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

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

#1 Post by zzffnn » Wed Nov 11, 2015 1:20 am

Does anyone here use COL+Pol routinely? Have you tried to improve it further, say, with an additional 1/4 lamda plate? Have you compared it with UGF or any other contrast techniques?

I found COL+ pol+ offset 1/4 lamda plate (or partial 1/2 lamda plate) gave very good and repeatable images/videos. Without lamda plate, contrast and resolution is not as great.

What I meant by offset 1/4 lamda plate is this:
I would place a 1/4 lamda plate over about 50% of the aperture of microscope field diaphragm (and polarizer). Result seems best with firstly crossed polarizer and analyzer. If the lamda plate covers over too much (~70%) or too little (~20%) of the field aperture, the benefit is lost. Then I move the lamda plate back and forth slightly (while looking into eyepieces) to enhance contrast and resolution. 45 degree angle of placment seems to work best for retarder.

With the above method, using Olympus E-PM2 camera in video mode, I was able to kind of resolve striae of Frustulia rhomboides diatom with a water immersion NA 1.23 70x objective:
https://m.youtube.com/watch?v=vJDKo9YNggw
https://m.youtube.com/watch?v=31EzUBdV0pw

My UGF filter (see ref #1) can somewhat resolve Amphipleura pellucida in photos (when used with water immersion, see ref#2), but it is not as repeatable at NA 1.23 and light loss is more (in reality, I have to use flash). Oftentime, videos made with UGF were too dark, when high degree of oblique and dark field was dialed in for more contrast and resolution. Sometimes with small and transparent ciliates, my camera cannot keep up with the light loss.

For videos, I think I still prefer COL+pol+offset 1/4 lamda plate, as it is more repeatable and gives brighter image.

What are your thoughts? Thank you in advance for your kind comments!

References:
1) http://www.photomacrography.net/forum/v ... fca3642cff

2)
http://www.photomacrography.net/forum/v ... hp?t=29096
Last edited by zzffnn on Wed Nov 18, 2015 2:24 pm, edited 8 times in total.

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Re: COL + Pol + offset retarder vs UGF

#2 Post by 75RR » Wed Nov 11, 2015 2:16 am

Hi zzffnn, interesting idea.

Would like to test this but was wondering if using Pleurosigma angulatum, also from Kemp's 8 form slide, rather than Frustulia rhomboides might be better.
The punctae of Pleurosigma angulatum can be distinguished with a 40x/0,65 objective which would be more representative and perhaps encourage more people to participate as everybody has one.

Might we say that the test is about which illumination method provides the best image/resolution in video?
Testing Oblique and Circular Oblique with and without Pol and with and without a retarder?
What do people think?
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Re: COL + Pol + offset retarder vs UGF

#3 Post by zzffnn » Wed Nov 11, 2015 2:38 am

75RR,

Let us not restrict ourselves just to videos. We may discuss anything related to UGF and COL+Pol. Edit: Trying oblique+Pol would work too, though in my rig it did not work as well as COL+Pol.

Also any test diatom would work, as long a difference is seen during comparison. I did not pick Pleurosigma as it is kind of easy to resolve, even with UGF and 40/0.65, or COL+Pol used the same way (I did not see much difference, between different lighting techniques, when using Pleurosigma as resolution target).

Once you resolve Pleurosigma at NA 0.65, there is nothing else intermediately difficult, as the next more difficult one (and the 2nd most difficult of all 8) is the Frustulia at NA 0.95-1.1.
Last edited by zzffnn on Wed Nov 11, 2015 4:13 am, edited 2 times in total.

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Re: COL + Pol + offset retarder vs UGF

#4 Post by 75RR » Wed Nov 11, 2015 3:11 am

Agree stills would be easier to compare. Still think Pleurosigma is a good choice as it is not just about resolving the punctae but about how well one can resolve them.

Adding UGF + UGF with Pol is a good idea, as it would allow those who do not have Phase, and hence are not able to try COL (Circular Oblique Lighting), to participate.

In fact anything that can improve resolution should be "on". The only requirement should be the use of Pleurosigma as the subject and a 40/0,65 objective to ensure a valid comparison.

For those of you who are wondering who or what is a Kemp 8 Form Test Plate See: viewtopic.php?f=11&t=346

Link to Klaus Kemp website: http://www.diatoms.co.uk/
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Re: COL + Pol + offset retarder vs UGF

#5 Post by zzffnn » Wed Nov 11, 2015 3:55 am

75RR wrote: Adding UGF + UGF with Pol is a good idea, as it would allow those who do not have Phase, and hence are not able to try COL (Circular Oblique Lighting), to participate.

