Dark field illumination: Questions and answers

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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apochronaut
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Re: Dark field illumination: Questions and answers

#31 Post by apochronaut » Mon Mar 05, 2018 1:13 pm

Until the development of plan optics, when the entire lens pack was down in the nose of the barrel, it was a fairly easy task to apply an iris diaphragm to an objective. The Leitz, example above is so simple and straightforward, replacing the top barrel section of the objective , with one containing an iris. If a calibration for the N.A. was deemed necessary, then that could be accomplished and applied to the barrel as well.

However, once the lens formulas expanded, to included higher degrees of correction and plan performance, especially at over a 20 F.N.( apparent field of view), it was not any longer possible to just slip an iris into the barrel, in most cases. The entire objective would have to be formulated in order to accommodate the presence of the iris diaphragm, so some iris equipped objectives , all of a sudden got very expensive. There was a limited market for a special purpose built objective.
An example from my own experience but I am sure there are lots of others, are the various planachro 100X objectives made by AO.
There are in fact; 6 of them, with 5 being 100X 1.25 N.A. and one being a .90 no cover objective. Three of the 5 cover slip corrected versions, are clearly improvements over time, with higher degrees of colour correction and the moniker "advanced planachro" appearing to describe those born in the 80's. However, going back to the early 70's, when the plan optics first became de rigeur, there was the grandfather version cat.# 1024 and then the iris equipped version cat.# 1014. Later, when the term advanced planachro began to be bandied around, # 1014 was given that title too. It was not simply, a # 1024 with an iris, for the performance of the two is widely different. In side by side tests, the # 1014 outperforms the # 1024 by a country mile and is clearly a whole other optical arrangement, the price of which reflects that.

Hobbyst46
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Re: Dark field illumination: Questions and answers

#32 Post by Hobbyst46 » Mon Mar 05, 2018 9:46 pm

MicroBob wrote: This Edmund optics part would not be difficult to copy. It is only for low power darkfield work, offers probably less then a normal abbe condenser with dark field stop. More interesting would be a condensor made from an acryl glass body with LEDs shining into it from the side. I had a look for my prototype but couldn't find it. It would be quite a bit of work and precise machining to make one of these in a form ready for everyday use. So it's not that interesting if there is a used market that supplies dark field condensers for acceptable money.
I was frustrated by the inability to get acceptable DF with my 25x/0.45 and 40x/0.75 objectives. So I followed someone's post somewhere about using a strong desk lamp, and I positioned a cheap 9-LED flashlight above the stage, at an angle of about 45 deg, the front of the flashlight nearly touching the tip of the objective. DF appeared with the 25x/0.45, 40x/0.75 and somewhat even the 40x/0.95 (dry). Naturally the quality falls in that order, none of them has a really black background and are not worth a post. But it gives hope that, given proper sideways illumination, DF up to NA 0.75 can be achieved even without the pricey rare condensers.
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apochronaut
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Re: Dark field illumination: Questions and answers

#33 Post by apochronaut » Mon Mar 05, 2018 9:58 pm

Try a slit lamp, obliquely through the slide.

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Re: Dark field illumination: Questions and answers

#34 Post by Hobbyst46 » Mon Mar 05, 2018 10:29 pm

Exactly what was on my list... thanks! As I matter of fact I am trying to shine into the slide while shading the space between the objective and slide with a piece of black plastic from an obsolete floppy disk...and even better if I crudely imitate the reflective dedicated DF condensers, by lowering the condenser, inserting the flashlight between the condenser and stage, and shining upwards at the specimen from a distance of 8cm at a very shallow angle.
Discussions of special reflective condensers motivated amusing primitive experiments and re-inventing the mirror, yet...
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apochronaut
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Re: Dark field illumination: Questions and answers

#35 Post by apochronaut » Tue Mar 06, 2018 12:20 am

I thought I should provide a couple of examples of some of the things I mentioned in a couple of posts. This, I think is the most recent post I made based on DF images, going back to June . It happens to have an example of a hybrid DF backlit/sidelit image, a couple of quite flat DF images and two where the depth of field is enhanced partially by closing the iris diaphragm some.

The subject is Wallemia Sebi, an odd Xerophilic fungi, that is tolerant of severely low water activity and can grow on substrates as diverse as maple syrup and salt. The Sporophangium are around 5 um and the sporangiophores around 2.

First pic, backlit /sidelit hybrid. Second, with some depth showing the topography of the substrate. Third, some depth in a matrix of sporangium. Fourth, a very flat 2 dimensional sporangium/sporangiophore isolate.

