameters (range -2 to +2): Contrast +1, Sharpness +1, Saturation 0; Noise filter LOW, ISO 200.

In some cases I took a second image (I refocused). Those images are designated (2).

images: the images were cropped using Photoshop Elements, but were not otherwise edited.

[zzffnn]

gekko,

I often use good camera and produce poor images

I did some experiments today and found that there is no perfect universal solution for all applications and all subjects. For example, filter setting/position/size/iris stop optimized for one diatom A does not necessarily work best for diatom B.

COL+POL+retarder provides better latitude contrast and resolution. But it is picky with filter size, position and iris. I actually did not have optimal central stop size for NA 0.65 I had one stop that is too big and another that is too small - I ended up using the bigger one decentralized slightly - when not decentralized, I got interference colors or a dark spot). But if stop is decentralized slightly too much, resolution would be reduced. Likewise for iris opening.

UGF is more universal, at the cost of flattening latitude resolution and contrast. For example, UGF works well even with Petri dish holding 1 mm of pond water (without cover slip). Whereas COL+POL+retarder produced unnatural interference color.

[zzffnn]

Thank you gekko. I found your post very informative.

Your oblique and DIC look best among those photos, to my eyes. COL is not far behind the two. Minor difference may be caused by different focus points. Maybe full size uncropped photos will tell more difference, especially at the edge and borders?

Did you try to close down your condenser diaphragm slightly, especially with COL? That helped a lot in my rig.

I never thought of using PH + POL + FW Ret. That seemed to take away too much light in your setting. Have you tried using flash?

[75RR]

This was a better comparison I think, using cropped and unedited Pleurosigma angulatum. (Less potential processing variation)
I used Navicula lyra instead, fearing that the Phase and particularly Phase + Oblique would be too muddy with a 40x/0,65
Would have been nice to see results for Off-axis oblique. Though I would not expect any surprises there.

I think the results so far have shown that COL (Circular Oblique Lighting) bears more attention as does Off-axis oblique.
Two good additional reasons to add Phase to one's system.

[gekko]

Thank you gekko. I found your post very informative.

Your oblique and DIC look best among those photos, to my eyes. COL is not far behind the two. Minor difference may be caused by different focus points. Maybe full size uncropped photos will tell more difference, especially at the edge and borders?

Did you try to close down your condenser diaphragm slightly, especially with COL? That helped a lot in my rig.

I never thought of using PH + POL + FW Ret. That seemed to take away too much light in your setting. Have you tried using flash?

Thank you. Full size, uncropped photos will have to be posted as a large number of small pieces or require a wall-sized monitor, so it would not be practical, I think. Closing down the condenser diaphragm more eliminated all details as the resolution fell below what was required to show the striae, and in COL, where I used the phase annulus of the phase condenser, the condenser diaphragm is not available.
Sorry about the PH + POL + FW Ret: it was a misunderstanding on my part-- I thought it was one of the things you wanted. I still do not have the ability to use flash-- sorry.

[gekko]

This was a better comparison I think, using cropped and unedited Pleurosigma angulatum. (Less potential processing variation)
I used Navicula lyra instead, fearing that the Phase and particularly Phase + Oblique would be too muddy with a 40x/0,65
Would have been nice to see results for Off-axis oblique. Though I would not expect any surprises there.

I think the results so far have shown that COL (Circular Oblique Lighting) bears more attention as does Off-axis oblique.
Two good additional reasons to add Phase to one's system.

Thanks for your comments. You were right about phase results being muddy. I tried to use the Phase + Oblique as defined by zzffnn. I'm not sure what Off-axis oblique is, though. Actually, I have never liked the COL results I get, although I must admit that you do get very good results.

[75RR]

I'm not sure what Off-axis oblique is

It is what David Walker calls offsetting the brightfield ring in the phase condenser to achieve oblique. Thought it would be best to use the same name for it as he does.

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

[zzffnn]

Closing down the condenser diaphragm more eliminated all details as the resolution fell below what was required to show the striae, and in COL, where I used the phase annulus of the phase condenser, the condenser diaphragm is not available.

^ That may depends on specific diatom and specific microscope system. In my system with COL+POL+offset retarder, closing down condenser iris very slightly gave significant contrast boost and 3D effects to Navicula and Pleurosigma, but ruined resolution for Frustulia.