In fact anything that can improve resolution should be "on". The only requirement should be the use of Pleurosigma as the subject and a 40/0,65 objective to ensure a valid comparison.
^ Did you mean UGF vs oblique+Pol? UGF+Pol would be way too dark. But yes, any contrast light technique can be threw into the comparison mix. We like to find a few good ones for everyone to try! I have edited title and OP accordingly.

I don't have phase contrast or phase ring actually. For COL, I just used black center stops that are slightly too small for true darkfield. One can use a circle template (http://www.amazon.com/gp/product/B00290 ... detailpage) to DIY opague plastic stops (then glass slide and Blutak can be used to hold center stops under condenser).

It would be nice if everyone can use Klaus Kemp test diatoms as test targets, so that results are more comparable. But please don't let that stop anyone from trying - I think any small still subject, such as an immobilized ciliate, would work. Just keep in mind that if one's subjects of interest has low natural contrast, then resolution targets should be that way too.

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Re: COL + Pol + offset retarder vs UGF

#6 Post by 75RR » Wed Nov 11, 2015 4:20 am

I just used black center stops that are slightly too small for true darkfield.
Should that not be rings?

I was wondering if one could make a home made Phase kit. Apparently one can.

See link: http://www.microscopy-uk.org.uk/mag/ind ... hase2.html

It is even easier and cheaper to make phase rings for COL as Phase objectives are not needed.

See link on how COL works: http://www.microscopy-uk.org.uk/mag/ind ... pjcol.html
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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#7 Post by zzffnn » Wed Nov 11, 2015 4:36 am

If you push COL stops to close to maximum aperture of your objective (border of objective front lens becomes outer ring of COL in that case), then you only need a center stop (instead of a hollow ring), even condenser diaphragm is not needed (I left my condenser diaphragm completely open). Or you can close down condenser diaphragm slightly (and use diaphragm blades as outer ring for COL) at the cost of some resolution.

I think making a good hollow phase ring and align it with phase objectives ring is a bit more difficult that making DIY COL. I actually tried a quick-and-dirty DIY condenser annulus today with a 30x NA 0.55 DLL phase objective. It did not work, as my hollow ring is not perfectly circular and the two rings did not match very well (even though they look that way at phase objective's back lens). I think I can make it work, but lots of time is needed - I would rather ask a machinist to make me a few precise hollow rings.

Here is another way to DIY condenser phase annulus:
http://www.microscopy-uk.org.uk/mag/ind ... phase.html

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#8 Post by gekko » Wed Nov 11, 2015 12:43 pm

Interesting ideas, but, at least to me, rather complex. One difficulty might be related to definition: a lower resolution, high contrast image may look better than a higher resolution, low contrast image. Also, showing the full frame (uncropped) image at web resolution will, I think be the limiting factor: I think it will be essential to display a small crop from the full resolution image produced by the camera (i.e. no resizing, only cropping). I have an inkling that this may be at least part of the explanation why people often say that the view through the eyepiece was so much better (more detailed) than the (resized) image they view on the monitor screen. I will try to "participate" to the extent I can after I see some of your results with the details of how you obtained them.

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#9 Post by zzffnn » Wed Nov 11, 2015 12:53 pm

gekko,

This Amphipleura diatom photo (photo#4) of mine, although not a good one (when focused onto striae, the rest of the diatom did not seem be in focus), was a small crop:
http://www.photomacrography.net/forum/v ... hp?t=29096

My post on that thread mentioned imaging details.

I guess the main reasons why eyepieces look better than images on screen are camera shutter vibration (at high magnifications) and camera light sensitivity, at least in my case. If one manages to compensate those factors (by using flash or more light), then views would be similar.

The above linked diatom shot actually looks similar to eye view. There I used long exposure to eliminate shutter vibration and provide more light. As a result, photo looks brighter and has more striae resolution once digitally magnified. Eye view looked slightly more contrasty and sharper, probably due to eyes' higher light sensitivity (even though camera got overall more light by exposing much longer). I had a flash shot that look slight better (flash also eliminate vibration and provides more light), but I lost it.

In contrast, in camera's video mode, I did see better eye view than camera recording such as this one: https://m.youtube.com/watch?v=31EzUBdV0pw
This is because although there is no vibration, light sensitivity issue is still there. And in video mode, flash or long exposure would not work. It is all up to how much light camera can capture.