The sample was aqueous.
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MicroBob
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Re: Dark field illumination: Questions and answers

#36 Post by MicroBob » Wed Mar 07, 2018 6:34 pm

Hi together,

today I machined a new body for an 18W LED to counteract the light loss from the Zeiss Jena pancratic condensor.
Now I get a nice bright dark field image even with this condensor that uses only a small part of the light coming out of the base.
I tried it with a Zeiss West 100:1 1,25 and got a medium gray background.
Then I had a look around in my diatom strew with the Leitz NPL 50 1,0 Oel. I got the same perfectly black background as with my old 10W LED and took a stack of images. It is only a bubble, but I like it! :lol:
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Re: Dark field illumination: Questions and answers

#37 Post by Hobbyst46 » Wed Mar 07, 2018 6:43 pm

Very nice photo. This 50x objective is without an iris, correct?

I am still hoping for a decent DF for the 40x/0.75 objective by adding an illumination source.
A strong radial LED light positioned below the stage at the same height as the normal top condenser lens (nearly touching the slide from below) should do it.

There are flexible adhesive LED strips, ~10W/m, that carry powerful SMD LEDS and are fed from a 12V power supply. These strips are used for household cabinet lights, decorative illumination etc. If 10cm of such strip could be fitted inside a thin walled shallow cylinder, they would form an inward radial illumination lamp. How to survive without a special DF condenser...
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MicroBob
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Re: Dark field illumination: Questions and answers

#38 Post by MicroBob » Wed Mar 07, 2018 8:46 pm

Yes, the Leitz objective is without iris. The test with the n.a. 1,25 shows that the light cone of this condensor must be something like 1,25 to 1,4, so very sharp cut off between light and dark.

When we look at your nice n.a. 0,75 objective I would say that you need 0,85 as a minimum for the lower value, this with a sharp cut off. At the same time you would need a couple of watts of light and a cooling for the LEDs. All together not a simple project. And probably the first versions will give you nice light at n.a. 0,85 ut to 0,95, but at the same time below 0,85 - so no black background.

Your phase condensor probably has a position with an aperture - am I right? I think when you look into the tube, preferrably with a phase microscope, you would be able to see, at which aperture setting the back lens of the objective is just completely lighted. If you then look at the aperture itself you would see the diameter of opening, that fits to the aperture of your lens. Your darkfield stop then would have to have a disc in the middle that is somewhat larger than this aperture diameter with a free ring around it. Do the darkfield stops you use fit to this explanation?

There are Zeiss Jena dark field condensors that are more frequent on the market (here). On ebay they go for about 50€ It would probably be possible to machine or 3D-print a condensor dovetail ring to adapt it to your GFL. If you contact the seller in advance you could provide him with a halfway cheap way of shipping. A "Maxibirief International" from Germany to Israel is 7€ for up to 1 kg.

https://www.ebay.de/itm/CARL-ZEISS-JENA ... 7675.l2557
https://www.ebay.de/itm/CARL-ZEISS-JENA ... SwuHdaah2P

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Re: Dark field illumination: Questions and answers

#39 Post by Hobbyst46 » Wed Mar 07, 2018 9:52 pm

MicroBob wrote: At the same time you would need a couple of watts of light and a cooling for the LEDs. All together not a simple project. And probably the first versions will give you nice light at n.a. 0,85 ut to 0,95, but at the same time below 0,85 - so no black background.
Perhaps; I think it will be ~1 watt or less; cooling - yes, problematic unless I can add aluminum cooling fins; chances are indeed not high. The NA might be varied by raising or lowering the carrier of this fixture - that is, the condenser. Not great but maybe decent DF. I am sure of it since I placed a 5mm narrow-beam LED horizontally below the slide and experimented.
Your phase condensor probably has a position with an aperture - am I right?
Yes.
I think when you look into the tube, preferably with a phase microscope, you would be able to see, at which aperture setting the back lens of the objective is just completely lighted
Yes.
If you then look at the aperture itself you would see the diameter of opening, that fits to the aperture of your lens.
Certainly.
Your darkfield stop then would have to have a disc in the middle that is somewhat larger than this aperture diameter with a free ring around it. Do the darkfield stops you use fit to this explanation?
I never checked it seriously, counted on Zeiss instead: there are all three: D, Ph2 and Ph3 stops. I can check them. There are empty openings as well (for DIC) but centering a DIY stop inside any of these would be difficult.
There are Zeiss Jena dark field condensors that are more frequent on the market (here)....
That might be a possibility. Actually I own a simple brightfield condenser (flip-out lens) that I am not using at all and perhaps I can take it apart and use the dovetail on a Jena Kondenser.
With your permission I will PM.
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Hobbyst46
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Re: Dark field illumination: Questions and answers

#40 Post by Hobbyst46 » Sun Mar 18, 2018 1:27 pm

In continuation of the subject I start a new post about DIY-DF under the same topic.
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Re: Dark field illumination: Questions and answers

#41 Post by Hobbyst46 » Sat Mar 24, 2018 11:28 am

A little late update about condenser-less darkfield:

Someone had thought about it a long time ago. There is a commercial optic fiber slide holder that does it. The slide
mechanical holder is placed on the stage, it replaces the ordinary slide holder. The very thin optic fiber - apparently an array of them - is horizontal, so the beam enters the slide sideways, and most of the light inside the slide undergoes total reflection, except below the specimen itself, where it provides the angle illumination and produces darkfield even for very high NA. Apparently with a very dark black background.
Alternatively, a similar device but with an array of very thin LEDs that shines directly, sideways, into the slide.

