A recent research article about Oblique Illumination

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Hobbyst46
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A recent research article about Oblique Illumination

#1 Post by Hobbyst46 » Thu Jun 28, 2018 3:18 pm

A recent article:

"Oblique illumination in microscopy: A quantitative evaluation"
by Carlos Sanchez et al, of Spain
in: Micron 105, November 2017
DOI:10.1016/j.micron 2017.11.006

Using objective mathematical criteria (the details of which can be ignored by the casual reader, who only wants figures and bottom-lines!) of image quality, they provide evidence for the advantage of oblique illumination over brightfield, phase contrast and DIC, especially for some diatoms.
Last edited by Hobbyst46 on Fri Jun 29, 2018 8:54 am, edited 1 time in total.

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Re: A recent research article about Oblique Illumination

#2 Post by MichaelG. » Thu Jun 28, 2018 3:46 pm

Thanks for that ^^^

First search found Elsevier 'Science Direct' offering to sell it to me.
... Then I found it on Research Gate:
https://www.researchgate.net/publicatio ... evaluation

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Re: A recent research article about Oblique Illumination

#3 Post by 75RR » Thu Jun 28, 2018 4:53 pm

Thanks for the heads up Hobbyst46

and thanks for the link MichaelG.

Have just skimmed it so far.

Glad to see he is promoting COL use with Diatoms. See page 7
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A-COL.jpg
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Re: A recent research article about Oblique Illumination

#4 Post by apochronaut » Fri Jun 29, 2018 8:42 am

That is a very interesting and useful paper. Thank you for pointing it out and thanks for the easy link, MichaelG.
It seems that the BF research microscope achieved maturation sometime in the 1920's, probably brought about through necessity: W.W.I , the Spanish Flu etc. By that time, the major microscope makers were capable of producing fully functional, achromatic through to apochromatic optical systems of high numerical aperture, with high intensity versatile multi fiterable illuminators and stands displaying refined engineering and precision of function. All else since that golden pinnacle of microscope manufacture has been to tweak what already existed and often on what was already known. Illuminators have been tweaked, lens formulas have been tweaked by virtue of the availability of an expanding array of glasses and coatings, stands have been made more ergonomic and photography through the microscope has been simplified. By and large though, in the case of BF, many microscopes made in the 1920's and quite a few made prior to that can produce images that could easily have come out of a fine BF microscope made in 2018, better, in many instances. Where the principal difference might lie when noticeable, would be in the contrast and of course planarity and field of view, all of those being defects, that the optical engineers of the 20's were quite aware of. For sure, those makers of such refined beauties of engineering and craft, were well aware of those shortcomings and eagerly awaited the day when glass would arrive that would allow them to perform the tweaks that they knew, were just beyond their grasp.
One of the tools those early instruments were provided with however, that went a long way towards compensating for those failures, and with which to enhance image quality, was an offset diaphragm, a commonly found accessory on high end microscopes up in some cases into the 50's. In fact, they were so relied on, that along with an achromat condenser, the presence of an offset diaphragm or a variable leaf "crescent" diaphragm is one of the signs that a pre , roughly 1955 microscope is a cut above others. While these are designed for a more simple type of oblique, it indicates the level of use that a stand was engineered for and it might be considered that stands carrying such condensers were the DIC scopes of their day. It is quite noticeable too, that the practical advent of phase microscopy, emerged and developed in an ascending overlap with the descent of the use of oblique condensers, over about a 10 year period between about 1947 to 1957. Lomo is one mfg. that seems to have maintained them to any degree, in later years.

Another tool from the past was the " rosettes" or variable stop attachment. I have only ever seen a couple of these, with the one in my possession coming from the Leitz Canada stock , when they sold everything Leitz off and became Leica. Subsequently I have seen them being mfg. in India but I cannot remember where. This is a reverse iris , with a set of opaque leaves that can be opened and closed with a lever. They reside in the filter tray , under the condenser, providing progressive COL, quite cheaply and with any microscope that it can be stuck on to. Pretty much the way of the Dodo, I am afraid but worthy of a resurgence.

