Oil immersion or higher power eyepieces?

[Amoeba]

I am weighing up the pros and cons of purchasing a couple of oil immersion lenses, a x54 and a x100 in addition to a Leitz SM-LUX Microscope which already comes with 4 objectives up to x40. The necessary additional 1.3 condenser and the two oil objectives will effectively double the total outlay. I have already been informed that the x100 is rarely used, but one of the things it is used for is Diatoms which I am interested in. I have been carefully considering the information that I have been given, supplementing it with a bit of on-line research and I came across these two articles:

http://www.micrographia.com/tutoria/mic ... cb0700.htm

http://www.microscopy-uk.org.uk/mag/ind ... pjoil.html

Both of these are very informative and both mention using dry objectives along with a higher power eyepiece (e.g. x15 or x20) as an alternative to oil immersion, the practical plus being no messing about with oil and the added complexity of swapping between dry and oil objectives. Apparently less time wasted, while still achieving reasonably high magnification power. One example quoted states that a 15x eyepiece with a x40 objective gives 600x magnification while still providing acceptable resolution. Since I have no experience using either, I wanted to ask members what the experience is like with using higher power eyepieces when compared to using oil immersion objectives?

Since the Microscope I am thinking of buying is a Leitz, I was looking for Leitz eyepieces on eBay, but could only find 10x. Did they ever manufacture Periplan eyepieces in any other magnification factor? There are at least a couple of variants of cheaper Chinese versions (e.g. 15x with 23.2mmdiameter - not counting other types for glass wearers and extra wide field) but would they be suitable for the Leitz SM-LUX? How much compromise would there be in terms of image quality when using such a cheaper eyepiece?

[billbillt]

I have Amscope oculars here to 20x and like all of them.. I notice little if any image degradation.. Bear in mind I am not a perfectionist, and use what I have instead of attempting to obtain complete perfection pouring endlessly over matching ocular and objective combinations. I am a hobbyist and all of it works well enough to suit my everyday enjoyment....

The Best,
BillT

[MichaelG.]

One example quoted states that a 15x eyepiece with a x40 objective gives 600x magnification while still providing acceptable resolution. Since I have no experience using either, I wanted to ask members what the experience is like with using higher power eyepieces when compared to using oil immersion objectives?

Yes; the overall magnification will be 600x, but the contribution from the eyepiece is 'empty magnification'.
What constitutes "acceptable resolution" is an observer's choice; but resolution is directly related to the Numerical Aperture of the objective, and therefore highest resolution demands oil-immersion.

As an aside: Personally, I have yet to find any high magnification eyepieces that provide comfortable viewing.

MichaelG.
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Edit: Having just seen BillT's post, I must emphasise that my preference for low power eyepieces is a personal matter, and I envy those that can enjoy using high powers.
.
P.S. This interactive tutorial is worth a look:
https://www.microscopyu.com/tutorials/i ... ion-airyna

[Charles]

One example quoted states that a 15x eyepiece with a x40 objective gives 600x magnification while still providing acceptable resolution. Since I have no experience using either, I wanted to ask members what the experience is like with using higher power eyepieces when compared to using oil immersion objectives?

Yes; the overall magnification will be 600x, but the contribution from the eyepiece is 'empty magnification'.

Actually using a 40X objective with a NA of 0.65 used with a 15X eyepiece will not give you empty magnification. An objective with a 0.65 NA theoretically should give you good resolution up to 650X.

Older scopes commonly used 12.5X eyepieces as well as with the use of a multiplier like the Zeiss Optovar would give you added magnification from 1.25X-1.6X-2X so you didn't need to swap out eyepieces.

[MichaelG.]

Actually using a 40X objective with a NA of 0.65 used with a 15X eyepiece will not give you empty magnification. An objective with a 0.65 NA theoretically should give you good resolution up to 650X.

I bow to your wisdom, Charles.

... I may not have expressed myself sufficiently well.

MichaelG.
.

P.S. This, from Leica, provides a useful summary:
https://www.leica-microsystems.com/scie ... ification/

[apochronaut]

Stick to magnifications that do not exceed 1000X the N.A. with each objective and you will be fine. 20x eyepieces can be used with low magnification objectives....even 25X with most 10x because the N.A. is usually .25 or higher. The practicality of using 20X or higher eyepieces ends around a 30X objective. I have one Bausch & Lomb that is .65 and a CBS, that is .55, so you can see what their possibilities are.

