X40,x60,x100 objective différence in use

[Mindwarp]

Hello everyone,

I’d like please like to know what is the best choice for an objective when the n.a. is almost the same >> Should I go for the highest or lowest magnification ? In the same brand and quality level.

Thanks for your answer

Ps : For example, now I have to decide between both neofluar plan infinity : the 40/1.3 oil Ph3 (my actual choice) or the 63/12.35 oil Ph3

[PeteM]

The lower magnification objective (say, 60x vs. 100x but the same NA) may have a bit more working distance - which is a plus for seeing deeper below the cover slip. Worth checking the specs to know for sure.

[Mindwarp]

Thanks for the information PeteM

So I might better favor objectives with the lower magnification and the higher n.a. If I’m right.

So by the way, May I ask what is the real necessity to have 100x for example when a 63x reach the same n.a ?

[PeteM]

I don't think there is a good reason to have both, for most of us.

Someone like a pathologist might have wanted to higher magnification for visual ID, but take a 60x image and blow it up to 100x digitally and (with the same NA) it should be pretty much the same. At least that's my impression.

[Hobbyst46]

So by the way, May I ask what is the real necessity to have 100x for example when a 63x reach the same n.a ?

The resolution is determined by the NA of the objective.

In addition to the working distance, depth of focus etc:

A higher magnification means that a larger portion of the field of view is occupied by the object of interest. This is relevant to the sensor - human eye or camera.
So the 100X1.3 and 63X1.25 yield almost the same resolution (theoretically only, because in practice there are other factors), yet the same object looks bigger under a higher magnification.
On the other hand, the lower magnification catches a larger number of objects in the same view.

Example: when I look at a layer of animal cells, I might prefer a 20X objective since it shows many cells, over a 100X that shows only a few cells - IF resolution is not the absolute target.

Another aspect: in epi-fluorescence, the brightness depends much much more on the NA than in brightfield. A 40X1.3 is a very useful combination of field coverage and NA (and is very expensive!).

[Mindwarp]

Very interesting all these precious informations @PeteM and @Hobbyst
Thanks you a lot, that make me think how to complete my setup!

[deBult]

A good read on the subject: sharpness index:

http://bettermicroscopy.blogspot.com/20 ... ts-of.html

[apochronaut]

One has always to factor in what they are actually doing with the sample. The original question was whether one should go for the highest or lowest magnification in the same brand and quality level , if the N.A. is the same.

If the purpose of the observation is to make a photo, the answer is a little different than if the purpose is visual observation.

A photomicrograph made through a lower magnification objective must be amplified digitally in order to achieve the same overall magnification for viewing as a photomicrograph made through an objective of higher magnification. If they are to be viewed on the same system and monitor, the image taken with the low magnification objective would need to be cropped in order to match the field of the high magnification objective and then increased in size in order to appear identical in terms of field size and magnification . The cropped and amplified image will have lower resolution. Manipulation of that is obviously possible with programs but it would seem that in that event, both the digital and optical resolution of the images are going to be slightly different irregardless.

In the case of visual observation , in theory a 50X 1.25 objective should in theory should have the same potential resolution as a 100X 1.25 objective. However, there are a number of factors that enter into the situation that disproportionately affects the 50X objective more than the 100X. Firstly, the seemingly carved in stone rule that an objective can be used effectively up to a magnification of 1000X it's N.A. , is not always the case with every objective. Sometimes, it is pushing the envelope because there are many factors that might reduce an objective's applicability to that condition. Fluorites and apochromats, tend to be a little more adapted to being challenged in that way as well. I've done a few direct comparisons and I think in each case, the image from a pushed objective was just ever so slightly less resolved. It was noticeable once one started to nudge that 1000X zone, sometimes somewhat before.


We often forget the role that eyepieces play. They magnify too and a 15X or 20X eyepiece magnifies any imperfection in the system above the objective 1.5X to 2X that of a 10X eyepiece. They also have more glass, more curvature in the lenses and provide a darker image. They can also have poorer eye relief and a smaller exit pupil. Some people do really great work with lower magnification higher N.A. objectives that rivals imaging with an objective of a higher magnification; 75RR's images on this forum with his Zeiss 63X 1.4 oil plan neofluor( hope that is correct) comes to mind but that is more the exception than the rule, especially if one is trying to achieve higher magnifications than are the norm for such objectives.
Generally, in order for an objective to be so overtly flexible, it also takes a little extra out of the preparation and one's time too even when it is a great lens. In general working between 1/3 and 2/3 of the N.A. potential with most objectives is the sweet spot and if you need to go higher, choosing another objective is usually a good idea. For this reason, I usually don't recommend eyepieces above 15X or 16X but they need to be made to the same standards at least as the 10s one uses, with an appropriate apparent field increase that gives close to the real field presented through the 10X. Some are close.

[PeteM]

A good read on the subject: sharpness index:

http://bettermicroscopy.blogspot.com/20 ... ts-of.html

Thanks for that link. About as good an explanation as any of why you might not need a 100x 1.4na objective if you already have a 60x 1.4na objective; especially in an age of digital imaging. Though, the author's computation of "sharpness index" does seem a bit arbitrary to me. Of greatest interest were those subjective ratings -- which are similar to my own experience.