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Re: A recent research article about Oblique Illumination

#5 Post by wporter » Fri Jun 29, 2018 3:47 pm

Apo,

I find the idea of a 'reverse iris' fascinating. Found nothing on the web except this Olympus darkfield writeup (with a picture of one, about halfway down the article):

https://www.olympus-lifescience.com/en/ ... darkfield/

Obviously, if the outer perimeter of the iris was made as circular as possible, it would lend itself to being a better variable darkfield stop than one with a rosette edge (less glare?). If regular irises can be made that are very circular, why not also for the reverse kind? And combine that with the ability to be offset in various directions & amounts, and you also have a nice variable oblique stop.

Apo, if you find yourself with your camera in your hand someday, please snap off a shot of yours and post it for us!

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Re: A recent research article about Oblique Illumination

#6 Post by Hobbyst46 » Fri Jun 29, 2018 7:00 pm

Thanks Apochronaut and wporter for your comments.
IMO, the results of Sanchez et al in that paper benefited also from their use of blue LED light (442/450nm; they also used UV, but even blue light was benefical). An LED is quite monochromatic, say to +/- 10-20nm, although the paper did not specify the range. Both the narrow range of wavelengths, which minimized chromatic aberrations, and the short mean wavelength (blue), which improves resolution by itself, contributed to image quality. The nice point is that with the same light, oblique was a tough competitor or even winner... (Mondial spirit now :lol: )

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Re: A recent research article about Oblique Illumination

#7 Post by zzffnn » Fri Jun 29, 2018 7:50 pm

Thanks for sharing the nice paper. I read it word by word.

I did not learn anything new from the paper though. The take home message, for me, is that for author's test subjects, crescent moon filters #3 and #4 in Figure 1A performed the best.

But I think your subject matter determines which filter + illumination works best. For example, Amphipleura pellucida works well with filter #6 in Figure 1C (per author) and extreme COL using Heine condenser (my own experience). But a rotifer works best with the crescent moon filter in between #1 (BF) and #2 in Figure 1a, which looks closest to my general purpose oblique filter as well.

I have been asking Apochronaut to find me that Indian manufacturer for reverser condenser iris, which I think would be really useful for oblique illumination. But he has not been able to find it yet. Anyone here wants to do a group buy to increase our chance of securing an order from the manufacturer, if we do find it? Or anyone wants to make a few and sell one to me? Please PM me, thanks.

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Re: A recent research article about Oblique Illumination

#8 Post by Hobbyst46 » Fri Jun 29, 2018 9:13 pm

@zzffnn
Some of the folks here have already posted beautiful comparisons of 3-5 illumination modes. The point I like in that paper is that by objective judgement of images, using statistical methods, they could show the benefit of a method (oblique) for certain objects (=diatoms; brain slices). I agree with your comment, yet this paper sort of motivated me to try oblique. I can do some with the rotating phase contrast condenser (thus far "my" diatoms are too small to see the effects).

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Re: A recent research article about Oblique Illumination

#9 Post by zzffnn » Sat Jun 30, 2018 3:54 am

Hobbyst46,
Yes, I agree. As the title of the article suggests, "quantitative evaluation" was main goal of the authors. I was just trying to summarize useful information for hobbyist application, in case members here don't have time to read it entirely.

