Effect of Cover Glass on image quality

[NikonUser]

EDIT: title changed from '2 Buy or not 2 Buy'

A few times each week I salivate over the Nikon and Olympus microscope pages on ebay.
But do I really need a decent microscope? I essentially have no need to photograph things I cannot see.
And, photography through a microscope seems to be a real hassle.

Rik has shown what can be done with a simple setup
HERE

My question to myself is 'could I get a similar result with a camera/bellows/objective'? (stuff I already have).

Taking a leaf out of Rik's book, the answer seems to be yes; but I still want a 'scope!

Nikon 60x ELWD CF M Plan Achromat; about 210mm extension; 28 frames at 2 microns; ZS PMax.
small JPG = 2144 pixel wide frame
Top: full frame; botton: actual pixels


NU2010-8&9

[rjlittlefield]

Very nice! Your specimen was holding up a lot better than mine. My chloroplasts were starting to come apart.

My question to myself is 'could I get a similar result with a camera/bellows/objective'? (stuff I already have).

There was a microscope focus block in there too, I think?

My little investigation was prompted by a question of what to do, starting from scratch and with little or no custom mechanics, just stuff that could be easily bought and plugged together.

I'm not surprised to see such a nice result as yours coming from a superb lens under control of a microscope focus block.

But I am curious about your comment that you still "want a 'scope!"

Can you spell out the reasons why? I can think of several for myself, but I'd be interested to hear yours.

--Rik

[Charles Krebs]

NU,

I essentially have no need to photograph things I cannot see.

This is a big part of my answer

If you are dealing with stationary subjects and you know the section you want to photograph... and... you find that you very rarely desire to exceed the 60X on sensor you have shown here then I would say no. But...

Where a "full-fledged" microscope is wonderful is when you are exploring and examining subjects and have no pre-conceived "target" area. It's also great with live subjects. Even though I have my camera hooked up to a HD screen, and the screen image looks great, I find I still greatly prefer to observe and study subjects through the optical eyepieces. So often you will find details and subjects that were not obvious to the eye or through a stereo scope.

As far as image quality goes, you will actually do as good or better with direct projection (as you are now doing) up to roughly the 60X magnification you are using. However, if your subjects are in fluids then a cover slip is desirable (as the liquid surface can act as a distorting lens). And with a 60/0.7 M Plan (designed for no cover-slip) you are getting into the NA range where, if you shoot through a cover, image quality will start to decline. (I've got the "numbers" for this at home but I'm traveling this week).

And, photography through a microscope seems to be a real hassle.

The actual photography is probably simpler in many ways. The hassle however, is in initially getting everything set up right. The connection (mechanically, and more important, optically) between the microscope and camera can be tricky, or expensive, or both. If you are not using electronic flash (another "hassle" to initially set up) then your camera body selection is important, as a microscope mounted camera that creates any significant vibration will require special techniques.

[NikonUser]

But I am curious about your comment that you still "want a 'scope!"

Can you spell out the reasons why? I can think of several for myself, but I'd be interested to hear yours.

--Rik

Nothing rational, I just like precision scientific instruments with dials and lenses and stuff; if they can be used to take a photograph, even better.
Leaving out the focus block in the setup was a critical error, thanks for pointing that out. It was actually a vertical setup with my butchered Olympus BHM stand.

Charles: the leaf was on a slide in water under a coverslip; perhaps that helps explain the less than crisp image.

[Charles Krebs]

NU,

the leaf was on a slide in water under a coverslip; perhaps that helps explain the less than crisp image.

Two things in play here. The coverslip is one. Check out the small section I clipped out of a Nikon objective brochure (Specified Coverglass Thickness) and is found in this post:
http://www.photomacrography.net/forum/v ... c&start=15
While this chart is for biological objectives designed for a 0.17mm cover and then used with the wrong thickness coverslip, you can clearly see the effect. Quite severe at NA 0.70!

The second is something that really starts showing itself at the higher magnifications. Diffraction effects become very noticable. On sensor, the 60/0.7 will only have about half the resolution of the 10/0.25 and about 1/3 the resolution of the 4/0.13. So with light microscopy you just have to resign yourself to the fact that the highest mags will simply not look as crisp as something done at lower magnification, even with the finest optics.

[ChrisR]

Somewhere I saw it stated, or implied, that incorrect coverslip thickness causes spherical ab which can be corrected by changing the tube length. It was a while ago and I've been hoping to come across it again. It may not be true of course, though there's probably something in it.

I think it was in an image of text from a book.

[Charles Krebs]

Chris,

Correct. Some old microscopes had tubes that could be varied in length to correct for spherical aberration. Shortened if the SA was caused by too thick a coverslip and lengthened if too thin.

I've never seen such a scope. All the ones I've ever used are finite, fixed tube lengths, or more recent "infinity" scopes. The ("high/dry") objectives that are very sensitive to cover-slip thickness have adjustment collars that are turned (while observing) to correct SA.

[rjlittlefield]

Google search on tube length spherical aberration will find many references about this effect.

One microscopy-uk article talks about cover slip thickness versus tube length. It says that having too thick cover glass and/or mountant can be compensated by reducing the tube length, but that with typical microscopes this approach is pretty limited due to the mechanical design. Of course with bellows there would be more flexibility. The corollary is that having too thin cover glass could be compensated with a longer tube. Presumably there is some formula to say how much additional tube is required to compensate for any particular excess or deficiency of cover glass, but offhand I don't know where to find that. The article also discusses how to tell when the spherical aberration has been eliminated, so presumably this could be investigated experimentally. Judging from the images in the article, it should be pretty easy to evaluate using magnified Live View.

