Please help - 100x objective not working

pwnell · Post by **pwnell** » Wed Oct 19, 2011 8:25 pm

Well those two posts certainly answers a lot. For one, I am thinking now - am I not resolving the maximum amount of detail already at 40x when taking a photo on a high resolution sensor? Blowing it up full resolution - does it then not contain all the optical detail that a 100x objective would capture on the same sensor?

pwnell · Post by **pwnell** » Wed Oct 19, 2011 8:42 pm

Continuing my tests... Here is a copepod at 40x magnification - no cropping.

And with 100x oil, also not cropped, just resized to 800x600 (though I did process it - sharpening etc):

Is this "acceptable"? Look specifically at the hairs and fine details that I am trying to resolve, and the included scale.

Charles Krebs · Post by **Charles Krebs** » Wed Oct 19, 2011 9:21 pm

Waldo,

Always a bit difficult to judge from a single 800 pixel image... but yes (and unlike your very first attempt) this does look to be in the ballpark for a 100X objective.

For one, I am thinking now - am I not resolving the maximum amount of detail already at 40x when taking a photo on a high resolution sensor? Blowing it up full resolution - does it then not contain all the optical detail that a 100x objective would capture on the same sensor?

You can pretty accurately make such an assessment based on the NA of the objective. "Optical detail", or resolution is directly related to the numerical aperture (NA). For example, when "enlarged", a 60/1.40 image will provide a picture that displays the same amount of resolution ("detail") as a 100/1.40. And since by the time you get to a 60X objective nearly all DSLR sensors are significantly "overkill" (even with direct projection) some people prefer to use such a 60X over a 100X. But if your 40X has a NA of 0.65 then the 100/1.25 will provide a picture that can reveal significantly smaller details on the subject (but again, even though you may be able to discern smaller subject details the overall appearance of the full image may not look as "crisp"as the non-enlarged 40X shot). On the other hand, if you have a 40/0.95 the choice gets more difficult, especially if you are not oiling a high NA condenser. In this case you may be hard pressed to see much difference in detail between a 40/0.95 and a 100/1.25 (but there are sometimes other non-microscopy image characteristics to keep in mind when digitally "enlarging"... such as digital noise).

If you have not already done so, you might find it interesting to play around with this spreadsheet. (For "direct projection" the Relay Magnification should be entered as "1"):
http://www.krebsmicro.com/scale_bars.xls

pwnell · Post by **pwnell** » Wed Oct 19, 2011 10:21 pm

I have a 40/0.65... So would a 60/0.7 Air be better for photography than my 100/1.25 oil? I presume from your explanation that that 60x/0.7 would yield more usable resolution than a 100x/1.25. Asking since I see a Nikon 60x/0.7 Phase Contrast on their web site that looks useful.

EDIT: Or do I have the NA the wrong way round?

ChrisR · Post by **ChrisR** » Wed Oct 19, 2011 11:04 pm

So would a 60/0.7 Air be better for photography than my 100/1.25 oil?

Other way round. Higher NA should lead to more resolution.

Charles - if "everything" is oiled between an oil objective and subject, coverslip presence/thickness is not relevant, yes?
But with an air objective at 0.7 you'd expect its thickness to be fairly critical?

Charles Krebs · Post by **Charles Krebs** » Wed Oct 19, 2011 11:06 pm

The higher the NA (numerically... in effect it runs the opposite direction of a camera lens f/number) the smaller the subject detail that can be resolved. So an objective with a NA of 1.25 will be able to resolve, on the subject, much finer detail than a 0.70 objective. A 60/0.70 would not be able to resolve details much smaller than your 40/0.65.

pwnell · Post by **pwnell** » Thu Oct 20, 2011 8:59 am

Thanks Charles, and everyone else for the great feedback.

Charles Krebs · Post by **Charles Krebs** » Thu Oct 20, 2011 9:45 am

ChrisR,

Charles - if "everything" is oiled between an oil objective and subject, coverslip presence/thickness is not relevant, yes?
But with an air objective at 0.7 you'd expect its thickness to be fairly critical?

Yes, that is essentially true (although "not relevant" is perhaps overstating it.

) And this is the reason why "high-dry" (high NA, non-immersion) objectives very often have correction collars built into them to accommodate for varying coverslip thickness. While some people don't care to mess with oil, sometimes it almost seems easier, since there is no need to fiddle with the correction collar. A consideration is that once a slide has been "oiled" you really can't go back and use a lower power, dry objective effectively until the slide has been cleaned. (And with a temporary wet mount you are often stuck with using oil immersion objectives. These are typically 40X and up).