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Re: A recent research article about Oblique Illumination

#10 Post by 75RR » Sat Jun 30, 2018 5:04 am

I can do some with the rotating phase contrast condenser ...
Offsetting the brightfield port in a rotating phase condenser may seem a rough and ready way to implement oblique, and yet it can prove to be surprisingly subtle when used in conjunction with the condenser iris as that alters the diameter of the crescent 'stop'.
The rotating phase contrast condenser can thus give us both size and X axis position of the crescent 'stop'. Additionally, offsetting the condenser itself also gives the crescent 'stop' Y axis movement.
The use of a rotating stage adds another level of control to oblique lighting as it allows the subject to be illuminated from the optimal angle.
(thus far "my" diatoms are too small to see the effects).
You should consider getting an 8 Form Test Plate from Klaus Kemp
It is not only very useful but also a kind of right of passage for microscopists.

http://www.diatoms.co.uk/index.htm
Last edited by 75RR on Sat Jun 30, 2018 8:41 am, edited 1 time in total.
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Re: A recent research article about Oblique Illumination

#11 Post by MichaelG. » Sat Jun 30, 2018 7:13 am

zzffnn wrote:... I have been asking Apochronaut to find me that Indian manufacturer for reverser condenser iris, which I think would be really useful for oblique illumination. But he has not been able to find it yet. Anyone here wants to do a group buy to increase our chance of securing an order from the manufacturer, if we do find it? Or anyone wants to make a few and sell one to me? Please PM me, thanks.
I recalled reading something by Conrad Beck ... and have just found this:
https://www.worthpoint.com/worthopedia/ ... -831877837

The search is on, for details of the Traviss device !

MichaelG.
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http://www.microscope-antiques.com/VHtraviss.html

https://www.flickr.com/photos/interneta ... 0762341581

https://ia800208.us.archive.org/17/item ... 07roya.pdf
This is a big download, but it's worth the trivial effort involved ... The description starts on p362
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Re: A recent research article about Oblique Illumination

#12 Post by 75RR » Sat Jun 30, 2018 8:11 am

75RR wrote:
I can do some with the rotating phase contrast condenser ...
Offsetting the brightfield port in a rotating phase condenser may seem a rough and ready way to implement oblique, and yet it can prove to be surprisingly subtle when used in conjunction with the condenser iris as that alters the diameter of the crescent 'stop'.
The rotating phase contrast condenser can thus give us both size and X axis position of the crescent 'stop'. Additionally, offsetting the the condenser itself also gives the crescent 'stop' Y axis movement.
The use of a rotating stage adds another level of control to oblique lighting as it allows the subject to be illuminated from the optimal angle.
(thus far "my" diatoms are too small to see the effects).
You should consider getting an 8 Form Test Plate from Klaus Kemp
It is not only very useful but also a kind of right of passage for microscopists.

http://www.diatoms.co.uk/index.htm
Nice search skills! Thanks
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Re: A recent research article about Oblique Illumination

#13 Post by wporter » Sat Jun 30, 2018 2:45 pm

Thanks, MichaelG, for the great find; here are the three pages for everyone's convenience:
Attachments
traviss stop  j of roy mic soc 1907 p362.png
traviss stop j of roy mic soc 1907 p362.png (403.93 KiB) Viewed 10345 times
traviss stop  j of roy mic soc 1907 p363.png
traviss stop j of roy mic soc 1907 p363.png (235.74 KiB) Viewed 10345 times
traviss stop  j of roy mic soc 1907 p364.png
traviss stop j of roy mic soc 1907 p364.png (228.73 KiB) Viewed 10345 times

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Re: A recent research article about Oblique Illumination

#14 Post by 75RR » Sat Jun 30, 2018 3:17 pm

It would be interesting to be able to combine Traviss' Expanding Spot with the movable crescent stop of a rotating Phase Condenser.
In the meantime I wonder if one could simulate this scenario with a selection of darkfield stops in the filter holder.
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Re: A recent research article about Oblique Illumination

#15 Post by Hobbyst46 » Sat Jun 30, 2018 9:45 pm

MichaelG, bravo to finding this old document!
Personally, I somehow like the term "expanding stop" more than "reverse iris". Although in truth, the shrinking/expanding/reverse iris is doing the job, and the stop just helps it doing its job :roll:

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Re: A recent research article about Oblique Illumination

#16 Post by zzffnn » Sun Jul 01, 2018 12:43 am

75RR wrote:It would be interesting to be able to combine Traviss' Expanding Spot with the movable crescent stop of a rotating Phase Condenser.
In the meantime I wonder if one could simulate this scenario with a selection of darkfield stops in the filter holder.
Decentering undersized darkfield stops is what I have been doing. Diatom dotting is pretty good that way and can resolve Frustulia to dots, at NA 1.1 water immersion. But I think decentering a Traviss stop will perform better and be much more convenient for different objectives and subjects.

One should be able to Blutak a Travis's stop on microscope slide, then attach that slide near condenser back focal plane (though some DiY modification may be needed, as some condenser BFP is not easily accessible).

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Re: A recent research article about Oblique Illumination

#17 Post by MichaelG. » Sun Jul 01, 2018 6:40 am

Eagle-eyed readers of the 'detailed description' by Traviss will notice some inconsistencies between the text and the illustrations.

This modern graphic should help clarify the proportions: https://goo.gl/images/oMXn95
Source: https://www.nature.com/articles/ncb0800_E151

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Re: A recent research article about Oblique Illumination

#18 Post by Hobbyst46 » Sun Jul 01, 2018 7:46 am

Hobbyst46 wrote:Personally, I somehow like the term "expanding stop" more than "reverse iris"
Sorry, taking it back. In Google, "expanding disk" leads to the increasing the capacity of computer disks...

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Re: A recent research article about Oblique Illumination

#19 Post by apochronaut » Sun Jul 01, 2018 6:16 pm

Hobbyst46 wrote:
Hobbyst46 wrote:Personally, I somehow like the term "expanding stop" more than "reverse iris"
Sorry, taking it back. In Google, "expanding disk" leads to the increasing the capacity of computer disks...

Variable stop, or variable dark field stop. is often used.

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Re: A recent research article about Oblique Illumination

#20 Post by MichaelG. » Mon Jul 02, 2018 6:33 am

Those interested in experimental fabrication might find this modern 'Rudimentary Treatise' useful:
https://www.mccrone.com/mm/rudimentary- ... diaphragm/

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P.S. The Quekett 'USB Flash Drive' might be the bargain of the decade !!
http://www.quekett.org/about/books
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Re: A recent research article about Oblique Illumination

#21 Post by 75RR » Mon Jul 02, 2018 2:14 pm

MichaelG. wrote: .
P.S. The Quekett 'USB Flash Drive' might be the bargain of the decade !!
http://www.quekett.org/about/books
Anyone with a Mac try out the QMCQwek3.3 interface on the Quekett USB Flash Drive - will it work?
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Re: A recent research article about Oblique Illumination

#22 Post by MichaelG. » Mon Jul 02, 2018 6:33 pm

75RR wrote:
MichaelG. wrote: .
P.S. The Quekett 'USB Flash Drive' might be the bargain of the decade !!
http://www.quekett.org/about/books
Anyone with a Mac try out the QMCQwek3.3 interface on the Quekett USB Flash Drive - will it work?
.
Yes, and Yes
iMac .. running Snow Leopard

Yes, and Yes
MacBook Pro .. running el Capitan

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Re: A recent research article about Oblique Illumination

#23 Post by 75RR » Tue Jul 03, 2018 4:26 am

Thanks, will jump in then.

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Re: A recent research article about Oblique Illumination

#24 Post by 75RR » Tue Jul 10, 2018 9:41 pm

75RR wrote:Thanks, will jump in then.

Mac mini ...running Yosemite
Have just received the Quekett Journal of Microscopy 1868–2012 on a USB flash drive.

1). Neat 8 GB flash drive
2). Working well on my Mac mini 2012 running Yosemite
3). A pleasure to read well written articles on microscopy that date from the eighteen hundreds ... gives one a nice sense of continuity.
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