I think the best person to answer this would be someone from Zeiss. I have tweaked a lot in our lab's microscope and whenever I have doubts I contact the local technical experts of Zeiss. It usually resolves the problem most effectively.

Thank you, Biswajoy. My experience with Zeiss is opposite to yours. I could not find a knowledgeable enough Zeiss rep. Researchgate is my last resort. :-(

Hi Irina, I understand your experience with with the representatives, their level of knowledge differs a lot from place to place. According to the specifications at Zeiss catalogue, objectives with both threads are designed for the same optical system, thus should be interchangeable with the help of a thread adapter. The objective needs to match the tube lens of the microscope. Besides the focal length of the tube lens that affects the magnification, the performance depends on the aberration corrections in the tube lens that supplement the aberration correction in the objective lens. There's no doubts that the new M27 objectives will work for you, even if the tube lens in the old microscope is not exactly identical to the one in the newer microscopes. However, if that is the case, the performance may be slightly compromised (e.g. in chromatic aberration correction), though it could be still better than that of your old objectives, especially if they have been heavily used. You may try to post your question to Confocal List, where most microscopy experts go, and possibly get an opinion from somebody who has tested this or even from people from Zeiss factory.

Hi Irina, I suppose that Axiovert 200 origional utilize objectives with RMS threads. RMS is smaller in diameter than M27 which mean that it is easy to adapt high quality RMS objectives to niew (M27) microscopes. BUT the opposite is more tricky because the M27 side of the adapter will stick out of the turret and the lens tip of M27 objective will be more prominent. Therefore, the distance between the lens tip (objective lens) and the tube lens will be prolonged, as also Radeck M. explains. This might effect the whole optical system. May I ask, what type(s) of M27 objective(s) you want to acquire for your axiovert? What characteristics in the M27 objectives you are seeking? Regarding numerical aperture and chromatic abberation, it is very difficult to distinguish between newer RMS and M27 objectives...

Hi Radek:

Thank you very much for your response. It is clear now to me that buying the new objectives I was thinking about is not worth it for this old microscope. I will post a question on the website you suggested but for now it seems that the fact that the light aberration correction will be compromised due to elevated position of the objectives makes it worthless spending money on expensive advanced objectives I planned to buy.

Hi Olav:

Thank you for your response!

Our goal is to have phase contrast images which could be superimposed on the fluorescent images of stained cultured cells or tissues. So we need to have phase objectives that would give us the best possible fluorescent images.

We have good old objectives for fluorescent images (with RMS thread, Fluar 10x/NA 0.5 and Fluar 20x/NA 0.75). But those are not phase objectives.

I was thinking about Plan-Apochromat phase objectives (M27 thread): 10x/NA 0.45 and 20x/NA 0.8.

https://www.micro-shop.zeiss.com/en/us/shop/objectives/420641-9910-000/Objective-Plan-Apochromat-10x-0.45-Ph1-M27

https://www.micro-shop.zeiss.com/en/us/shop/objectives/420651-9911-000/Objective-Plan-Apochromat-20x-0.8-Ph2-M27

These objectives give phase images and still have the NA almost as good or even better than our old Fluar objectives.

I understand! I am working with similar issues... Even if the apochromat (ph) objectives have a slightly higher NA, I will consider the objectives you already have as something better regarding fluorescence microscopy, especially if the fluorescence signals are week. The phase ring and the vast number of objective lenses in the apochromats will "eat" a lot of photons. However, the apochromats is better when it comes to the correction of the "plan curvature". Have you consider to buy second hand nomarsky prisms and upgrade to differential interference contrast (DIC)? I think DIC + fluorescence is a superior combination due to the halo in phase microscopy. You might find prisms at reduced price at internet sites, even ebay.

Hi Olav! Thank you very much for your time and advices!

Can any type of objectives work with DIC?

No problems! I am interested in these issues... No, not all types of objectives can be used for DIC. I am pretty sure that these objectives could produce fine DIC-images. (Regarding plan-apochromat, only objectives that has "DIC" written on the barrel is ideal for DIC microscopy, due to optical tensions).

I do not know how familiar you are with DIC, but you need to invest in two polarization filter, (under the condenser and in the tube). You also need two DIC prisms/filters per objectives: one in the condenser and one DIC-prism slider above the objectives. This imply that the objective turret need to have DIC slider slots (Do you have such a turret?). This prism set have to mach the NA and magnification of the objective. In your case, you will probably need two sets, one set for each objectives. It is very very very expensive if you buy these components new at zeiss. I got it second hand to a fraction of the price, and it is really worth it. Brilliant images!!!

Check out these DIC prism sliders:

https://www.fluorescencemicroscopes.com/zeiss-microscope-dic-prism-slider-ii-for-fluar-10x-0-50/

https://www.ebay.com/itm/Zeiss-Microscope-DIC-Prism-Slider-III-for-Fluar-20x-0-75-Objective-/252267021285

Condensers for axiovert:

https://www.ebay.com/sch/i.html?_from=R40&_trksid=p2334524.m570.l1313&_nkw=Zeiss+condenser+axiovert+DIC&_sacat=0&LH_TitleDesc=0&_osacat=0&_odkw=Zeiss+condenser+axiovert

Turrets:

https://www.ebay.com/sch/i.html?_from=R40&_trksid=p2047675.m570.l1313&_nkw=axiovert+turret+DIC+&_sacat=0

To buy second hand phase objectives with RMS thread is also an option. My experience is that older zeiss objectives is comparable to new ones, if nicely used. Look forward to hear about your decision.

Best regards Olav

Hi Olav! Thanks a lot! We also have the following idea. What if we buy a phase objective and make fluorescent and phase images with a Fluar and Phase objectives, respectively? Is it possible to have exactly the same field of view with a fluorescent and a phase (but different) objectives?

Hi Irina, it won't be easy to make sure the two objectives are looking at the exactly same area. In my experience, people prefer to use the same objective and take fluorescence images with the phase contrast lenses, even though they loose some signal on the phase ring. Depends on how week your fluorescence signal is; in many cases this is not a big issue. Otherwise, I agree with Olav that DIC images tend to look way better than phase contrast and at the same time DIC doesn't have the issue with the loss of fluorescence on the phase ring, so it may something worth considering.

There are also advocates of brightfield, who use these arguments:

1. Brightfield reaches higher spatial resolution than DIC or phase contrast while being technically much simpler.

2. Phase contrast and DIC were developed to overcome the poor contrast in brightfield of samples that do not strongly absorb light. However, both were developed in times before digital image processing, which can be used to enhance the poor contrast in brightfield images.

There is quite an interesting discussion on this topic at Confocal List:

http://confocal-microscopy-list.588098.n2.nabble.com/Phase-Contrast-Microscopy-tt7590859.html#a7590872

As Radek explains, two pairs of objectives is not recommendable due to slightly different areas of field of view. (For the same reason you need to readjust köhler illumination when changing magnificatiion...) If your fluorescence signals is good, phase contrast objectives will do, especially if you get hold of plan-apochromatph (high NA). Plan-Neofluar ph 10x and 20x is also fine objectives for fluorescence microscopy even if the NA is lower. A smaller number of lenses means less photon absorption. These objectives is easy to acquire (RMS) and cheaper. If you go for digital contrasting, as Radek suggested, very good second hand plan-apochromats (RMS) are much easier to find.