From there on up, unless you are into dry apochromats where the N.A. can achieve the theoretical dry limit( .95), a 15X eyepiece is often a possibility and some of them can be really fine; offering a better apparent view than a 10X with some, because modern ones are designed to achieve close to the real field of a 10X , which makes the apparent field, out around your ears, 25mm or so.

N.A. x 1000 ÷ objective magnification = maximum theoretical eyepiece magnification.

Here is how to understand empty magnification. You walk into a movie theater and Humphrey Bogart is on the screen. You look at his cigar and you can't read the label. So, you walk closer to the screen, so you can read the label. That's empty magnification.

[charlie g]

Thanks for posting your thoughts on an initial outlay for your microscopy,'amoeba'. I sense the type of microscopy you will enjoy..will direct start with a stereo microscope vrs. a compound microscope with a turrent of objectives (four or five objectives on your purchased scopes turrent?).

As you will delve into diatoms sooner or latter, as your forum 'handel' is: amoeba...It seems a compound microscope is your need for wonderful microscopy.
So I suggest you only purchase a compound microscope with a proper substage condenser of: NA 1.25...or greater numerical aperture.

Why, as a start should you purchase an 'oil-immersion 50 X objective'? I suggest you share what type of microscopy you intend to start with...and keep all objectives: 4X, 10X,40X, 60X...non-immersion objectives ( dry objectives)..and also have an oil-100X objective. But you should start with a substage condenser permitting Kohler illumination with a specification of: substage condenser NA 1.25...or greater.

Oil objective 100X permits wonderful observation when you are up to the delicate treatments of the specimen glass slide/specimen/coverslip/oil droplet placement...then proper shut down and ckeaning of that oil-objective.

I think you need a basic compound microscope: proper substage condenser, turrent of dry objectives...and a quality 100X oil-objective...keep eyepieces to: 6X, 10X, 12.5X, 15X..but focus ( my pun?!!) on the basic compound microscope and 10X eyepieces to start. Charlie guevara

[MichaelG.]

I would like, if I may, to expand a little upon what I wrote [rather badly] last night:

Yes; the overall magnification will be 600x, but the contribution from the eyepiece is 'empty magnification'.
What constitutes "acceptable resolution" is an observer's choice; but resolution is directly related to the Numerical Aperture of the objective, and therefore highest resolution demands oil-immersion.

I was justly corrected for using the term 'empty magnification' in that wide context ... so a few more words are needed to express what I was trying to say:

Yes; the overall magnification will be 600x, but [*] the contribution from the eyepiece is simply to make visible the performance of the objective. Resolution is directly related to the Numerical Aperture of the objective, and therefore highest resolution demands oil-immersion.

What constitutes "acceptable resolution" is an observer's choice, but; to resolve the smallest details we need the objective to have the highest possible NA, and the eyepiece to provide an appropriate magnification of the primary image.
Leica, for example, currently suggests using an overall magnification in the range 500 to 1000 times the NA of the objective: Below 500 we are not 'seeing' the full performance of the objective [because the final image contains detail too small to resolve with our eye], and above 1000 we are getting 'empty magnification' where things look bigger, but there is no more detail to be seen.

MichaelG.
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[*] aside from any optical corrections that it might be providing.

[Amoeba]

Thank you for the various responses. As a hobbyist, I have a desire to make do with whatever is available at the lowest cost, but as a photographer (and previously hobbyist astronomer), I appreciate the advantage of good optics and good resolution when it comes to viewing and taking good images for the record.

I had to look up that term 'empty magnification' as I had not hear of it before. In addition to the kinks already posted, I came across this one:

https://www.microscopyu.com/microscopy- ... tion-range

It has a nice little table of useful eyepiece/objective combinations. It seems that x15 might be OK, although x20 might be pushing things too far.

I must admit that the choice of the x54 oil immersion is a bit of a whim because it is available to me (at a decent price) as part of the package if I want it and I have seen some very nice pictures produced with one. A dry x60 might have been my preference though if one had been available. I am deliberating on whether to give the two oil ones a miss and look for a dry x60 on eBay instead, although I suspect finding a Leitz for a reasonable price might be difficult?