Edit: I suggested uncropped photos, as your good photos look very similar to each other, except for slight variation in focus deph. I was wondering if borders of diatoms may tell a different story - in 75RR's photo, it was easier for me to tell difference by looking at diatom borders (borders showed difference in resolution and 3D effects). But I understand if you don't want to show borders.

[apochronaut]

One of the aspects of these resolution tests, I would like to see controlled, although I don't exactly know how to rule out the human factor in this, is that excessive post processing can skew the results so much. I am very much a traditionalist. I like microscopes and optics; I don't anywhere near as much like computers and silicon chips. If we are testing the optical resolution of various techniques, then we need to keep the post processing to that level which brings the image back to the visual view, because all too often the captured image is not as good as what the eye sees and a small amount of post processing helps to restore the original experience.
I agree that the test subject needs to be one of the more easily resolved ones , but one just on the edge with commonly available achromats, something like Frustulia Rhomboides, if using 100X 1.25immersion objectives and Gyrosigma Balticum if using 40X .65 dry objectives. Other specialized objective types, such as water immersion,higher N.A. ,fluorite or apo probably shouldn't be used , mainly because not everyone has them and they don't compare well. I also don't think we should be using easily resolved subjects, with lots of contrast because that also tends to skew the results and present a poorer technique in a better light.

[gekko]

zffnn, I agree that it is very difficult to determine the exact plane of focus using diatoms (they act as 2-D diffraction gratings and there is more than one plane where they appear in focus. I will repeat the test (not this week, though) focusing only on the edge as you suggested. Many thanks for your input.

75RR, from the link you provided, David Walker says, "Zeiss 40X NA1.0 apo at full aperture with 50% oblique using an offset brightfield port in phase condenser. Oblique 45° to long axis" and I still don't understand what that means. My best interpretation would be what zzffnn described, as blocking the light to the condenser from one side although I did it from the right ("east") rather than "south east" as I vaguely understand David Walker's "Oblique 45°" to mean. But then I am likely wrong. If someone can explain this to me for the next iteration, I would be grateful. Sorry if I am too slow!

apochronaut, in the test I conducted there was absolutely no post-processing, as I stated (I let the camera do the conversion from digital data to jpeg image using the highest quality jpeg, and I set the image parameters and those remained constant for all the images). As far as I can determine, the human variable was the choice of the focus plane, and the human-cum-camera variable was the exposure (I think that I should have adjusted the exposure using the histogram to obtain similar density since the phase images came out too dark). I think the diatom I used, Pleurosigma angulatum, would, more-or-less, satisfy your criterion: striae spacing of about 0.53 µm and I used a 40x/0.7 objective (in fact, closing the condenser aperture to about 90% of the objective aperture completely obliterated the striae), but please correct me if I am wrong (I guess I could have used Petrodictyon gemma with striae spacing of 0.50 µm as a more stringent test).
I would very much appreciate your suggestions for improving what I did, as I plan to redo the test, this time focusing on the edge of the diatom as zzffnn suggested which, I think, should give much more consistent focus planes.

Thank you all for your input.

P.S. In any case, I think phase should be lower in resolution than the other methods because the condenser aperture under phase is determined by the outer diameter of the phase annulus, which is always (at least in my experience) smaller than the objective aperture. The same would be true for COL if the outer diameter of the COL annulus used corresponds to a smaller NA than that of the objective used

[zzffnn]

Thank you gekko and Apo for your kind comments.

I tried to filmed all test diatoms in my video, but I previously broke Gyrosigma and Pleurosigma into pieces, so you cannot see them well there.

My scope setting was optimized for Navicula at the time (so we can try to match what 75RR got). But I found that setting did not work well for Nitschia or Frustulia, even though it worked well for Stauroneis and reasonably for Pleurosigma. Each diatom demands different scope setting, NA and resolution/contrast balance.

