Zeiss West DIC with Mismatched Objectives

75RR · Post by **75RR** » Mon Sep 05, 2016 8:56 am

That is great - and you haven't even oiled the condenser to the slide yet!

Makes me wonder what would happen with a 100x/1.3 Planapo

I have a delaminated one, as you say they are more affordable, which I will try with the Plan 40x/0.65 slider and the Planapo 63x/1.4 slider to see what might be seen.
Have a love/hate relationship with Zeiss at the moment.
By the way did they ever get around to shooting the person that came up with the dodgy adhesive? ;)

75RR · Post by **75RR** » Mon Sep 05, 2016 9:01 am

Have been rereading this thread and can't see/must have missed the part where you mention how you have attached your camera.

I am using a raised 12.5x kpl (and yes it also has some delamination) as a direct projection lens.

Pau · Post by **Pau** » Mon Sep 05, 2016 9:15 am

75RR wrote:I am using a raised 12.5x kpl (and yes it also has some delamination) as a direct projection lens.

Does it work better than your former afocal setup with a 50mm lens?

75RR · Post by **75RR** » Mon Sep 05, 2016 9:45 am

Hi Pau,
I was getting flaring with the 50mm 1.4 OM lens on the E-p2, not sure why, it might have been because my lens has a little fungus in it.
Also bought a Zeiss Mipro 63mm but did not see an obvious improvement - mind you it was a quick test. Will try again at some point.

zzffnn · Post by **zzffnn** » Mon Sep 05, 2016 5:12 pm

Very nice imaging, thank you for sharing, Dave.
Do you have a 8-form test diatom slide to try DIC on?
I heard a dry condenser, even an aplantic-achromatic one, cannot go over NA 0.95.

Cactusdave · Post by **Cactusdave** » Tue Sep 06, 2016 7:05 am

75RR -- I will post my fixed afocal coupling system in a separate post so as not to clutter this one. Flare can be an issue, you just need to know where to flock if it is.

zzffnn -- My understanding of Effective NA in a system where the objective has a higher NA than the condenser is that it is a function of the NA of both condenser and objective according to the formula: Effective NA = Objective NA + Condenser NA /2. So in this case Objective NA = 1.4, dry condenser NA, as you rightly state, is about 0.90-0.95, so Effective NA should be 1.4+0.95/2, so a bit over 1.1. If I'm wrong about this I'm glad to be corrected.

Yes I have the Klaus Kemp 8-form test slide, but as my rather extensive slide collection isn't very systematically organised, I must confess I can't find it at present.

Alan Wood · Post by **Alan Wood** » Tue Sep 06, 2016 9:01 am

David

I thought the effective NA of a condenser+objective could not exceed 1 unless oil (or another liquid) is used top and bottom of the slide.

Alan Wood

75RR · Post by **75RR** » Tue Sep 06, 2016 10:16 am

Image from: FUNDAMENTALS OF LIGHT MICROSCOPY AND ELECTRONIC IMAGING
Douglas B. Murphy

Cactusdave · Post by **Cactusdave** » Tue Sep 06, 2016 11:58 am

Thanks for correcting me both of you. If I had thought for a second about the importance of the refractive index of the oil, I would have realised my mistake. Even more to gain from trying DIC with this lens and the X40 slider with the condenser oiled.

Pau · Post by **Pau** » Tue Sep 06, 2016 3:27 pm

Cactusdave wrote: My understanding of Effective NA in a system where the objective has a higher NA than the condenser is that it is a function of the NA of both condenser and objective according to the formula: Effective NA = Objective NA + Condenser NA /2. So in this case Objective NA = 1.4, dry condenser NA, as you rightly state, is about 0.90-0.95, so Effective NA should be 1.4+0.95/2, so a bit over 1.1. If I'm wrong about this I'm glad to be corrected.

Nothing wrong here, it's the classic formula. Oil is computed in the objective NA specification.
I think it is not as accurate as it's stated in most books, but likely it will apply within certain limits for bright field microscopy. For DIC the used illuminated aperture is a bit lower because of the interference bands you use to make contrast but in practical terms in many cases resolution can be even higher than with BF because you don't need to close the aperture diaphragm to get contrast.

We had at the forum some interesting previous discussions on this subject, but I cannot find them at this moment.

rjlittlefield · Post by **rjlittlefield** » Tue Sep 06, 2016 10:27 pm

Pau wrote:We had at the forum some interesting previous discussions on this subject, but I cannot find them at this moment.

Around http://www.photomacrography.net/forum/v ... 4212#94212 , I think.