The turret has 4 positions I think and the Microcope itself will come with x4, x10, x25 and x40 objectives. I am thinking of buying the dry x16 that is available as well as the x54 and x100 oil immersion for a fuller range of magnifications but I am struggling to decide on oil immersion.

Do I gather from Charlie G that the 1.3 condenser would be a no-brainer anyway?

As regards the type of Microscopy of interest, as a beginner this is a rather difficult question as I don't really know which way my interests may take me. For the present I can only generally state that I am interested in looking at botanical specimens, aquatic life as well as Diatoms. We already do own a Nikon stereoscope which has up to 40x magnification which I use mostly for detailed Electronics work but we have also had fun examining a variety of objects in close up detail. However I am now interested in buying a higher power instrument for more detailed study of microscopic life.

[Charles]

I do a lot of diatom work and rarely use any objective requiring oil immersion, but I don't do a lot of photo work either. I do use my dry 40X and 63X quite a bit. But if you are trying to get the best resolution at the highest magnification for photographs, then you may want the oil immersion. As to the NA of the condenser. It depends on what objectives you are going to use. What condenser does your Leitz have already? Most Leitz condensers are dry 0.90 NA for use with dry objectives. To utilize the objectives over 0.9 NA, like their 100X 1.25 or 1.30 NA objective, you will need at least a 1.25 NA condenser, but better to get an 1.30 NA condenser if available. You could use a 1.25 NA condenser with a 1.30 NA objective, which will give you good resolution, but you would only get the max NA of 1.25 out of the combination. Also, to get the max resolution of an oil objective NA over 0.95, you will need to oil the condenser to the slide as well as oiling the objective to the slide since air has an NA of 1.0. I would suggest, work with what you have and get to know your system. High NA condensers and objectives show up for a low price, and then buy for the best price.

[apochronaut]

The condenser does not limit the resolution of the objective to the N.A. of the condenser. A dry achromat condenser with an N.A. of .90, will not limit the N.A. of an oil immersion objective to .90. The resultant N.A. of the objective will be a hybrid between the condenser N.A. and the objective N.A. For a standard 1.25 N.A. oil objective , that will be about 1.1 or thereabouts.

A number of years ago, it was standard practice to use a 1.25 abbe oil condenser and it still is for a lot of cookie cutter microscopes. The better microscope companies do listen to their customers and beginning in the 70's, it became more common to see the convenience of .90 or .95 dry condensers in increasing abundance but they were almost all achromats, a big step up from an abbe. Abbe condensers are not very suitable for use with plan well colour corrected objectives, especially if you are photographing. A dry achromat will in most cases achieve equal or better overall performance to an oiled abbe, despite it's lower working N.A. and the convenience factor cannot be overstated. If you are going to opt for an oil immersion condenser in order to maximize resolution , it would be false economy to not aim for an aplanatic or aspheric abbe condenser, or preferably a 1.3 or 1.4 achromat. OIling a .90 or .95 achromat condenser will degrade performance.

[Charles]

The condenser does not limit the resolution of the objective to the N.A. of the condenser. A dry achromat condenser with an N.A. of .90, will not limit the N.A. of an oil immersion objective to .90. The resultant N.A. of the objective will be a hybrid between the condenser N.A. and the objective N.A. For a standard 1.25 N.A. oil objective , that will be about 1.1 or thereabouts.

I'll disagree with your 1.1 thereabouts reasoning. The NA is limited to the lowest NA in the combination of condenser and objective. And also since air has an NA of 1 there is no way you are going to get an NA higher than 1 or actually in the case with a 0.90 NA condenser, over 0.90.

[Amoeba]

I believe that the condenser is the standard one that comes with the Microscope, probably the No. 601 L. From the manual this seems to be an aspherical condenser with an N.A of 0.90. The optional one I am being offered is a 1.3 (brightfield but design type unknown) with two top lenses for 0.9NA and 1.3NA respectively.

if I understand correctly then:

a) Air has an NA of 1.0 max, but the x60 objective might be 0.85 so the 0.90 condenser will suffice.

b) The best possible resolution would be achieved with oil immersion and then the 1.3 condenser becomes necessary.

c) I will get less than optimal results from oiling a 0.90 condenser in conjunction with an oil immersion objective.

d) A 15x eyepiece or a x60 objective should increase useful magnification but the resolution will still be limited to the NA of the objective/condenser combination (0.65 for a x40 and 0.85 for a x60).