Somewhat related, I think most of us have had the experience of finding a 20x objective just right for so many subjects. Cells large enough, good depth of field, good contrast, adequate working distance. Anyone know who decided 10x - 40x 100x should be the basic spec for school-level scopes, leaving that big gap between 10x and 40x??

[apochronaut]

The blog is a continuation of one Dave Jackson has had for a while. It went into liimbo for a year or so. The current incarnation is apparently by a new blogger, whose initials are also DJ.....
This index just says that you can expect a higher degree of performance from an objective with a high N.A. That's all. Because peak N.A. has technical limitations and is expensive and difficult to engineer at a high value into objectives with low magnification, those of moderately high magnification and high N.A. are granted the dual possibility of being easier to engineer a composite of a close to peak level of N.A. , plus relatively high magnification. The last bit would need be close enough that a reasonable eyepiece magnification could conceivably max out the N.A.

This index is about as useful as choosing an ice cream cone on the basis of how many scoops you can pile on cones of various diameters. Three inch diameter scoops can be piled on three inch cones quite high, before they topple. Three inch scoops will topple on a 4 inch cone at the same height but several scoops have sunk into the cone, so you get more ice cream. Three inch scoops on a two inch cone tople at the same height too but the first couple of scoops soften and spill over the edge, so you get less ice cream. It therefore looks like the mid size system is more efficient in achieving a higher amount of ice cream.

Ultimately, iregardless of index, you get the same resolution based on the N.A., you just didn't expect it to be so with certain lower magnifications.
However, the Rayleigh criterion predicts objective performance as well as condenser performance, so you get a total optical package scorecard, giving you an accurate measure of resolution based on more of the players in the system. I'm not sure it is time to give ip on the tried and true Rayleigh Criterion.

No point in reinventing the wheel, if your new design is square!

[Hobbyst46]

At first, the "sharpness index" seemed to me a "figure of merit", a feature that AFAIK is not of much concern in microscopy. But some math reveals more. What the blogger calls the "Sharpness index" can be related to the total useful magnification as follows. Call the sharpness index S. It is defined by the blogger as 1000 times the NA of objective and divided by the magnification of the objective:

S= 1000 x NA(obj)/M(obj)

But, the total useful magnification, call it UM(t), is the magnification of the objective { M(obj) } times that of the eyepiece { M(ep) }:
UM(t) = M(obj) x M(ep) = 1000 x NA(obj)
{well, sometimes suggested as 500xNA(obj), so just for the sake of the argument}
So,
S = UM(t) / M(obj) = M(ep)
So, the sharpness index is just a suggested theoretical "optimal eyepiece magnification"; reinventing the wheel, maybe or another way to avoid empty magnification, rather than a definitive figure of merit. I did not notice that the Blogger mentioned the role of the eyepiece though.

Anyway, citing from the MicroscopyU site:

"Exceeding the limit of useful magnification causes the image to suffer from the phenomenon of empty magnification (illustrated in Figure 1(b)), where increasing magnification through the eyepiece or intermediate tube lens only causes the image to become more magnified with no corresponding increase in detail resolution. In contrast, the image shown in Figure 1(a) was captured using the correct objective and eyepiece combination to effectively utilize the numerical aperture to achieve optimum resolution."

[deBult]

The role of the eyepiece was in the next blog article:

http://bettermicroscopy.blogspot.com/2019/11/

[Hobbyst46]

The role of the eyepiece was in the next blog article:

http://bettermicroscopy.blogspot.com/2019/11/

Thanks for point that out. So I only read the first part of the sharpness index blog presentation and did not go further...

[cd13]

Following this post

[apochronaut]

The role of the eyepiece was in the next blog article:

http://bettermicroscopy.blogspot.com/2019/11/

That's why most of the older apochromat systems used a 15X eyepiece as a default, rather than a 10X. It is also why they used 2mm objectives rather than 1.8mm objectives. A 2mm objective gives approx. 90X magnification and a 1.8mm objective gives approx. 100X magnification.

In the apochromat systems where the N.A.s are almost always the next logical increment higher than in achromat systems ; .30 and+ , .60 and+ , .85 and+ and 1.30 and+ for the 4 common objectives, a higher than 10X magnification eyepiece offers a better use of the native resolution of the objective. In the case of the high magnification oil immersion objective, using a 15X eyepiece could push the magnification beyond the practical limits of resolution but a 90X by 15X system gives 1350X, still acceptably within bounds for an apochromat at 1.30 but usually a 1.40 was also available and might have been preferred. Apochromat systems, since their great development in the era of the Spanish Flu epidemic , sought to offer maximum flexibility while matching objective N.A.s to eyepiece magnification. A typical system would have had 16mm, 8mm, 4mm, 3mm, 2mm and often 1.5mm objectives. The 3mm would be available in a sub 1.0 N.A. dry version and a greater than 1.0 N.A. oil version, often 1.4. Eyepieces would be something like 5X, 10X, 12.5X, 15X, 20X and 30X plus a 2.5X photo eyepiece, so matching the eyepiece in order to maximize resolution was always a possibility.

[Mindwarp]

Thanks a lot to you all who took that much time to answer my question and raise that much fascinating informations and tips.