(Note: Apochronaut has suggested to do such directed illumination earlier in the Post).

So this darkfield accessory can be used together with condenser-generated illumination.
The original name of the device was Darklite, produced by MVI.

Questions:
1. Is it still on the market, who makes it, how much could it cost?
2. How well does it perform?
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stillcool
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Re: Dark field illumination: Questions and answers

#42 Post by stillcool » Sat Mar 24, 2018 11:32 am

saw one on ebay US sold for 100 bucks

MichaelG.
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Re: Dark field illumination: Questions and answers

#43 Post by MichaelG. » Sat Mar 24, 2018 2:30 pm

stillcool wrote:saw one on ebay US sold for 100 bucks
Could you please provide a link to the listing ?

This is something I have been pondering for a while, and there is no point 're-inventing the wheel.

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MicroBob
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Re: Dark field illumination: Questions and answers

#44 Post by MicroBob » Sat Mar 24, 2018 3:32 pm

Hi Doron, nice find!

Here are pictures of the Darklite product: https://www.google.de/imgres?imgurl=htt ... mrc&uact=8

https://www.google.de/imgres?imgurl=htt ... mrc&uact=8

manual: http://www.mvi-inc.com/wp-content/uploa ... -SHEET.pdf

MVI homepage exists, but the product pages I tried were links to other manufacturers. May be their suppliers now sell the products directly.

The Darklite looks expensive, probably more expensive than a dark field condenser. So I can't see a place for this product on the market, as long as the microscope makers offer dark field condensers. The design looks good though and it should not be too difficult to make such a slide holder with LED illumination. In the manual they write that there should be no label on the slide between light entrance and object.

Bob

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Re: Dark field illumination: Questions and answers

#45 Post by Hobbyst46 » Sat Mar 24, 2018 6:08 pm

In the manual they write that there should be no label on the slide between light entrance and object.
Hi Bob
That is because the idea is total reflection of the light rays inside the slide, when the top glass surface of the slide is in contact with air.
If it were a glued label, the refractive index would have been different and internal reflection would have been incomplete.
In my "fun DIY" experiments I directed a 5mm-LED light into the slide, but the beam was too wide, so the slide was externally illuminated by "stray light" from the LED. Obviously wrong...

I tried the links, the first one was a dead end, the second one shows a used item that will not fit my microscope stage (The location of the screw holes in the original MVI depends on the specific microscope stage for which it is ordered from the manufacturer).

So the instrument is neither inexpensive (we think) nor commercially available (status yet unclear).
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Re: Dark field illumination: Questions and answers

#46 Post by MichaelG. » Sat Mar 24, 2018 6:22 pm

Thanks very much for that info. MicroBob
... Much appreciated.

MichaelG.
.
.
P.S. ... Not sure if it's just on my iPad. but the formatting of this thread seems to have gone haywire.

.

Edit: This paper is worth downloading
https://www.ncbi.nlm.nih.gov/pmc/articl ... 058344.pdf
... found via: http://www.photomacrography.net/forum/v ... 6f85b7386d
but, in my opinion, easier to read in the PDF version
Last edited by MichaelG. on Sat Mar 24, 2018 7:35 pm, edited 1 time in total.
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Re: Dark field illumination: Questions and answers

#47 Post by Hobbyst46 » Sat Mar 24, 2018 6:30 pm

MichaelG:

it will actually be "Reinventing the square wheel" :lol:
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Re: Dark field illumination: Questions and answers

#48 Post by MichaelG. » Sat Mar 24, 2018 7:47 pm

Hobbyst46 wrote:MichaelG:

it will actually be "Reinventing the square wheel" :lol:
I take your point :lol:
But do have a look at the LED mutant, in that paper I have just linked.

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Re: Dark field illumination: Questions and answers

#49 Post by Hobbyst46 » Sat Mar 24, 2018 8:08 pm

But do have a look at the LED mutant, in that paper I have just linked.
Yes, this paper is the one that told me that the idea had already materialized. The authors also claim that it can be superior to dedicated DF condensers in terms of resolution. But if it is not an off-the-shelf accessory to buy and use? Anyway, thanks. I will re-read it, to see how they combined DF with fluorescence.
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Re: Dark field illumination: Questions and answers

#50 Post by MichaelG. » Sat Mar 24, 2018 9:09 pm

Here is the datasheet for that 0.4mm LED
http://pdf1.alldatasheet.com/datasheet- ... asheet.pdf

and here the F2 [presumably its successor]
http://ce.citizen.co.jp/productse/info.php?no=72

... Looks useful.

