Zeiss West DIC with Mismatched Objectives

Cactusdave · Post by **Cactusdave** » Thu Aug 11, 2016 1:10 pm

I was going to go straight on to X40 after X25. There are some interesting X40 objectives that it would be nice to check for DIC compatibility, I had in mind particularly to check the Zeiss West X40, 0.75 water immersion objective. Thinking of water immersion objectives made me think of the Lomo X30, 0.90 EAF water immersion objective. I have used this before successfully with a X40 DIC prism on my Nikon Diaphot http://www.photomacrography.net/forum/v ... 8646#98646 and was pleased to find it also works well with Zeiss X40 DIC prism slider in the normal upright position. I used the Lomo objective without extension, though it is a short non-DIN length. I used it with the built in iris fully open, so it's at it's full NA. I used it as a conventional immersion objective with a drop of distilled water on the coverslip and the objective dipped into this. For an immersion lens this objective has a rather long working distance so when used like this a quite generous water drop is needed to keep contact between the coverslip and objective tip. DIC is good and full extinction is possible. There is significant chromatic aberration with this lens and the Leitz Periplan eyepiece I am using for afocal coupling, and as most images are of diatoms I have converted to black and white were this is an issue.

Some sample images all of diatoms from various vintage and contemporary mounts.

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All images are stacks of between 10-17 images. As I have discovered before this lens performs well producing bright images with good resolution as would be expected from its high NA. It works very nicely with the X40 DIC prism slider. It does have a problem with chromatic aberration which might have been lessened by the choice of an eyepiece with more compatible compensation, but I think some CA is characteristic of the lens.

Beatsy · Post by **Beatsy** » Thu Aug 11, 2016 2:14 pm

Nice images Dave and a very useful thread overall for benchmarking what I should expect from my mix-n-match attempts. Thanks for doing it. Are you going right through the series - as in 63x and 100x next?

Cactusdave · Post by **Cactusdave** » Fri Aug 12, 2016 1:37 pm

Thanks Beatsy. I do plan to go beyond X40, but I'm not anticipating much success. I don't have either the Zeiss X100 or the X63 prism sliders and I think one or other of those, designed for immersion objectives, would be desirable for anything involving oil immersion. I have slight hopes that I might get something workable with a dry X60 or X63, but the only dry Zeiss objective I have in that range is the Neofluar X63, 0.90 Ph3, and a phase ring pretty much kills any chance of DIC. I missed a chance of getting the Planapo X63, 1.4 slider for a reasonable price a while back, and I'm still kicking myself.

75RR · Post by **75RR** » Fri Aug 26, 2016 3:41 am

Have only just come across this thread - forum equivalent of a pot of gold for Zeiss 160 DIC custodians! :)
Nice to have both details and images. Many thanks for the effort you have put into it.
I will make another attempt to find matching objectives though I am somewhat hampered by only having a 40x and a 63x slider
and a limited number of objectives.

Cactusdave · Post by **Cactusdave** » Sat Aug 27, 2016 8:56 am

Thanks 75RR. I'm really pleased if anyone finds this little odyssey useful. I now have a very nice option at X40, the Zeiss X40, 0.95 planapochromat see for example http://www.photomacrography.net/forum/v ... 468#182468 and http://www.photomacrography.net/forum/v ... 205#183205 . I'll post a couple of more examples taken with this excellent lens. All taken from a diatom arrangement by Steve Beats. The first is a single unstacked image.

The second is stacked from 21 images with Zerene.

The final image is a 16 image Helicon stack of an old mount of an Aulacodiscus diatom mounted in Cassia oil, which has a refractive index of about 1.61.

I've also just found a nice option for DIC at X63 with a dry Leitz objective that I'll post shortly.

Pau · Post by **Pau** » Sat Aug 27, 2016 9:45 am

I'm really pleased if anyone finds this little odyssey useful

I also do, as you could guess.

I admire the quality of DIC you're getting, mine is some steps down.