I agree, a lower resolution high contrast image may look better than a higher resolution low contrast image. One resolution to that may be HDR, focus stacking and digital processing in post, if one likes to purse perfection. Personally, as a biologist video maker, I usually opt for a higher resolution low contrast image (if I have to choose) and change focus point in video to provide a deeper global view.

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#10 Post by 75RR » Wed Nov 11, 2015 4:32 pm

Here is a link to an interesting and relevant article by David Walker at Microscopy UK.
It has been posted before in another thread so some of you may have already seen it. Worth another look.

http://www.microscopy-uk.org.uk/mag/ind ... -test.html
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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#11 Post by JimT » Wed Nov 11, 2015 10:10 pm

75RR (and others), thanks for these great posts and tutorial. I tried one of the filters suggested in the Microscopy UK posts but it did not turn out so good. For now I will stick with my UGF and DIY DIC filters as I think the images with them were better than the ones with my 'not so good' COL filter.

Can't promise I won't try again however.

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#12 Post by zzffnn » Thu Nov 12, 2015 2:42 am

JimT,

If you take only photos with flash and UGF, you don't really need COL+POL+retarder. Benefit of the later is less light reduction, but since your flash can supply enough light and reduce vibration, you won't see big difference.

For videos, the more light provided by COL+POL would be useful.

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#13 Post by gekko » Sat Nov 14, 2015 12:30 am

I have the 8-form test slide. I am waiting for the first demonstration so I can try and emulate whomever posts it :) .

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#14 Post by zzffnn » Sat Nov 14, 2015 12:58 am

gekko wrote:I have the 8-form test slide. I am waiting for the first demonstration so I can try and emulate whomever posts it :) .
Amphipleura, sharpened photo, small crop, with UGF (photo #4):
http://www.photomacrography.net/forum/v ... hp?t=29096

Frustulia and Amphipleura video, unprocessed, with COL+POL+Offset retarder:
https://m.youtube.com/watch?v=vJDKo9YNggw
https://m.youtube.com/watch?v=31EzUBdV0pw

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#15 Post by gekko » Sat Nov 14, 2015 1:10 am

Thank you. Yes I've looked at those earlier. But I'm not certain as to what exactly the protocol is. You used a high NA water-immersion lens, which I don't have, and I cannot use a UFG effectively because the Nikon condenser does not allow access to the front focal plane. But I bet I'm not understanding exactly the purpose or the procedure. Sorry for having a very slow brain :( .

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#16 Post by zzffnn » Sat Nov 14, 2015 1:23 am

gekko wrote:Thank you. Yes I've looked at those earlier. But I'm not certain as to what exactly the protocol is. You used a high NA water-immersion lens, which I don't have, and I cannot use a UFG effectively because the Nikon condenser does not allow access to the front focal plane. But I bet I'm not understanding exactly the purpose or the procedure. Sorry for having a very slow brain :( .
Sorry, it was my slow brain that did not understand what you meant. I don't think there needs to be an exact protocol - please do whatever that works for you. We are only after good results.

Yes, you may not be able to use UGF with your condenser - you don't have to do that. You could try an oil objective with COL+POL+offset retarder, maybe? With mounted diatoms, NA 1.25 oil objective should outperform NA 1.25 water objective (I was just too lazy to use oil). You can cut out some center stops for COL, which does not require high precision.

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#17 Post by gekko » Sat Nov 14, 2015 9:59 am

So is the aim to try to get the best "visibility" of the diatom details by any means at one's disposal (best resolution, contrast enhancement)? Or are there "rules"?

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#18 Post by zzffnn » Sat Nov 14, 2015 12:42 pm

Any cheap means would work. No rules. Wait, maybe <$200 and no DIC :mrgreen:

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#19 Post by 75RR » Sat Nov 14, 2015 3:34 pm

Any cheap means would work. No rules.
If there are no rules i.e. a valid means of comparison using for example, 40x/0,65 and Pleurosigma angulatum, then I see little point in this.
There has to be common ground, a baseline that we can all use.
Otherwise we are talking DIC and $10,000 microscopes or an exotic objective nobody else has.

Note: I realize that not everyone has a Kemp 8 Form Test Slide, and though they are not cheap I do think they are well worth it.
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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#20 Post by zzffnn » Sat Nov 14, 2015 4:00 pm

I guess it would not hurt to see how cheap DIYs fair against DIC. I bet the difference is not as significant as price difference suggests. Theoretically, oblique or COL has better resolution than DIC, IIRC, though DIC's image may look better (more contrasty, brighter, ect). As an example, many diatmists hold the opinion that one does not have to have DIC or exotic optics to resolve striae of Amphipleura.