[apochronaut]

P.S. In any case, I think phase should be lower in resolution than the other methods because the condenser aperture under phase is determined by the outer diameter of the phase annulus, which is always (at least in my experience) smaller than the objective aperture. The same would be true for COL if the outer diameter of the COL annulus used corresponds to a smaller NA than that of the objective used[/quote]


Yes, phase can be disappointing as a method for high resolution, unless it is based on fluorite or apo objectives and then the outer diameter of the phase annulus would be proportionately consistent to the actual N.A. of the objective, which is usually quite high. It is however , one of the best methods for the separation of details that it can resolve. I will be posting some various phase images, when I get a moment.

[75RR]

75RR, from the link you provided, David Walker says, "Zeiss 40X NA1.0 apo at full aperture with 50% oblique using an offset brightfield port in phase condenser. Oblique 45° to long axis" and I still don't understand what that means. My best interpretation would be what zzffnn described, as blocking the light to the condenser from one side although I did it from the right ("east") rather than "south east" as I vaguely understand David Walker's "Oblique 45°" to mean. But then I am likely wrong. If someone can explain this to me for the next iteration, I would be grateful. Sorry if I am too slow!

My understanding is that the 50% oblique refers to how far the brightfield port is offset (in this case halfway across the field of view), the 45˚to long axis refers to the angle of the offset port to the diatom. Long axis refers to the length as opposed to the width of the diatom.
I believe he has a rotating stage which allows him to set the angle he wants.

I have a rotating stage on my xmas wish list.

Hope this diagram helps.

[zzffnn]

^ Interesting 75RR. I have not read that article, but I offset my retarder that way (I eyeballed my results and thought 45 degrees of around 50% offset works good as starting point, it helps when light hits at an angle I guess).

[75RR]

^ Interesting 75RR. I have not read that article, but I offset my retarder that way (I eyeballed my results and thought 45 degrees of around 50% offset works good as starting point, it helps when light hits at an angle I guess).

Well worth reading.

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

[gekko]

75RR, yes that was what I understood from zzffnn's description earlier, and then after I looked at the article you posted the link to, I learned that I should have used the 45-degree angle. Thank you for your excellent illustrations. I guess I need my i's to be dotted, and t's crossed to understand stuff.

All: I am still not sure what the purpose of this test is. My best, albeit vague guess is that it has to do with determining the method that gives best visibility to the diatom, i.e. the best compromise between resolution and contrast. But this is very complex, given that available choice of optics and diatoms-- too many degrees of freedom. With Lyrella lyra that 75RR used, one can use a number of objectives with different powers and different NAs to resolve them, and a number of contrast enhancing methods. Sorry again for being so dense, but I am still not clear what the purpose here is. Initially I thought it was that, given a certain diatom that can just barely be resolved by a given objective, to determine which contrast enhancement technique gave the clearest image. But now I don't think I was right, rather it is something much more complex, and my thinking about it is still very vague.

[zzffnn]

gekko,

My initial purpose was, using a transparent protist such as test diatom, determine:

1) compare various forms of oblique and COL vs. UGF. See what are pros and cons of each.

2) how resolving can phase contrast get, with or without oblique, taking consideration of halo artifacts?

3) if someone is using COL+POL, does he/she use retarder the same way or is there a better way?

4) is there image difference between optimal set-up's of COL vs oblique?

Those purpose may be too unique and personal to be universally appealing, so I did not want to restrict the thread just to those pursues.

I then thought generally comparing various contrast techniques would be a fun activity - anyone can do it based on his/her preference, draw his/her own conclusion or simply pass on experience (not necessarily providing photos/videos) here in this thread. That approach may not be rigorous or helpful to all, but if someone learns something here, it would be worth it.

Based on what I learned from this very helpful thread and my own experiments, my answers to the above questions are:

1) Compared to COL+POL, UGF is easier to set up but oftentime its image looks flat. I would likely use UGF for initial screening and filming, then switch to COL+POL+ offset retarder for better image if there is a chance.

2) many phase contrast seems to have limited resolution and halos affect phase images. However I am still waiting to see if Phil's B-Minus phase optics offer better contrast and resolution without many halos.

3) no one has used COL+POL+offset retarder.

4) Yes, there is subtle image difference between COL and oblique, but when set-up optimally, both can be highly resolving and contrasty.

[gekko]

zzffnn, many thanks for your additional comments. I am a bit puzzled by the COL + POL bit. I assume by POL you mean crossed polarizers. Would the result be strongly affected by weather what we are looking at is birefringent?

