Questions about DIC setup (Images added)

[Pau]

Against any good advice, I'm setting up an hybrid DIC microscope with parts of different makers and eras. Yes, I know, all the components MUST be matched and this way is highly risky, but I don't have the budget to buy a complete setup.

My scope is a Zeiss Standard, the objectives Leitz NPL Fluotar, the polarizer from a Zeiss Axio and the analyzer a Nikon. All them play well toghether for simple polarization work.
Some time ago I bought a Zeiss DIC (InKo) condenser with 3 prisms that seems designed to work with the dedicated DIC slider III, a very rare and expensive part (last one was sold in eBay for $1,600!) and a very restricted selection of planachromat objectives.
Unable to find this part and after being aware that the Olympus BX51 uses an apparently similar setup (Nomarski with only one common objective prism slider for all magnifications) when I was able to buy that slider (U-DICT) for a moderate price I decided to try. I'm going to mechanically modify the intermediate Zeiss tube with Telan lenses designed to allow the prism to fully allow mounting the Oly one.
After lots of tests and visits to Molecular Expressions website and some other sources, now I can get a reasonably good DIC effect

But the system is very far from perfect:
- The field of view background isn't fully uniform, with some light intensity gradient.
- The conoscopic image through a telescope eyepiece with the system set up at full extinction and both prisms in place is not dark but grey and with one diagonal band (similar to the view with only one dic prism in place although much less defined)
- The DIC effect is fairly good with the 40/0.75 and 50/1 objectives and the III prism, good but less uniform with the 25/0.55 with prism II and very subtle and uneven at 100/1.32. At lower mags I'm unable to obtain DIC

Following Molecular Expressions website http://micro.magnet.fsu.edu/primer/tech ... ation.html
the objective DIC prism must be situated in the back focal objective plane, a conjugate plane with the DIC condenser prism. In my setup it isn't but testing the microscope I find it isn't possible with the original Zeiss one because that plane is not imaged inside the intermediate tube. In the Olympus BX51, an infinite system, the objective DIC slider is placed in the infinite space, not very close to the objective, so I suppose that again it isn't at the back focal plane.
I suppose that my problems come, at least partially, from this inability to place the prisms at conjugate planes

I'm very confused.
I never did use a true DIC microscope, so I only have information about DIC from readings and images (like the magnificent micrographies posted here)

And now the questions for the DIC experts in the forum:
- When you have DIC properly setup, is the background fully even along the field of view or is there any gradient?

- I'm interested mainly in Olympus scopes and Zeiss older ones because Nikon, Leitz and most Zeiss systems use individual prisms for each objective. With that systems, is the condenser prism imaged in the position of the objective prism slider?. The easiest way to test it I found is to put a phase ring in the condenser and a painted microscope slide in the slider position and focus with a telescope eyepiece. Can you test it and tell me?

Waiting you comments, answers and suggestions...
...but please don't say "you're crazy, that system can't work", I already knew it

[Cactusdave]

I have a Nikon Diaphot inverted microscope with long working distace phase/DIC condenser with prisms for the Nikon X10 Plan DIC and X40 LWD Plan DIC objectives. In this inverted Nikon system, polariser, Wollaston prisms and variable Lamda plate are all in the condenser and variable analyser and second prism are mounted below the objectives. The objectives are strain-free and supposedly 'matched' to the prisms, but otherwise are not special.
It is possible to get good 'silver/grey' DIC background across the whole field quite easily with the X10 objective. The X40 is more tricky and getting good DIC and an even background depends on careful attention to condenser and analyser settings. The evenness of the background is also influenced by the thickness and quality of the slide and cover glass. This is particularly so for older permanent mounts.

I have tried several 'wrong' objectives in this system with varying degrees of success in getting DIC. I have a Nikon X20 Plan DIC objective, and this gives reasonable DIC across about 50% of the field with the X40 prism, but, though usable it is certainly not even across the field. I have also used a Nikon X4 0.13 Plan DL objective with the X10 prism. Here I get a 'band' of strong DIC-like or oblique contrast which can be moved across the field of view by rotating the analyser. This doesn't seem to be true DIC, but seems to be the effect that Graham Matthews has called 'VIOLA' http://www.photomacrography.net/forum/v ... ight=voila More interestingly I obtained a Zeiss X25 0.65 Planapo objective recently and thought, with out much hope I would try it with the Nikon DIC. Really this objective ought not to give any kind of DIC effect at all or at best give the 'VIOLA' effect. Imagine my surprise and pleasure when I discovered it gave very reasonable DIC with the X10 prism and essentially perfect DIC with the X40 prism with an even background. I can't explain this, but it certainly illustrates that wildly non-matched objectives can give excellent DIC and it is always worth experimenting.

It is worth noting that a certain lack of evenness of the DIC effect across the whole field is not completely disastrous, provided the subject itself is evenly covered, as a lot can be done to 'homogenize' the background in post processing.

