Photography through microscope: lack of sharpness

Starting out in microscopy? Post images and ask questions relating to the microscope and get answers from our more advanced users on the subject.

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nielsgeode
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Photography through microscope: lack of sharpness

Post by nielsgeode »

I managed to get my hands on a second hand microscope for a bargain. If you look through it, the view is extremely sharp and really good with lots of visible detail.

When I connect my camera with an adapter and photograph using a photo eyepiece, the images are not nearly as sharp as what I see when I look directly through the microscope. I focus using magnified live view on my PC.

The photos through the microscope were made using a Zeiss Plan 6.3x / NA 0.16 objective and an Olympus FK 3.3x photo eyepiece.

Here is an image of the microscope.
Image
Image

Here is a sample image with a 100% crop:
Image
Image


I am quite new to this kind of 'stuff' so I have not really an idea what is happening here. Help is highly appreciated.

Pau
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Post by Pau »

A nice old Zeiss GFL :D

Very likely the Oly FK is not well matched with the Zeiss for two reasons:

- The amount of compensation is different: I can see lateral chromatic aberration, most visible at the lower left corner (but this will not affect much the central area sharpness). This could be more or less corrected in post processing, best shooting RAW, that also will allow you to fine tune the color temperature

- The 3.3X magnification is too high, in particular if you use APSC or smaller sensor. For FF the recommended magnification is 2.5X and for APSC 1.6X. This will lead to magnify too much the central area, so the sharpness will be lower, much more noticeable if you use high magnification objectives.

The best way to couple the camera with those microscopes is usually the afocal method using Zeiss KPL eyepieces:
http://www.photomacrography.net/forum/v ... 9265#99265


There may also be some vibration blur.

Some questions:
- What camera do you use?
- Is the camera image parfocal with the viewing eyepieces?
- Is the camera frame comparable to the field of view through the eyepieces or much smaller?

Some other points:
The objectives I can see seem three Plan Achromats, one Neofluar or Planapo and an unknown black barrel one, am I right?

At the image I also can see some longitudinal chromatic aberration, this is quite frequent with microscope objectives but the best planapos and difficult to avoid or correct. The best way to deal with is focus stacking

In any case the full image doesn't seem too bad, 100% crop may be excessive to judge the sharpness. I usually magnify at 50% in most microscope images.
Pau

nielsgeode
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Post by nielsgeode »

Thanx for your reply Pau! I have a desire for "ultimate sharpness". For example, when I shoot portraits I prefer my Carl Zeiss Apo Sonnar 135 f/2.

I paid a 100 euros for this microscope, which I think is quite cheap.

A dumb question: is there other optics in there, other than the objectives and the eye pieces? Can you buy other (brand) objectives which are better and use them on this microscope? For example, I have a 40x NA 0.65. How much better can you get? Are there Plan Apo's which are extremely good with much higher NA (e.g. 0.9?)

The 2.5x olympus and the 1.6x olympus are already quite expensive (especially 1.6x) and difficult to get. I will check out the link in your post.

Pau
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Post by Pau »

Yours is a DIN standard microscope, so you can use any DIN components. Unfortunately, despite the dimensions and parfocal distance are standardised the optical correction is not, so each brand (and often series) of objectives must be paired the the matched eyepieces for best results. This mans that you can change the optical componentes from Zeiss to Olympus, Leitz or Nikon (and much others) but better change both objectives, eyepieces and photoeyepieces.

Plan apos have usualy wider NA than achromats, sometimes much higher in modern infinites. 40X 0.95 are made but they are very tricky to use. Immersion objectives have higher NA but do need immersion...

There a are plan apos from all four big brands, the Zeiss will work well with your viewing KPL eyepieces and are excellent but very often suffer delamination.

Olympus SPlan Apos are excellent (the ones that Ch Krebs use!)

Leitz also are very good but harder to find in some magnifications.

Nikon CF have the advantage that they don't need complementary corrections at the eyepiece and are very nice for sure. 2.5X CF projective photoeyepieces are easy to find.

In any case a single plan apo will cost you at least 2 or 3X the price of the microscope, so maybe you could do better looking for a more modern system.

Infinite corrected modern microscopes from the big 4 makers are nicer: better optics and wider field of view and BTW much more expensive, but if you want the ultimate quality....