And keep in mind that apple-to-apple comparison is not easy, since a slight turn of fine focus may change diatom details a lot.

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#21 Post by 75RR » Sat Nov 14, 2015 4:52 pm

And keep in mind that apple-to-apple comparison is not easy, since a slight turn of fine focus may change diatom details a lot.
Perhaps, but throwing oranges and pears into the mix does not help.
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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#22 Post by zzffnn » Sat Nov 14, 2015 5:44 pm

75RR surely has a valid point.

The discussion of apple-to-apple (rigorous) comparison comes up a lot in the audio hobbyists world too. I think it is a matter of preference, do you prefer to have:
1) some non rigorous but broad comparison;
Or
2) some limited, narrow and rigorous comparison.

I personally prefer 1), if I have to pick only one. But 2) is also nice to have. I do not want 2) to exclude 1) though.

Keep in mind that we are hobbyist and not research scientists working in professional environment. My suggestion is let us keep it fun and informal and let the audience decide by themselves.

If this has to be a rigorous comparison, then it has to be done by the same person, having access to all contrast teniques, with the same focus point onto the same diatom. Logistically, it may be difficult, tiring, time-consuming and not fun.

For example, if focus point is changed when switching from COL to phase, then comparison is not quite rigorous. One can only try.

As another example, as for comparing with DIC, only gekko and a few other members can do it. And gekko cannot use UGF. So technically, if we exclude DIC, gekko cannot play and won't have much else to compare to. But I am sure someone, myself included, would be interested in seeing DIC vs COL+POL, so that little piece of information would help someone, even if comparison is not scientifically rigorous.

Also, I have no phase. But I do want to see phase and/or phase+oblique thrown into the comparison with UGF and COL+POL. If someone does such comparison, even though not in a rigours manner, I would still find it helpful (and better than not seeing any comparison).

I was once a research scientist. What we concluded in journal club discussion was that, any paper published outside of most prestigious journals has flaws, if you look close enough. Sometimes significant flaws that can be picked out very easily. But we can still learn from those flawed papers.
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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#23 Post by zzffnn » Sat Nov 14, 2015 5:44 pm

And this is what I meant by phase + oblique:

One does not have to offset phase ring to get oblique.

One can center and focus phase as one normally does it. Then add DIY oblique by partially blocking the light entering condenser, at any point below the condenser, with any means. Carlos, over at the other forum, does it with a coin over his field diaphragm. I do it with a glass slide partially blocked with black tape (then I Blutak the slide under my condenser - Blutak and slide can be moved back and forth to adjust oblique).

Phil has used oblique + phase to very good effects, though I don't know how he did it.

I would highly appreciate it, if anyone, preferably with a test diatom, can give oblique+phase a try and comment (report findings) on this comparison thread.

I don't have phase myself, so I cannot do the comparison myself.

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#24 Post by 75RR » Sat Nov 14, 2015 9:52 pm

I would highly appreciate it, if anyone, preferably with a test diatom, can give oblique+phase a try ...
40x/0,65 * 1.25, Brightfield, Oblique (home made UGF), Circular Oblique Lighting, Offset Phase Brightfield Ring Oblique (2/6, 3/6 and 4/6 - 0/6 being fully open), Phase, Phase + UGF (which is a basically a fine tuned Oblique shader)
Diatom: Navicula lyra 110µm width
Treated all images the same where possible. Converted them to Black and White.
Include the two phase in original colour as well.

Image Image Image
Image Image Image
Image Image
Image Image
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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#25 Post by zzffnn » Sat Nov 14, 2015 11:00 pm

Outstanding comparison and great images! I like them a lot! Thank you very much.

I do prefer phase+oblique over phase alone. Though phase halo is obvious with this thick diatom. Does your phase objective also have NA of 0.65? Or is it 0.75?

I am surprised how well offset phase ring performed. I will try it in the future, once I make them. So 3/6 is more open (brighter) than 4/6, correct? 4/6 oblique looks stunning and seems to be the best of all those photos.

Is it possible that you were too conservative with COL and UGF? What if you make COL central stop bigger and/or close down condenser diaphragm slightly (there might be some flare)? COL+POL+offset retarder would also cut down flare and increase contrast.