Since I have no idea whether the dry objectives will be sufficient for photography it seems I might as well try the suck-it-and-see approach and purchase the extra oil immersion accessories, although maybe I can pass on the x54 objective and just stick with the x100? If I don't use the oil immersion kit then it then I can always sell it on. A pair of Far East 15x eyepieces are cheap enough to buy later. Of course, the other factor will be the imaging device..... a subject for another thread!

[apochronaut]

Yes Charles, I know it seems illogical due to the air to slide gap the lightpath travels through but the N.A. of the condenser would only have absolute dominance over the potential N.A. of the objective , if the light traveled through a completely consistent medium : 100% air between the condenser and the objective.

This is never the case. The light refracts,diffracts, reflects and scatters, by passing first through air, then glass, then water or balsam + sample, then glass and finally the immersion medium. This results in the angle of incidence at the objective being somewhat different than the angle leaving the condenser.

The sample type and thickness, has a degree of effect on this, so the final incident angle would be tricky to calculate.

A little test to see what is going on, is to set up an iris equipped oil immersion objective , with N.A. markings on the adjustment ring, and illuminate it through any sample from a dry condenser. The condenser should have a known N.A.( .90 achromat for instance) and the objective should be at least 1.25 N.A., better if higher but with N.A. markings on the adjustment collar. View the sample with a full objective aperture and then with an aperture that matches the condenser.
There will be a resolution difference, resulting from the objective being able to work at a higher N.A. than that of the condenser, with the iris fully open.

[apochronaut]

A pair of Far East 15x eyepieces are cheap enough to buy later.

Don't waste your money on them. First of all, eyepieces are usually as specific to the brand of microscope as the objectives are. With Leitz it is even more so because they changed from 170mm to 160mm and the intermediate image distance changed. I don't have a Leitz microscope but I have a lot of Leitz eyepieces and I went through a complete testing routine with them, to determine if any of them would work well with the objectives I was planning on using for my dry high magnification field microscope. The objectives comprised a 10x .30 CBS(Conrad Beck & Sons) 160mm t.l. 37mm parfocal, a 30x .55 CBS 160mm 37mm parfocal, a 62x .85? E. Leitz Wetzlar( 170mm , 37mm parfocal and an 80x .90 Hacker 160mm 37mm parfocal. I figured I would try Leitz eyepieces in order to comply with the requirements for the 62X. Surprisingly, the Leitz eyepieces were all over the place in their degree of a correction match for that old 62X( pre-w.w. II). In going over the eyepieces in detaIl, I found that I had eyepieces that had been generated out of three different programs; 170mm 37mm parfocal, 170mm 45mm parfocal and 160mm 45mm parfocal. Luckily, one set of very old 10X periplan worked well and also luckily with the other objectives too but what that test showed was that you will need eyepieces quite specific to the task. Generic 15X will almost for sure exhibit lateral ca.

Another aspect of lens design that doesn't get talked about too much, is how carefully the lenses have been made. Ideally, they should be polished to 1/4λ but speeding up production, as is the case with cheap instruments and even some that are considered better, can result in that, being more but not less. Some companies, Wild is known, and probably most of the older major companies would have been careful about this but it would be easy for bulk produced cheap optics to vary by a 1/4λ or maybe more, not to mention cheap machining and a lack of centering.

Look for the correct 15X eyepieces( possibly 16?). They won't be that expensive.

[Amoeba]

... it would be easy for bulk produced cheap optics to vary by a 1/4λ or maybe more, not to mention cheap machining and a lack of centering.

Look for the correct 15X eyepieces( possibly 16?). They won't be that expensive.

Point well made and taken. I would imagine that there should be no problem with eyepieces that are designed for a 170mm tube, limited NA notwithstanding. I did have concerns about those cheap eyepieces not only in terms of quality but also in terms of the focal length as they are 160 rather than 170. Would they even focus? Any such purchase would naturally be a risk as one can never be sure of compatibility nor of sufficient quality. I guess it is a bit of an unknown quantity for all of the reasons you mention and more.