Another interesting reference I ran into from the Google search is HERE, titled "Increased resolving power in the presence of spherical aberration", a 1927 article by R.Kingslake et.al., in which it is discussed that when dealing with point sources (stellar astronomy), one can actually get enhanced resolution by having a specific and fairly large amount of spherical aberration. Quoting from the summary:

This result is very interesting, as it shows that a lens with a decidedly undesirable amount of spherical aberration can actually give better resolution on close double stars than a perfectly corrected objective. It should, however, be carefully noted that such a lens could not give a clear image of an extended object. The resolution of close double stars must occur within the broad bright diffraction ring which surrounds the small central dot of light, and, owing to the large amount of diffused light, an extended object such as a planet or the Moon would appear so fogged that clear definition would be impossible.

This is new to me. Fascinating!

--Rik

[Charles Krebs]

Chris....
After I sent the previous message I googled a bit. Here's a good di article:
http://www.microscopy-uk.org.uk/mag/ind ... jtube.html

Whoops... crossed with Rik...

[rjlittlefield]

Hah! Found the same article!

--Rik

[ChrisR]

No it wasn't either of those - I Googled too!

I'm bothered by the Nikon graph's suggestion that a significant percentage of image quality would be lost at say NA 0.75, versus the concluding remark from the article you both cited:

In the final analysis, my personal thoughts on this matter suggest that of all the choices of x40 dry objective that are available, the most satisfying in all respects is the standard x40 fluorite whose aperture at around 0.75 na, excellent colour correction and relative insensitivity to coverslip and mountant variations scores highly in imaging quality and convenience of use. Lacking a correction collar has rarely ever been an issue in its use.

I don't know what Nikon mean when they suggest losing, say, 50% of the image quality.

I must look harder for the article or whatever it was.

[Charles Krebs]

Chris,

"relative insensitivity" is the key.

I can assure you from experience that a 0.70 objective designed for a 0.17mm cover-slip will look very bad if no cover-slip is used. But most microscopists would never be that far "off". Ideally, in a box of #1.5 coverslips it would be nice if each were 0.17mm thick... but they vary between about 0.16 to 0.19mm. This is not too deadly with a NA of about 0.70 but quickly gets critical as NA increases, and can be a real issue with a 0.95 NA objective (if the correction collar is set wrong). Correction collars are great, but frankly they are a bit of a pain since it's something that should be set carefully for each situation, which takes time and concentration. So I think what the author of that article meant was that, with a typical box of #1.5 cover-slips he need not be too concerned with a 0.70 or 0.75 NA objective. But he would surely never attempt to use such an objective without a cover-slip.

This doesn't get into the fact that there are many times where there is some separation between the subject and the cover, and of course there are mounting media of varying RI's. So it's not that cut and dry. But compared to a dry 40/0.95 objective, it is not unresonable to call a 40/0.75 "relatively insensitive" to cover-glass thickness. (And even more so a 40/0.65... as long as an attempt has been made to use an appropriate cover-slip).

[rjlittlefield]

I don't know what Nikon mean when they suggest losing, say, 50% of the image quality.

I've wondered about that too, ever since Charlie posted that snippet.

The best I can figure is that the brochure came from a two-step process. First, somebody who knew what they were doing picked some fine level of detail near the resolution limit of each lens under ideal conditions, and computed answers to the question "What contrast will this detail have, with the indicated cover slip error?". Second, somebody else who wasn't quite as sharp as the first person stripped out what the numbers actually meant, and added the words about "no image formation occurs without cover glass". What remains in the brochure is only a fuzzy picture of what actually happens.

You might also be interested in this graph of tolerance to tube length errors, as a function of NA:

http://www.science-info.net/docs/etc/Tube-Length-na.gif

I don't know the assumptions or the math behind this graph. As shown in the graph, tolerance is inversely proportional to NA to the fourth power for dry objectives and NA to the sixth power for immersion objectives. These are very steep. Even with the dry objectives that have the most interest to us, the tolerance is shown as 30 mm of tube length for NA 0.40, but only 4 mm at NA 0.70 .

--Rik

[Charles Krebs]

Some cover glass reading for a Saturday morning.

http://www.olympusmicro.com/primer/anat ... ction.html

http://micro.magnet.fsu.edu/primer/java ... index.html

http://www.zeiss.de/4125681F004E2140/Em ... ection.pdf

http://www.mediacy.com/pdfs/Spherical_Aberration.pdf

[rjlittlefield]

Re-reading this thread, I'm now surprised by how good NU's image looks. If I understand correctly, it was shot through a cover glass but using a 60X NA 0.70 objective intended for use with no cover glass. This amounts to a 0.17 mm deviation from the design conditions.

I don't have one of those Nikon 60X ELWD's.

I do, however, have a 40X NA 0.65 achromat designed for 0.17 mm cover glass. Here is a moth scale shot with and without cover glass, shown as animated GIF. I didn't think labels were needed.



These were shot with my current afocal setup, which makes about 80X on sensor, and these are actual pixels from an image resized to be 1600 across the frame. The two images were carefully focused on the center scale, using 5X Live View. The weight of the cover glass deformed the wing slightly, so other elements did shift focus between the two shots.

--Rik