What if you dial in more oblique for UGF (it looks slightly flat)? When you look into objective back lens, were black parts completely black (no peripheral light leaking in)?

All are single shots (without stacking), correct?

Thanks again!

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#26 Post by 75RR » Sat Nov 14, 2015 11:35 pm

You are welcome. Hope you find them useful.

All images are single shots taken with a Zeiss Ph2 40/0,65 (I was too lazy to change the objective)

I also like Offset phase. David Walker calls it Off-axis oblique. He reckons there is no resolution loss.
See his article: http://www.microscopy-uk.org.uk/mag/ind ... -test.html

I divided the field of view in six sections by eye. 0/6 is all open, 3/6 is half way closed.
Is it possible that you were too conservative with COL and UGF?
Might have been, with more time I am sure I could do better with UGF. The COL is pretty good. Lots of detail! It can however be improved with photo editing software. I kept the editing to a minimum.
What if you dial in more oblique for UGF (it looks slightly flat)? When you look into objective back lens, were black parts completely black (no peripheral light leaking in)?
Did not look at the objective back lens when I did UGF, just looked through the eyepieces.
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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#27 Post by zzffnn » Sun Nov 15, 2015 12:07 am

75RR wrote:Did not look at the objective back lens when I did UGF, just looked through the eyepieces.
Please look at objective back lens with UGF next time. You would see a significant improvement. At least I did. Sometimes, especially with flash, back lens view+eyepiece view let you position UGF optimally. If flash output angle is very wide, you can push UGF to completely dark with eye view, but still got a bright photo.

Original inventor of UGF, 'Litonotus", intended UGF to be truly universal and fits any condenser, so he did not optimize the filter shape/size to perfectly fit his condenser or higher magnification objectives. He noted that his UGF did not perform well at 40x NA 0.65 (images looked like plain oblique). That was probably because he had light leaking from the black side of oblique (his original filter was not big enough at the oblique side to block light leaking). Once I blocked that leaking light, my >40x images got a bit better (I saw good UGF effect even at 70x NA 1.23 and that performed at least as good as my COL+POL+offset retarder, if not for more light loss).

I found your posts very helpful! Than you!

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#28 Post by zzffnn » Sun Nov 15, 2015 2:15 am

Attached is a condenser filter ring template. You can use it for COL, darkfield or as phase condenser annuli. Please note that ring size may or may not be correct, depending on printing size. And you need to change ring size based on your needs.

But since it is an editable PowerPoint file, you can change any subject's size (by right-licking it and select "size"), including the hollow ring sizes. In PowerPoint, the only way to accurately generate hollow ring sizes seem to be making circles' solid outlines to sizes according to an arbitrary point " pt" system. Take a look at the file and you will see what I meant.

Print it using a laser printer onto a laser transparency at highest possible DPI resolution. Please remember to print as "color" with a black-and-white printer (otherwise those white rings won't show up as hollow rings. I have to make those rings white in PowerPoint, otherwise they won't appear clearly in black background). Then you can use it as condenser filter.
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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#29 Post by zzffnn » Tue Nov 17, 2015 10:53 pm

https://youtu.be/mLFR88hXAes
https://youtu.be/xhicFS5ToMY

I filmed 75RR's test diatom (Navicula) using COL+POL+offset retarder, UGF and a 40x NA 0.65 objective. For videos, I prefer COL+POL+retarderfor for its better 3D effect and brightness. UGF seemed to flatten out thick diatoms. But UGF is easier to set up for myself. Please view videos in 1080p and read their notes for details.

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Re: COL + Pol + offset retarder vs UGF vs other contrast techniques

#30 Post by gekko » Wed Nov 18, 2015 12:22 am

zzffnn, I looked at your videos (is there a difference between the two videos? sorry I couldn't quite tell-- my memory is not that good :( ). Is there a reason why you use video rather than still? With my camera, video mode resolution is less than 1 Mpix, just about the minimum required for use with my 40x/0.7 objective**. In still mode, the camera's resolution is 12 Mpix. I have done a preliminary run using Pleurosigma angulatum (striae spacing of 0.53) rather than N. lyra (spacing of 1.25 µm) that 75RR used because it is closer to the theoretical resolution of the lens (0.48 µm**). As I said this was just a preliminary test and I will repeat it after I get your feedback on what to change. I hope post the results tomorrow.
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** figures obtained from Charles Krebs' spreadsheet: http://krebsmicro.com/relayDSLR/relay_micro.xls

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