I've updated my objectives collection and I plan to do this kind of testing post when done

75RR · Post by **75RR** » Mon Aug 29, 2016 6:48 am

Great results on the Zeiss 40x/0.95 Planapochromat, congratulations on this addition to your stable.

I have a delaminated Zeiss 40x/1.0 Planapochromat on its way - hoping it will give me better images than the Plan 40x/0.65 despite this. We shall see.

Tested a Zeiss Neofluar 25x/0.60 Pol and found to my delight that though it lacks extinction (as you have mention on some combinations),
it works quite well either side of it, towards the positive and negative bias.
I used the 40x slider, right side up in position II

No joy on the Plan 16x/0.35 nor the Neofluar 6.3x/0.20
I think I will have to get the 16x slider for them.

Cactusdave · Post by **Cactusdave** » Tue Aug 30, 2016 6:26 am

Thanks Pau and 75RR, glad you are finding this useful and interesting.

75RR, the Microscope Italy article http://www.microscopeitaly.it/2009/11/3 ... -versione/ claims to have good DIC with a Zeiss Jena X6.3 Apochromat and the X40 slider upside down and the condenser in the I position. I have been unable to repeat that observation, but have got good DIC with the same objective with the X16 slider upside down and the condenser in the I position, as posted in this thread. It's always worthwhile without a slide in place, to take one of the eyepieces out and look at the back focal plane of the objective. A narrow dark band with sharp edges is not what we want. What we want is something perhaps with a darker centre, but with a more gently graduated darkening on either side (positive and negative if you like).

With your X25 Neofluar, try dropping the condenser a little below what would be optimal for brightfield while watching the DIC or the back focal plane image. It may help to give a more even DIC effect at the expense of a rather darker image overall. If you do get a better effect try re-optimising the two diaphragm apertures and the condenser DIC controls at the new condenser height. All these factors together and separately make a big difference to the quality of the DIC image.

If you want a alternative lower powered objective that will work with the X40 slider, consider trying to get a Lomo X20, 0.65 Apochromat. As I have shown, this works well. I suspect the equivalent Zeiss Jena X20 Apochromat would also work, as it is likely optically identical to the Lomo, but I don't have one to try to confirm this.

Cactusdave · Post by **Cactusdave** » Tue Aug 30, 2016 7:14 am

I have tried two options so far for DIC with a high power dry objective. One, the Leitz X63, 0.85 EF objective was not good, but the other, the Leitz NPL Fluorite X63, 0.90 with correction collar proved very good with the X40 DIC slider in the normal upright position and the condenser in the II position. Almost full extinction is possible. This objective is a big fat lens, much bigger and fatter than the Zeiss Neofluar X63, 0.90 with correction collar. I would like to test the Zeiss objective, but I only have a Ph3 version that's pretty badly delaminated, so trying that would be a waste of time. Anyway, this is what the Leitz objective looks like.

It is worth mentioning a couple of things that apply when using high power dry objectives. One, they are champion coverslip smashers. They have very little working distance, so little it may be impossible to use them with older vintage mounts having thick coverslips. Without the discipline of using a liquid drop that you get from an immersion objective, it's all too easy to casually rack the objective down too far in search of focus. Secondly if there's a correction collar, it's setting really matters. With the Leitz NPL Fluorite X63, accurately setting the correction collar for the coverslip by rotating it carefully while watching the image shows a significant improvement in sharpness when it is exactly correct for that individual coverslip.

Some example images with this objective. All from a Steve Beats diatom arrangement with a 0.17 coverslip (as verified by the correction collar!)

The first is a 6 image stack with Helicon Method B

The second is stack of 9 images with Helicon Method B.

The final image is a single unstacked shot.

This objective gives nice DIC and good resolution. The condenser needs to be just a fraction below maximum height, and the correction collar needs to be adjusted carefully.

Ilikebiscuits · Post by **Ilikebiscuits** » Tue Aug 30, 2016 7:59 am

Wonderful results

Cactusdave · Post by **Cactusdave** » Tue Aug 30, 2016 8:24 am

Thanks Ilikebiscuits. I have responded to your PM.