[RogelioMoreno]

Pau,

- When you have DIC properly setup, is the background fully even along the field of view or is there any gradient?

With my current Nikon setup there is some gradient, with some objectives you see the background darker at the corner and lighter at the center. Nikon claims that the new DIC system has homogeneous background, I can not confirm that yet; but checking the DIC objective slider (for the new system) that I have I can tell you that is true.

With that systems, is the condenser prism imaged in the position of the objective prism slider?

Yes, I checked my system with a phase ring and the objective slider dark band was seen at the back focus plane of the objective together with the phase ring image.

Remember that the DIC prism used at the objective side are Nomarski type (the focal plane is outside the prism, at the back focal plane of the objective). You are using a Olympus slider that the Nomarski prism was made with the focal plane to be below it some distance (the exact distance that there is from the prism (when it is inserted in the slot) to the back focal plane of the objectives, all the Olympus objectives that work with this DIC slider should have back focal plane located at the same point (or very close) inside the objective. You should be able to move the olympus DIC slider up/down to put the focal plane of the prism at the back focal plane of the objectives that you are using (the condenser DIC prism image is already at the back focal plane of the objectives).

Rogelio

[Pau]

Many thanks Dave and Rogelio for posting your experiences and knowledge

Remember that the DIC prism used at the objective side are Nomarski type (the focal plane is outside the prism, at the back focal plane of the objective). You are using a Olympus slider that the Nomarski prism was made with the focal plane to be below it some distance

It's an important point I passed over!, and it may explain my partially good results. I've tried putting the objective prism head down and its effect is very different (but much worse: the interference bands are clearly imaged)

(the exact distance that there is from the prism (when it is inserted in the slot) to the back focal plane of the objectives, all the Olympus objectives that work with this DIC slider should have back focal plane located at the same point (or very close) inside the objective.

Inside the objective like phase objectives?. I'm a bit more confused...

You should be able to move the olympus DIC slider up/down to put the focal plane of the prism at the back focal plane of the

objectives that you are using (the condenser DIC prism image is already at the back focal plane of the objectives).

I cant't move it freely, only can switch betwwen two locations in the intermediate tube. Te one that works better is the one more far of the objective

I have tried several 'wrong' objectives in this system with varying degrees of success in getting DIC. I have a Nikon X20 Plan DIC objective, and this gives reasonable DIC across about 50% of the field with the X40 prism, but, though usable it is certainly not even across the field. I have also used a Nikon X4 0.13 Plan DL objective with the X10 prism. Here I get a 'band' of strong DIC-like or oblique contrast which can be moved across the field of view by rotating the analyser. This doesn't seem to be true DIC,

This behavoir is very similar to what I obtain with the 25/0.55 and the III prism (good but uneven) and the 16x, 10X and 4X objectives (a dark band with some DIC effect at its borders).

More interestingly I obtained a Zeiss X25 0.65 Planapo objective recently and thought, with out much hope I would try it with the Nikon DIC. ...
...I discovered it gave very reasonable DIC with the X10 prism and essentially perfect DIC with the X40 prism with an even background. I can't explain this, but it certainly illustrates that wildly non-matched objectives can give excellent DIC and it is always worth experimenting.

Perhaps the objective NA (0.65 in both ogjectives) is more important than the magnification

I would like to know the experiences of other DIC users

[Pau]

First test photos with this setup. Diatom test plate. Monocular tube, 10X Periplan eyepiece and Olympus OM 50mm 1.8 lens. EOS 7d "unprocessed"

1. 40/0.75 prism III

2. 25/0.55 prism II[/img]

3. 40/0.75 prism III 50% crop

4. 40/0.75 prism III, more contrasted diatom frustules


What do you think?

(edited to add images)

[RogelioMoreno]

Inside the objective like phase objectives?. I'm a bit more confused...

Yes, you should see the black band (of the objective prism, for the test remove the condenser prism from the light path) inside the objective (in the same point where you would see the phase ring if the objective were phase objective)..

I cant't move it freely, only can switch betwwen two locations in the intermediate tube.

I know you can not move it freely (on Olympus microscope you can not move it freely vertically); but if you want you use the U-DICT slider on other microscope then it will be good to put the slider at the right distance from the objective.

Rogelio

[Cactusdave]

Your test diatom shots don't show particularly convincing DIC to me with the exception perhaps of the third shot, which does look pretty good with the background rendered in a distintive DIC way. Certainly there is some contrast enhancing effect in the other images, but it looks more like the type of oblique effect seen with mismatched DIC components that Graham Matthews dubbed 'VOILA' http://www.photomacrography.net/forum/v ... php?t=4386 . In this link there is a revealing shot of the back lens of the objective during operation of 'VOILA' which might interest you, also some diatom images obtained using the technique showing how 'DIC-like' they can be. The diatoms you are using as test objects are perhaps not the best choice for exploring DIC and oblique effects. Something with more 3D structure like Graham's diatom specimen, which I think is Actinoptychus heliopelta will make it easier to assess your effects.