In terms of objectives, the manual says it is possible to use Leitz 160 and 170 objectives as the focal distance only varies by 2mm but they will not be parafocal. It is interesting to note that there exists a 62x 0.85NA although I have no idea whether the existing 170 eyepieces will be 37mm parafocal, but it seems maybe one to look out for on eBay.

[MicroBob]

I think one point has not been mentioned: Ernst Abbe found out that the magnification should not exceed 500-1000 times the n.A.. This is magnification of objective, eyepiece and tube factor. Binocular tubes usually have a tube factor between 1,0 and 1,6, so may be a 10x eyepiece and a 40x objective already offer 640x magnification. Often the tube factor is printed on the binocular tube.

Before buying mixed parts I would recommend to look at the diagramm on Leitz-compatibility I posted a link to. Old Leitz eyepieces picked up the image 18mm down the tube. This is about the maximum value of all manufacturers. So if you have a 170mm microscope with 170mm objectives it would have to be such an eyepiece. Do you have the complete information on the microscope and additional parts available?

When your plan is to somewhen get to the point where you can take really good microscope pictures you might think about planar objectives. For viewing they are not really important, but for photography they are a big plus. If you don't want to spend the money right now, you could start out with normal achromats and upgrade later. Camera lenses are usually more or less planar. Standard microscope lenses not.

[MichaelG.]

... Ernst Abbe found out that the magnification should not exceed 500-1000 times the n.A.. This is magnification of objective, eyepiece and tube factor. Binocular tubes usually have a tube factor between 1,0 and 1,6, so may be a 10x eyepiece and a 40x objective already offer 640x magnification. Often the tube factor is printed on the binocular tube.

Yes, MicroBob ... A point well-made.

MichaelG.

[apochronaut]

. I have no idea whether the existing 170 eyepieces will be 37mm parafocal, but it seems maybe one to look out for on eBay.

Eyepieces do not have a parfocal measurement but are manufactured with specific focal distances, with which to pick up the intermediate image. The parfocal measurement of Leitz objectives were related to the tube length, so older Leitz eyepieces, designed for the 170mm t.l. should work well. Bear in mind though, that Leitz, just like everybody else, was constantly improving their f.n. and planarity as their glass technology and engineering advanced. The eyepieces designed for the microscope you have would have been the state of the art for a 170mm t.l. microscope, with an intermediate image distance of 152mm.
With Leitz, Alpha, was 170mm t.l. with 37mm parfocal and 152mm to the intermediate image : Beta, was 170mm t.l. with 45mm parfocal and 152mm to the intermediate image : Gamma, was 160mm t.l. with 45mm parfocal and 150mm to the intermediate image( Wild pattern) : Delta was infinity t.l. with 45mm parfocal and 200mm to the intermediate image(AO/Reichert pattern) This is where the problem lies with Leitz eyepieces. There is only a 2mm intermediate image distance difference between the 170mm t.l. systems and the 160mm t.l. systems because the older eyepieces designed for the 170mm tubes pick up the image 18mm down the tube( 170-18= 152 but since the eyepieces designed for the 160mm system pick up the intermediate image 10mm down the tube (160-10= 150), that 8mm difference causes unacceptable ca and spherical aberration. Eyepieces designed for 160mm systems, conforming to DIN standard 58887, cannot be used on Leitz 170mm systems.

With respect to your higher mag. eyepieces, I took a look and the correct ones are in fact 16X. They are marked Periplan GF 16X Ernst Leitz GmbH Wetzlar.

[MichaelG.]

It probably doesn't add much to the discussion, but ...
Here is an interesting recent page by Leica on the subject of Immersion Objectives.
https://www.leica-microsystems.com/scie ... esolution/

MichaelG.

[einman]

I am late to the party but I wanted to support a couple of comments made by Apochronaut:

"The resolving power of an optical system is computed by averaging the N.A. value of the objective and the working N.A. of the condenser."- This is per Cargille a manufacturer of immersion oils

If you read the information presented by Leica at the link you will see the following:

"Placing immersion liquid on the lens of the condenser is usually not necessary. If the microscope is correctly set up and aligned to achieve optimal contrast and illumination across the specimen (see the article on Koehler Illumination), then the position and settings of the condenser will be optimised so as to contribute to the overall NA of the microscope system."

This supports the contention that it is not necessary to oil the lens on the condenser. Although you can do so if the condenser is designed as such. Whether the improvement in na is worth the effort is questionable.