Here is a pertinent section from the Nikon site:
http://www.microscopyu.com/articles/for ... ction.html

When using oil immersion objectives, it may appear that coverslip thickness is of only limited concern, because its refractive index approximately matches that of the immersion oil. That is true when the specimen is mounted in Canada balsam or other mounting media with refractive indexes similar to that of the coverslip. However, it is no longer true when the specimen is mounted, for example, in physiological saline or other aqueous media whose refractive index is significantly different from that of the coverslip. In these conditions, even focusing through a thin layer of water only 10 microns thick can lead to significant aberrations and a point spread function (PSF) that is no longer symmetrical above and below the focal plane. This is because oil immersion objectives are designed assuming that the specimen slide is homogeneously immersed. Unless the specimen region is adjacent to the coverslip (even in an aqueous environment), optical assumptions employed to calculate the aberration corrections are no longer valid.

pwnell · Post by **pwnell** » Thu Oct 20, 2011 9:54 am

So based on that, would it mean a 60x/0.7 dry objective would be more versatile? Seeing that there is no need to clean the cover plate, and mounting a specimen has much fewer requirements? It seems to me like I can get good results if my mounting technique is perfect, but that is not always possible.

I was thinking of this objective:
http://objectives.nikoninstruments.com/ ... hp?c[]=119

Charles Krebs · Post by **Charles Krebs** » Thu Oct 20, 2011 10:51 am

Waldo,

That is likely an excellent objective. It has characteristics that indicate it is designed for slightly specific usage... "tissue culture". It has long working distances for a 60X. As a result the numerical aperture (0.70) is pretty modest for a high end 60X, which is often more around 0.85 and up. The correction collar is made to provide correction for the fairly thick containers used in tissue culture (0.1 to 1.3mm). A more typical correction collar is designed to accommodate for coverslip variations and usually cover the range 0.11–0.23mm.

These characteristics (long working distance, wide range of thickness correction) may seem desirable, and there is no question that they are nice, and are actually essential for some uses. (You do pay more for them, and that puts you in the price range of what I would consider more desirable 60X objectives for most uses). As far as resolution is concerned I doubt you would see any increase over your 40/065. (However, a Plan Fluor is more highly color corrected than a Plan Achromat). But unless you anticipate really needing the working distance and correction for thick containers (much more often needed on inverted microscopes rather than upright) I would really be looking for a NA higher than 0.70 on a 60X. If I wanted a "dry" one with correction collar I would look at something like the CFI Plan Fluor 60/0.85 or CFI Plan Apo Lambda 60/0.95.

Bob S · Post by **Bob S** » Sat Oct 22, 2011 1:40 pm

If you have a condenser with NA 0.9 and no oil, then your objective lens cannot resolve better than NA 0.9. If you want to get the full resolution of your lens, you need the correct condenser for it.

Pau · Post by **Pau** » Sun Oct 23, 2011 1:28 am

Bob S wrote:If you have a condenser with NA 0.9 and no oil, then your objective lens cannot resolve better than NA 0.9. If you want to get the full resolution of your lens, you need the correct condenser for it.

Not exactly, the system NA is stated in many references as (NAobj+NAcond)/2 , so in Waldo's scope the max. NA will be 1.075.

But in brightfield illumination you usually need to close a bit the condenser aperture to get enough contrast, so the real NA will be lower.
I'm not sure if a 1.4 oil condenser would increase significatively the resolution in brightfield in normal conditions if you need to close its aperture.

(While typically low cost micoscopes are equipped with Abbe 1.25 NA condensers many high quality ones have swing out 0.9 NA ones)

Charles Krebs · Post by **Charles Krebs** » Sun Oct 23, 2011 2:51 pm

Not exactly, the system NA is stated in many references as (NAobj+NAcond)/2

Many of the detailed, authoritative references I've seen give this, and I have put it on my spreadsheet page as well. But I have never found a really good discussion on this relationship. (Seems that by using reductio ad absurdum you can come up with some values that just seem impossible

)

Here's an interesting page to read:
http://tinyurl.com/6fwug93

Also read here: (search for "condenser", choose page 595 in search results)
http://tinyurl.com/6khv6t2

rjlittlefield · Post by **rjlittlefield** » Sun Oct 23, 2011 2:56 pm

This is probably a good place to say again what I inappropriately put in another thread:

One way to think about it is that two kinds of light end up hitting the objective: an undiffracted component direct from the condenser, and a diffracted component that has interacted with the specimen. The undiffracted component strikes only the central part of the objective, using whatever NA the condenser has, while the diffracted component expands as a sphere around the subject and thus uses the whole NA of the objective.

Because the final image is formed as usual by interference between the two components, the effective NA is somewhere between the condenser and the objective. Exactly where depends on how heavily diffracting the subject is, so there's no one "correct" formula.

--Rik

Pau · Post by **Pau** » Sun Oct 23, 2011 3:28 pm

Charles, thanks for the comments and references, the first one is very interesting, the second one (595) seems unavailabe.

When you say "with some values that just seem impossible", are you referring to low NA objectives with very high NA condensers?

Rik, I think you did a good condensed explanation, but yes, here it seems fully in its place