Would it be helpful if I simply repeat what I did, but focus on the edge of the diatom? Do you suggest any changes? I will try to repeat the experiment in a week or two, so there is time for thought.

Thanks again.

[apochronaut]

I'm curious why you used monochrome , Gekko. I know you have mentioned reasons in other threads but in this case , since this is a comparison of techniques in their native form( or am I wrong in that?) , the removal of colour artifacts , alters the comparison somewhat doesn't it? I'm thinking that diffraction affects resolution and by de-colourizing the images, the chroma associated with the diffraction gets downplayed as a reduction factor in resolution and diffraction would not be consistent across methods, would it.
I suppose I see the value of such a comparison as a visual comparison , moreso than a photographed comparison, although clearly to be presented here, it must be by photographic record.

[gekko]

Many thanks, apochronaut, for your input. You are right, of course. My thought was that we are comparing the different techniques "within" a person, not among us. Using color and no green filter would, I thought confound the effects of the lighting method with the aberrations of the optics. But I guess it depends on the purpose of the exercise, and, although a bit clearer, the purpose is still rather vague to me. However, I will do it both ways. The CA fringes with my CF achromatic lenses when observing diatoms when viewed at full size is quite disconcerting, but it should be instructive. So, as I understand it, what I should be doing is repeating what I did before but focusing on the diatom edge (I'll have to see though, if then the striae will be in focus). Then repeat the exercise using color without the green filter. If that sounds good to everyone, I will proceed accordingly, otherwise, please let me know what to modify.
Thanks again, apochronaut and zzffnn for your inputs.

[zzffnn]

zzffnn, many thanks for your additional comments. I am a bit puzzled by the COL + POL bit. I assume by POL you mean crossed polarizers. Would the result be strongly affected by weather what we are looking at is birefringent?

Would it be helpful if I simply repeat what I did, but focus on the edge of the diatom? Do you suggest any changes? I will try to repeat the experiment in a week or two, so there is time for thought.

Thanks again.

Yes, I always use crossed polarizers. Image color is sometimes affected. In one case, 1mm thick pond water showed up as red (instead of dark grey/blue/black) with crossed POL. In another case, a sand particle showed birefringent colors.

Also COL+POL+offset retarder seems to offer less focus depth than UGF. Not sure if this is rralted to POL.

You do not have to repeat what you did, unless you want to do so. I got good information from your posts. I have no other suggestions, except what you have already knew - playing with filter positions, size and condenser iris may make a diffetence, sometimes a slight closing-diwn of iris may actually reveal more details.

[zzffnn]

Yes, I think comparison "within the same person" makes more sense, as between different persons and scopes, there are too many variables.

gekko,
Sorry if I confused you - I did not ask you to focus onto diatom edge though. I said it would be nice to show what your diatom edge looked like, when you focused onto central dots or striae.

[zzffnn]

Phil,

For me personally, visual observation by an expert like you and some verbal comments are good enough. You do not have to document everything with photos or videos. Though it may be easier to just upload a photo than describing a photo, I guess?

[zzffnn]

I prefer to let thread participants to choose their own comfortable perspective, whether it be focused, rigorous and objective, or broad, casual and subjective. It is up to you. I have received some useful information herein, for my own quest.

I did not want to ask for a rigorous study (which means lots of investment of time) from a hobbyist group. For example, if one is enduring Canadian winter and has crops in the field to take care, then I would feel bad to ask for that much tiime investment from said person.

For those who have energy and want to do serious science though, I invite you to join me in drafting a comparison manuscript for submitting to a peer-reviewed microscopy journal. We will do it the right way, in a rigorous manner. Sky is the limit, depending on how much you want to/can invest.

This is how rigorously I did science as a professional : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2729133/ . I have authored a few peer-reviewed publications and NIH-styled grant proposals in the pathogenic microbiology/immunology field (search my name with my mentor's name "Zhang F Chopra AK" and you will find some of mine work at NIH's PubMed: http://www.ncbi.nlm.nih.gov/pubmed).

[gekko]

Many thanks for the further explanation. Next week I"ll post the edges, and later I can repeat the test focusing on the edges if that is desirable. I will ask for suggestions on methodology, etc., before I do any more though.