If you are just starting in microscopy my advice is to use your GFL as learning tool and later define the best way to upgrade. With your macro skills you'll make a fast progression :D
You can take fine images with the GFL an your Plan achromats, but of course not the best possible resolution and sharpness.

Like in other kinds of photography, and even more, in Microscopy illumination is often more important than optical quality. Speciallized illumination techniques are a most important aspect to contemplate. Some ones will be cheap like oblique an darkfield at low magnification while others like DIC are very expensive and need specialized components.
Pau

Charles Krebs
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Post by Charles Krebs »

Pau has (once again) offered excellent advice.
I have a desire for "ultimate sharpness". For example, when I shoot portraits I prefer my Carl Zeiss Apo Sonnar 135 f/2.
If this is the case, then you want to use Plan Apo objectives. (Modern Plan Fluorites are excellent as well, and perhaps just as "sharp" (related to NA), but the Apos will show somewhat better overall color correction.

There is one option that has not been mentioned.... direct projection. I do not know what camera you are using has, but if you are able to place the camera body on the trinocular tube so that the image into the camera is in sharp focus at the same time as the viewing eyepieces then you can place the objective's image directly onto the camera sensor with no other intermediate optics. (This will require some DIY work, and it may not be a possibility with your trinocular head and camera body, I just don't know). There would be too much vignetting on a full frame camera (24x36mm sensor) and still likely quite a bit on an APS sized sensor, but the quality could be superb. The other big consideration with this method is that you would need to use objectives that do not require additional color correction via eyepieces and photoeyepieces. With the stand you show above that would mean Nikon CF finite objectives. They can still be found at prices that are certainly not "bargains" but are far less than the newer infinity type objectives (which need a tube lens).

nielsgeode
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Post by nielsgeode »

Direct projection sounds very interesting to me. I have removed the head of my microscope and somehow placed the photo tube with my DSLR above what is normally the place where the head connects. It should definitely be fixed in some way, but this was just a first attempt to see what I can get. FOV looks pretty goed. Later, I will post some images of the setup and the results

Meanwhile, I found, what I think is a nice plan apo on a secondhand Dutch auction site:

Zeiss Plan apochromate 25x NA 0.65
Image

How good would this objective perform and how much would it be worth in good condition?

Pau
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Post by Pau »

- The Zeiss Planapo 25/0.65 is excellent. I have one with a bit of delamination at the rear element and the font element showing one scratch and even in this suboptimal condition it outperforms the otherwise very good Leitz NPL Fluotar 25/0.55 (plan fluorite) in perfect condition I also own.
BTW at second hand market there is not a fixed value, you could expect to get it from $150 to $300 if in perfect condition (I bought mine for about $150 but did get a significative partial refund). If you buy it, first carefully check it for delamination, a good method is under a steromicroscope illuminated with very oblique incident light (for example wit a Ikea LED lamp)

- Direct projection only would work well, as Charles points, with Nikon CF objectives. With the Zeiss the image center will be nice but outside it will be horrible: they need a big amount of correction done at the KPL eyepieces. With your trinocular head you'll need to remove the phototube to place the sensor at the right distance (10 mm under the phototube end) and even then maybe you could have some vignette.
To mount the camera without the micorscope head is easy but you loss the eyepieces vision, a most desirable thing to have, almost indispensable if you work at high magnification and to fine tune the illumination.
Rafcamera makes an adapter with a Zeiss dovetail and a male M42X1 thread that will facilitate mounting the camera with M42 tubes or bellows. I did help him to desing it and works nicely.
http://rafcamera.com/en/adapters/m42x1/ ... m-to-m42x1
Pau

nielsgeode
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Post by nielsgeode »

Thanx a lot for your reply Pau!

So this Zeiss 25x 0.65 is horrible in the corners? This would be a major disadvantage for me unless it is fully fixable with the proper eyepiece.

I think the center sharpness will always be reduced by an eyepiece, or am I wrong?

Is there objectives out there that are overal sharper without vignetting on APS-C and which are superb without eyepiece, but also excellent with the proper eyepiece (in case I wish extra magnification)?

Pau
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Post by Pau »

nielsgeode wrote:Thanx a lot for your reply Pau!