--Rik

Cactusdave · Post by **Cactusdave** » Wed Sep 07, 2016 2:00 am

Thanks for the link Rik.

I found this link helpful also ; http://www.olympusmicro.com/primer/anat ... rsion.html

To quote from it:
' A factor that is commonly overlooked when using oil immersion objectives of increased numerical aperture is limitations placed on the system by the substage condenser. In a situation where an oil objective of NA = 1.40 is being used to image a specimen with a substage condenser of smaller numerical aperture (1.0 for example), the lower numerical aperture of the condenser overrides that of the objective and the total NA of the system is limited to 1.0, the numerical aperture of the condenser.

Modern substage condensers often have a high degree of correction (see our section on condensers) with numerical aperture values ranging between 1.0 and 1.40. In order to effectively utilize all the benefits of oil immersion, the interface between the substage condenser front lens and the underside of the microscope slide containing the specimen should be also be immersed in oil.'
This system has been termed a Homogeneous Immersion System and it is the ideal situation to achieve maximum numerical aperture and resolution in an optical microscope. In this case, the refractive index and dispersion of the objective front lens, immersion oil, substage condenser front lens, and the mounting medium are equal or very near equal. In this ideal system, an oblique light ray can pass through the condenser lens and completely through the microscope slide, immersion oil, and mounting medium undeviated by refraction at oil-glass or mounting medium-glass interfaces.

When using high-power achromat oil immersion objectives, it is sometimes permissible to omit the step of oiling the condenser top lens. This is because the condenser aperture diaphragm must often be reduced with lesser-corrected objectives to eliminate artifacts and provide optimum imaging. The reduction in diaphragm size reduces the potential increase in numerical aperture (provided by oiling the condenser lens) so the loss in image quality under these conditions is usually negligible.

Glycerin, refractive index 1.47 may be a viable alternative as the liquid between the upper surface of the condenser top lens and the underside of the slide, with a rather easier clean up as it is water miscible. Even water, refractive index 1.33 would presumably be better than air. However the full advantages of truly homogenous immersion would then be lost.

75RR · Post by **75RR** » Wed Sep 07, 2016 5:21 am

When using high-power achromat oil immersion objectives, it is sometimes permissible to omit the step of oiling the condenser top lens. This is because the condenser aperture diaphragm must often be reduced with lesser-corrected objectives to eliminate artifacts and provide optimum imaging. The reduction in diaphragm size reduces the potential increase in numerical aperture (provided by oiling the condenser lens) so the loss in image quality under these conditions is usually negligible.

Was wondering where the prevalent "I don't oil the condenser to the slide" vogue had originated. Now we know.
Thanks for setting the record straight.

Charles Krebs · Post by **Charles Krebs** » Wed Sep 07, 2016 9:37 pm

Dave,

Great thread, great results. Now that you are into the high power oil immersion objectives I'm going to pose a question or two.

If you check the back of the objective carefully with a phase telescope or Bertrand lens, do the DIC condenser inserts in the Zeiss condenser constrict the rear aperture at all, or can you still see the full clear aperture when the prism is moved into place?

I seem to remember that in some cases the diameter of the condenser prism (deliberately) limited the aperture to some degree.

I've seen a comparison of an Olympus 60/1.4 Plan Apo shot with the BH2-UCD condenser (0.90) and an oiled Olympus 1.40 condenser (same "60/1.40 oil" prism). There was extremely little, if any, noticeable difference even with significant pixel peeping.

I would love to see if you, or 75RR, really see any difference between the the condenser oiled and dry. (With DIC that is... I know it can make a difference in brightfield).

Cactusdave · Post by **Cactusdave** » Thu Sep 08, 2016 1:04 pm

Thanks for the compliments Charles. I'm gearing up for a comparison of DIC with the X40 slider and the X63, 1.4 Planapo +/- oiling the condenser by locating some suitable test diatom slides that will be a sterner test.

If you check the back of the objective carefully with a phase telescope or Bertrand lens, do the DIC condenser inserts in the Zeiss condenser constrict the rear aperture at all, or can you still see the full clear aperture when the prism is moved into place?

I seem to remember that in some cases the diameter of the condenser prism (deliberately) limited the aperture to some degree.

I've had a careful look at the back focal plane and I can't detect any difference in the clear rear aperture diameter with or without a DIC slider in place. The diameter of the circular prism window in the 160mm Zeiss objective slider slider is quite small though, 10mm. The sliders for the Axio series are rather longer and wider with a different shaped window. I normally restrict the rear aperture to about 80% with the condenser diaphragm in any case to help with control of glare if this is a problem.