MichaelG.
Last edited by MichaelG. on Sat Mar 24, 2018 9:34 pm, edited 1 time in total.
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Re: Dark field illumination: Questions and answers

#51 Post by Hobbyst46 » Sat Mar 24, 2018 9:25 pm

MichaelG. wrote:Here is the datasheet for that 0.4mm LED
http://pdf1.alldatasheet.com/datasheet- ... asheet.pdf
... Looks useful.

MichaelG.
Thanks MichaelG. The site is alive and active. Inquiry is in progress...
Last edited by Hobbyst46 on Sun Mar 25, 2018 4:30 pm, edited 1 time in total.
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Re: Dark field illumination: Questions and answers

#52 Post by Hobbyst46 » Sun Mar 25, 2018 4:26 pm

IF the price is reasonable, AND IF these tiny bits can be handled... the claims in the article are really tempting...
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Re: Dark field illumination: Questions and answers

#53 Post by MichaelG. » Sun Mar 25, 2018 5:48 pm

Hobbyst46 wrote:IF the price is reasonable, AND IF these tiny bits can be handled... the claims in the article are really tempting...
Glad to see your appetite is whetted
I'm in the UK, and have not yet found the devices for sale ... hopefuly you will have better luck.

Incidentally; I located the patent for the 'system' last night
https://worldwide.espacenet.com/publica ... cale=en_EP
... free to view & download.
I don't think it adds anything to what's in the article, about the illuminator, but it's nice to have.

Please do let me know if you find a stockist of the Citizen lamps.

MichaelG.

Note: Cree has a similar device, with an excellent datasheet:
http://www.cree.com/led-chips/media/doc ... CPR3EF.pdf
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Re: Dark field illumination: Questions and answers

#54 Post by Hobbyst46 » Sun Mar 25, 2018 7:00 pm

About the Cree product: two obvious differences between those and the CL-435:
(1) The Cree are fairly monochromatic, they are either blue, or slightly blue green or green, which means that the DF will have the same colors as well. Not so bad, yet...
(2) The Cree come in three sizes! choose one - or try them all?! The ordinary slide thickness being 1-1.1 mm, I would prefer the 0.6mm LEDS, but Kawano et al succeeded with 0.4mm, which might better prevent "stray light"... 0.8mm I think is too much. So now inquire about the physical appearance of Cree LEDS, availability and prices.

There may be another trick: To attach a 0.6mm fiber optic to a 5mm superbright LED and sideways illuminate the slide, as Apochronaut suggested. I have some components so may give it a shot. To check whether 0.6mm is better than 0.4mm. This becomes a divergent adventure...
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Re: Dark field illumination: Questions and answers

#55 Post by MichaelG. » Sun Mar 25, 2018 7:33 pm

:idea:
Here is another 0.4mm white 'sideview' package
https://everlighteurope.com/index.php?c ... hment=5763

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Re: Dark field illumination: Questions and answers

#56 Post by Hobbyst46 » Sun Mar 25, 2018 8:08 pm

Here is another 0.4mm white 'sideview' package
https://everlighteurope.com/index.php?c ... hment=5763
Very orderly and detailed information, including some about the soldering. Higher luminosity than the CL-435 but a wider beam angle.
They are white light and 0.4mm.
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Re: Dark field illumination: Questions and answers

#57 Post by MicroBob » Sun Mar 25, 2018 9:33 pm

It seems to be a 0,15W LED strip.

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Re: Dark field illumination: Questions and answers

#58 Post by MichaelG. » Sun Mar 25, 2018 10:42 pm

MicroBob wrote:It seems to be a 0,15W LED strip.
Yes ... and suitably diminutive for the proposed purpose.

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Re: Dark field illumination: Questions and answers

#59 Post by Hobbyst46 » Mon Mar 26, 2018 7:26 pm

Hi all, I did some more experiments, see the DIY DF post.
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Bryan
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Re: Dark field illumination: Questions and answers

#60 Post by Bryan » Fri Apr 24, 2020 12:11 am

I'm bringing back this old thread because I just obtained a Leitz Immersion dark field condenser the same as one mentioned earlier in this thread. It came with the iris adapter pictured below and mentioned on the last page of the linked instructions. What I don't have is the objective head to attach to the iris adapter. I checked all my objectives and I don't believe they will work with it. I would like to use the dark field condenser at a medium power as described in the instructions. Can anyone point me to the appropriate objective for this? My microscope is a 1957 Leitz SM Medical Microscope.

http://microscope.database.free.fr/Acce ... oscopy.pdf


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