So this Zeiss 25x 0.65 is horrible in the corners? This would be a major disadvantage for me unless it is fully fixable with the proper eyepiece.

I think the center sharpness will always be reduced by an eyepiece, or am I wrong?

Is there objectives out there that are overal sharper without vignetting on APS-C and which are superb without eyepiece, but also excellent with the proper eyepiece (in case I wish extra magnification)?
- No and yes: the Z Planapo, like any other objective not full corrected just needs an adequate compensating eyepiece to complete the optical correction. With the matched KPL eyepiece it's really excellent up to the corners. I will work badly without the eyepiece.

- Well, the photoeyepiece adds some magnification, so the image will be more cropped. Logically the objective image could more easily be outresolved by the sensor so it can look less sharp, but you don't loss actual resolution at the subject side, the first goal in microscopy. In fact if the sensor doesn't outresolve the objective you're losing some resolution even if the image looks sharper.

- Yes, the Nikon CF series as previoulsy commented. Many (most?) of them can cover APSC without much corner degradation. This is a subject very often discussed at the macro and equipment forums about the use of microscope objectives for "macro" work, ouside the microscope frame. They use Nikon CF (no compensatig) eyepieces. I have no direct experience with them, but other members have.
Pau

nielsgeode
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Post by nielsgeode »

Pau wrote:
nielsgeode wrote:Thanx a lot for your reply Pau!

So this Zeiss 25x 0.65 is horrible in the corners? This would be a major disadvantage for me unless it is fully fixable with the proper eyepiece.

I think the center sharpness will always be reduced by an eyepiece, or am I wrong?

Is there objectives out there that are overal sharper without vignetting on APS-C and which are superb without eyepiece, but also excellent with the proper eyepiece (in case I wish extra magnification)?
- No and yes: the Z Planapo, like any other objective not full corrected just needs an adequate compensating eyepiece to complete the optical correction. With the matched KPL eyepiece it's really excellent up to the corners. I will work badly without the eyepiece.

- Well, the photoeyepiece adds some magnification, so the image will be more cropped. Logically the objective image could more easily be outresolved by the sensor so it can look less sharp, but you don't loss actual resolution at the subject side, the first goal in microscopy. In fact if the sensor doesn't outresolve the objective you're losing some resolution even if the image looks sharper.

- Yes, the Nikon CF series as previoulsy commented. Many (most?) of them can cover APSC without much corner degradation. This is a subject very often discussed at the macro and equipment forums about the use of microscope objectives for "macro" work, ouside the microscope frame. They use Nikon CF (no compensatig) eyepieces. I have no direct experience with them, but other members have.
Thanx Pau!

Could you maybe post a link to the KPL eyepiece that will match the Zeiss 25x 0.65?

I have 3 finite CF plan objectives:
-20x NA 0.40 ELWD 160mm
-40x NA 0.65 ELWD 160mm
-60x NA 0.70 ELWD 210mm

But these are
-long working distance, so lower NA
-corrected for use without cover glass

So I think these are definitively not optimal for microscope work with cover glasses?

Pau
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Post by Pau »

Could you maybe post a link to the KPL eyepiece that will match the Zeiss 25x 0.65?
Any Zeiss KPL will match. As viewing eyepieces I think that the more convenient ones are the 10X with eyeglasses symbol (high eyepoint)

As photoeyepiece the best one (and much more expensive) is the S-KPL 10X, but for afocal any KPL high eyepoint will work nicely. There is a very rare PK 2.5X projective to use without relay lens, like the Oly NFK and FK
A most important point is the magnification to match the sensor

- afocally the formula is eyepiece magnification*camera lens FL /250. For APSC a good match is 1.6X, for FF 2.5X
...
But these are
-long working distance, so lower NA
-corrected for use without cover glass
So I think these are definitively not optimal for microscope work with cover glasses?
The 20/0.40 will work quite well but not optimally and will not be terribly sharp
The others will show noticable sperical aberration, i.e. poor sharpness
The 60X will be to avoid: low NA, not matched tube lenght....
Pau

Charles Krebs
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Post by Charles Krebs »