I have read pros and cons on both sides concluding that oiling the condenser helps a little, but the effort provides very little gain in image quality. If you absolutely must extract the highest possible NA then you need an aplanatic-achromatic condenser na 1.4 oiled and a planapo oil immersion objective with a matching na of 1.4.


For most of us an aplanatic-achromatic condenser with an na 0.90-1.3 without oil will suffice.

[mrsonchus]

Hi einman, what do you think about the use of my planapo x100 1.32 with 1.25 aplanatic condenser?
I routinely use oil on both slide's surfaces but may give it a quick look with just the slide/objective oiled?

John B.

[einman]

I was thinking about you and your work when I read this discussion. I would try it without oiling the condenser. With so many variables experimentation is sometimes simply the most accurate way of assessing the end result. Theoretically, even if you oil the condenser your max na would be somewhere between 1.25 and 1.32 by some discussions, assuming all other components were optimized, whereas others would indicate the max would be 1.25.

I am only commenting on articles I have read and do not purport to be an expert. I was simply acknowledging Phil's basis for his comments.

There are articles that disagree. For example: http://micro.magnet.fsu.edu/primer/anat ... rsion.html says, "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."

If you read through the technical explanation for obtaining maximum NA in the Leitz brochure entitled, "Image forming illuminating system" they also mention that the condenser is "theoretically" a limiting factor and should meet or exceed the na of the highest powered objective, when explaining optimal NA. I believe it comes down to Theoretical vs Practical. I believe Phil's comment better relates to what we see in practical use.

It could be that oiling the condenser while being theoretically correct is just not practical when assessing to what degree it contributes to the finished na.

For me the reason for seeking a condenser of 1.4 na for my objectives is to insure my resolution is not being limited by my equipment. Keep in mind that the refractive index of the oil can't be overlooked nor its other inherent properties such as wavelength absorbance etc. Each component acts as a variable. For example, you can optimize all aspects of the optics but if your specimen lies too deep within the mounting medium, beyond the objectives optimal working distance, then these discussions are a irrelevant as you will not get good resolution regardless. What is the refractive index of your mounting medium? That is another variable. Again when all is said and done oiling the condenser may not play an insignificant role.
We have discussed cover glass thickness in other threads. With oil immersion objectives it is not as critical but how much oil you put can be..so many variables. I prefer to go the NIKE route and "Just Do IT" then compare the results.

[mrsonchus]

Most definitely agree my friend, with so many variables the most effective way is to try and assess. As a generalisation I will say that my oil objectives perform extremely well, as does my water-immersion 1.0 objective. They're all very conspicuously bright, colourful and have great contrast.

I also have a dedicated 'dry' nosepiece and 'immersion' nosepiece for my Orthoplan - safer that way

[MicroBob]

Without oil immersion the condensor cant't go beyond n.A 1.0. This is quite a difference to the full n.A. of an oil immersion condensor. The effect will probably be the same as when I use a 40x objective and close the condensor diaphragm to an n.A of 70% of the nA of the lens. The contrast and depth of field will be increased and the resolution will be slightly reduced. So it is not a big problem to not use oil but it also can't be said that the oil immersion of the condensor is superfuous today.

[einman]

Without oil immersion the condensor cant't go beyond n.A 1.0. This is quite a difference to the full n.A. of an oil immersion condensor. The effect will probably be the same as when I use a 40x objective and close the condensor diaphragm to an n.A of 70% of the nA of the lens. The contrast and depth of field will be increased and the resolution will be slightly reduced. So it is not a big problem to not use oil but it also can't be said that the oil immersion of the condensor is superfuous today.

Quite correct based on the RI of air. That is not the discussion. If you oil the condenser and the other variables are are not properly tuned the system is compromised. So to determine if theoretical improvement in na by oiling is actually being obtained it is a simple experiment to do it with and without the oil. Much like using a planapo objective without using the collar. You end up with resolution potentially compromised and potentially less than that of a regular 40X planachromat. Even using the collar if you don't know the
coverglass thickness it is a trial and error (experimentation). Or, if the specimen is mounted beyond the DOF of the planapo, once again all is for naught. So we can talk theoretical all day. It is the practical application that matters and that is dependent on everyone's individual equipment etc.

Just do the experiment.