Pau · Post by **Pau** » Tue Aug 30, 2016 8:42 am

Dave, any advantage of the NPL Fluotar 63X 0.90 over the Z. Planapo 40X 0.95?

At your posted samples I prefer the Planapo, IMO the small magnification difference could be easily compensated cropping the image as at this magnification most camera sensors outresolve the objective.

I'm just now testing my Planapo 40/0.95 against a Pl Fluotar 40/0.70 and the Planapo wins in all but easy of use as you well pointed: not big but noticeable higher resolution and much less axial CA. Both provide acceptable DIC.

Cactusdave · Post by **Cactusdave** » Tue Aug 30, 2016 9:21 am

That's a very good point Pau. The same thought had crossed my mind. The Zeiss West X40, 0.95 Planapo is an exceptionally good lens and I have not checked with a really demanding test diatom, to see whether there is any worthwhile increase in resolution with the X63 compare with a cropped image from the X40 Planapo. I would say that my X40 planapo is not an absolutely perfect example. There is a tiny bit of delamination.

Cactusdave · Post by **Cactusdave** » Sun Sep 04, 2016 2:11 pm

I want to post one last image with the Leitz Fluotar X63, 0.90, correction collar. I think I squeezed a little better image out of it.

I forgot to mention, when talking about the usefulness of examining the back focal plane of the objective, about using it to check the centring of the condenser diaphragm aperture. If the condenser diaphragm is closed a little, the aperture should be visible and central in the back focal plane view. If it is offset to one side this can be corrected by a small rotation in either direction, of the condenser DIC position changer while looking at the back focal plane view. A tiny rotation should be enough. This optimisation is worth doing as it improves the DIC image.

Next I want to look at the Zeiss X63, 1.4 oil Planapochromat used with the X40 slider.

Cactusdave · Post by **Cactusdave** » Mon Sep 05, 2016 8:29 am

Although the Leitz 'high dry' X63 Fluotar, 0.90, correction collar gives very nice DIC images and has the advantage of no messy oil, it should be out resolved comfortably by the Zeiss West X63, 1.40 Planapochromat. This was my 'go-to' lens when I did immunofluorescence as a professional cell biologist, and I've always regarded it as one of the finest 160mm objectives Zeiss West ever made. It has its own dedicated objective slider for DIC, and as 75RR has shown, the combination can produce stellar images. http://www.photomacrography.net/forum/v ... 030#196030 I am not lucky enough to possess that slider so I thought the time had come to see if it would produce any kind of workable DIC with the X40 slider which I do have. My example of the Zeiss X63 Planapochromatr isn't perfect. It has visible though fairly minor delamination, which helped to bring it into my price range. Nevertheless I was delighted with the DIC images I got with the X40 objective slider in the normal position and the number II position condenser DIC prism. These illustration images are without the underside of the slide oiled to the condenser top lens. As 75RR has pointed out, http://www.photomacrography.net/forum/v ... 248#196248 it would almost certainly improve the image to do so as it would allow expression of the full NA of the lens. Used with the condenser dry I am limiting it to an NA of a bit over 1.1. I will get around to repeating the test with the condenser oiled at some point soon.

Anyway here are some images of diatoms from the arrangement by Steve Beats that I have used for a lot of my tests as I know the coverslip is spot on 0.17mm. The first image is a three image stack with Helicon method B.

The second diatom is a 6 image stack with Helicon B

The third image is a single unstacked shot without a lot of background cleanup, just levels, sharpening and Gaussian blur on the background.

The final image is my favourite, a fairly deep stack, 22 images Helicon B of a nice challenging 3D diatom.

DIC is generally good, with a pretty even background, though getting something as pretty as 75RR's required a bit of work. Resolution is also very good. Considering I now don't feel I have to shop for a £00 special slider, I was very pleased. It may be even better when I oil the condenser as well.