So this Zeiss 25x 0.65 is horrible in the corners? This would be a major disadvantage for me unless it is fully fixable with the proper eyepiece.
You should realize a few things. Microscope "tubes" (where eyepieces are inserted) are of two sizes... 23mm and 30mm. The vast majority of existing upright compound scopes (especially pre-infinity optics) have 23mm diameter tubes. Objectives therefore never needed to produce an image larger than about 20mm diameter since that was the largest size that an eyepiece inserted into a 23mm tube could "see". In reality many high-end finite objectives were designed to provide a "high quality" circular image of about 18mm diameter. As design and optics improved this moved up to about 20mm, and was often referred to as "wide-field". Although still not that common, as 30mm eyepiece tubes became more available, the objectives were designed to produce a larger image (~25mm to 26.5mm diameter). These are commonly referred to as "super wide-field".

The diagonal of an "APS-C" sized sensor is about 26mm to 27.5mm. As a result, if you wish to utilize "direct projection" with such a sensor, you really want to consider objectives that were designed so that they could be used with "super wide-field" heads.

I am not familiar enough with the Zeiss objectives to know which were designed to cover "super wide-field". I do know that the last couple incarnations of the Nikon CF (biological) objectives were used with super wide-field heads. (As were the Olympus S Plan Achromats and S Plan Apos... but these, like the Zeiss, utilized corrective eyepieces).

If you were to use an objective designed to cover 18-20mm diameter in direct-projection onto an APS-C sensor then the corners (out beyond that 18-20mm diameter) will likely drop off in image quality, often quite severe (not always, but likely). Pau knows more about Zeiss than I do. If the Zeiss Plan Apos of the series you referenced were not designed for super wide-field use, then you could expect less than stellar corner results. (I also do not know how much, if any, the Zeiss eyepieces perform field flattening).

I have three Zeiss Plan Apos. I use them on my BHS with the Olympus corrective eyepieces (23mm eyepiece tubes). I would not say that the Zeiss and Olympus chromatic corrections are the same (they are not), but I am very pleased with the results. The 160mm Zeiss Plan Apos are superb, but when purchasing used, the internal "delaminating" issue is quite real and you need to check for that carefully. (Personally I would avoid purchasing one unless I had a full return possibility).

As a result, if you are considering "direct-projection" with 160mm tube length finite objectives (and are after the best results) you really should, IMO, only consider the last couple versions of the Nikon CF Plan Achromats and/or Nikon CF Plan Apos.

So these are two quite different approaches. If you want to utilize Zeiss objectives you need to provide final chromatic corrections, and the "afocal" method is a common and good way to do this.

If you want to utilize direct projection, you will need to be very selective about the objectives you choose (super wide-field coverage and no needed chromatic correction), and thus probably forgo using any of the Zeiss you already have..

nielsgeode
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Post by nielsgeode »

Thanx a lot Charles for your very helpfull reply!

I think I will skip the Zeiss 25x NA 0.65 and try to find some SPlan Nikon's. If not too much effort, could you make s short summary of the different series of plan apochromat objectives from Nikon that are excellent for direct projection and that also cover complete APS-C?

Furthermore, I came across a series of four objectives that are for sale on a Dutch auction site. The info is limited (and the photo is poor quality). The seller doesn't know a lot about the objectives, but she wants about $100.

Information on how they perform is welcome.

Image

Alan Wood
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Post by Alan Wood »

The E A100 and E A40 might be from the Olympus E (education / economy) range, but I cannot be sure without seeing if they are marked Olympus on the other side. This range was designed for FN 18 compensating eyepieces (not widefield), and they are not flat field.

The NEA 10 might be from the later Olympus NE education / economy range, but I do not have a picture of the real thing.

The photo of the 4x does not show enough information.

Alan Wood

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Post by ChrisR »

Splan are Olympus, not Nikon.
The objectives illustrated look to me like low-end Oly achromats, requiring CA correction.

Since the Nikon CF series started (around 1990?), they've been free of need for correction.
There were finites and then infinites, and version of both with were designed for use with, or without, a cover glass. M meant Metallurgical, though it wan't always marked on the objective, then they changed to LU, for no cg.
There's a picture of some finite CF Nikons here:
Chris S.'s Bratcam final picture


Olympus finites, and their earlier infinites, needed CA correction. So there are no Olympus finites for direct projection, which are free of CA.

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