[Pau]

Dave and Rogelio, thanks again!!

- I tried the approach of focus the objective prism interference band toghether with a objective phase ring and the result is unexpected: in the lower vertical position of that prism it's very near but not totally coincident and in the upper position it's way more defocussed, but in the lower one it isn't any DIC effect and in the upper one it is

- Dave, are you refering to the third or fourth image?. The thirth one is very similar to the first (but with the prism shear in the opposite direction) I posted it to show the resolution.
I tried "VOILA" approach and with my system the relief effect is way less visible.
Of the conditions that Molecular Expression says about how to properly align the DIC microscope, with the 40X objective I accomplish all but the black background with no shear (mine is dark grey with some more light in peripherical zones

[Cactusdave]

It was the 'more contrasted' X40 image that I thought was pretty good DIC, Pau, so that would fit. The first X40 shot does look like DIC, but on my setup if I saw it, I would want to try to get a bit of a more contrasty DIC effect, exactly as you have done with the fourth image.

[RogelioMoreno]

- I tried the approach of focus the objective prism interference band toghether with a objective phase ring and the result is unexpected: in the lower vertical position of that prism it's very near but not totally coincident and in the upper position it's way more defocussed, but in the lower one it isn't any DIC effect and in the upper one it is

The direction of the interference band is very important and it must match with the direction of the condenser prism's interference band, if you are inserting the DIC slider in inverted position you changing the direction of the interference band (unless you are inserting it through the other end (assuming the intermediate attachment allows it) of the intermediate attachment). I remember when I was testing my DIC setup, with some combination of condenser prism and objective prism, the direction of the condenser prism's band and objectives prism´s band where the same (when seen through of the Bertrand lens); but no DIC effect, then I had the idea to rotate the condenser prism 180 degrees and I got DIC effect.

Rogelio

[Pau]

Thanks again for your help

I've retested all the mechanically possible objective Nomarski prism positions and I only can get DIC at one of them: SW-->NE in the upper slot (in my condenser the interference band is oriented NW-SE (corrected) and not NE-SW that seem more usual). I can place it NE-->SW but it doesn't deliver DIC.
With maximun DIC effect the background is fairly uneven. I will need to learn to live with it.

Some more test shots, now with the NPL Fluotar 50/1 oel.
What do you think about the DIC effect?

1.- Bucal mucose cells, a bit cropped


2.- Navicula


3.-Pleurosigma


4.- Crop of the same picture

[Cactusdave]

To me that looks like a very nice DIC effect, with, as you say, a rather uneven background. Actually the images are really very good, the objective lens seems to be performing very well with excellent resolution. I have to say with a hybrid system such as yours, I'd be pretty happy with those images. The uneven background is less important at this higher magnification and it could always be made more even with judicious use of Photoshop.

[lauriek]

I have to say I think the gradient background particularly on the third shot I'm your last post is quite aesthetically pleasing!

Really good work getting this stuff working, I think a great many of us would love a DIC setup but simply can't afford a conventional 'set' of equipment...

[RogelioMoreno]

Nice results!

Because your are using a hybrid system the best option is to try all the posible prisms postions (as you are actually doing). The NE-SW and NW-SW (I think you wanted to said NW-SE) that you mentions are when seen at the back focus plane of the objective? Remember that there is two ways to put the condenser prism NE-SW direction (after you put one the other is got rotating the prism 180 degrees), try both.

Rogelio

[Pau]

Thanks all for your comments,

Dave, yes I'm moderately satisfed with my results, perhaps can be a bit improved when I had finished the mechanical modifications and was able to work more comfortably with the trinocular head (I'm performing the tests with the intermediate piece partially dismounted and an upright monocular tube: each five minutes I need to stop to relax my neck).
The Leitz 50/1 is a really sweet objective, I also like it for darkfield with the ultracondenser

The uneven background is less important at this higher magnification and it could always be made more even with judicious use of Photoshop.

Any advice to beguin? Sometimes I use a gradient tool but here some kind of substractin of a background picture would be better, but I never tried this approach.

Laurie, I agree the image looks good (and Pleurosigma is a beauty) although I would prefer to get a even background

....Because your are using a hybrid system the best option is to try all the posible prisms postions (as you are actually doing). The NE-SW and NW-SW (I think you wanted to said NW-SE) that you mentions are when seen at the back focus plane of the objective? Remember that there is two ways to put the condenser prism NE-SW direction (after you put one the other is got rotating the prism 180 degrees), try both.Rogelio

I corrected the mistake an have put arrows to clarify that I've tried both senses in the same direction as you sugest, and yes, I'm refering to the black interference band at the (approx) objective back focal plane in conoscopic image.