Need advice for 10X microscope objective

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redaipaul1
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Need advice for 10X microscope objective

Post by redaipaul1 »

I would like some advice for an 10 X microscoscope objective based on your experience.
I new in the microphotograpfy world and any help will be much appreciated.

I made a test with a 10X microscope objectivie that I currently have but i not pleased on the rsults. Please see picture bellow

10 Microscope objective
Image

The best result so far
Image
Redai Paul

JKT
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Post by JKT »

With low NA like this 0.25 it may not be that important, but this objective is meant to be used with coverglass as indicated by the "0.17".

Macro_Cosmos
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Post by Macro_Cosmos »

10x with 0.25NA is alright. Deals with a 36mp camera just fine. (Assume it's from a good brand!)

However, yours is intended to be used with a cover glass that has a thickness of 0.17, without that, you'll see haziness, or spherical aberration.

You will see better results with a Nikon M Plan 10x finite objective, those are pretty cheap. The reference for this category is the Mitutoyo 10x M Plan Apo, NA 0.28, a really good objective lens.
Last edited by Macro_Cosmos on Wed Apr 08, 2020 12:42 pm, edited 1 time in total.

redaipaul1
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Post by redaipaul1 »

Thank you guys for your answers.

About thickness of 0.17 but I never thought that would influence tresult so much .

This means as for finite objectives I should search Plan M (metalurgical) for better results. As far as I know those are " / 0" .
Redai Paul

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Post by Pau »

redaipaul1 wrote:...
I new in the microphotograpfy world and any help will be much appreciated.

I made a test with a 10X microscope objectivie that I currently have but i not pleased on the rsults. ...
Of course there are better objectives, but first be sure that you're doing the technique rightly.
Please tell us your camera and illustrate details of your setup, your stacking technique...it's very difficult to give good advice without good data.
Macro_Cosmos wrote:...However, yours is intended to be used with a cover glass that has a thickness of 0.17, without that, you'll see haziness, or spherical aberration.

I disagree, as JKT says at 0.25 NA this is not important (often this kind of objectives are labelled 0.25/- meaning that cover glass doesn't matter)
Standard cover glass mismatch only matters at more than 0.40 NA
You will see better results with a Nikon M Plan 10x finite objective, those are pretty cheap. The reference for this category is the Mitutoyo 10x M Plan Apo, NA 0.28, a really good objective lens.
Here I fully agree, but the objective is only one part of the question.
Pau

redaipaul1
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Post by redaipaul1 »

@Pau thank you for your replay.
Please see below some pictures with my current set-up

Image

Image

more details regarding the set-up in the linck below ( also with set-up upgrades)

https://www.photomacrography.net/forum/ ... highlight=

Looking forward for your feedback.
Redai Paul

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Post by rjlittlefield »

Redai, thank you for the additional information.

I notice that
(a) you are using LED illumination, and
(b) your objective is stuck on the end of a long overhang.

This combination makes your setup very sensitive to any vibration. For example any slight "bobbing" of the camera, caused by shutter movement, will cause the objective to move a lot across the subject.

Because you are using a Canon camera, you should be shooting with EFSC enabled (Electronic First Shutter Curtain), so that there will be no shutter vibration when the exposure begins. With many Canon cameras, EFSC can be enabled by just shooting from Live View, with no flash connected. You can check whether EFSC is being used by making exposures with a long shutter time, say 1 second or more. If EFSC is being used, then at beginning of exposure there will be no "click" sound, only perhaps a quiet "zing" like a tiny alarm chiming once.

Regardless of what 10X NA 0.25 objective you are using, the center of the image should be crisp and clear. If it is not, then almost certainly the image is being blurred by vibration.

To avoid vibration blurring, it is best to shoot with flash, preferably using a "speedlight" style flash at low power so that you get short pulses of light. Shooting with flash is always the first recommendation when somebody reports soft images at high magnification.

--Rik

redaipaul1
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Post by redaipaul1 »

@ rjlittlefield thank you very much for the value information I never thought that LED light would influence the results in microphotography so much.

Than I will go back to the board and redesign my set-up to reduce as much as possible the vibrations.

I use the same setup for regular macro stack and the resuls are perfect ( to me of course), se picture blow.


Image

Image
Redai Paul

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Post by rjlittlefield »

I use the same setup for regular macro stack and the resuls are perfect ( to me of course), se picture blow.
I saw your good results in the other thread. Lovely images!

The difference is a matter of magnification. However much blur you might have gotten at 1X, it will be 10 times worse at 10X. So, 1/4 pixel blur at 1X looks fine, but 2.5 pixels blur at 10X looks quite soft!

--Rik

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Post by Macro_Cosmos »

Pau wrote: I disagree, as JKT says at 0.25 NA this is not important (often this kind of objectives are labelled 0.25/- meaning that cover glass doesn't matter)
Standard cover glass mismatch only matters at more than 0.40 NA
You will see better results with a Nikon M Plan 10x finite objective, those are pretty cheap. The reference for this category is the Mitutoyo 10x M Plan Apo, NA 0.28, a really good objective lens.
Here I fully agree, but the objective is only one part of the question.
I see, noted. Is there any literature to look into?
Besides corner fuzziness, I do see blur. Even if it's a high NA objective used improperly without the cover glass, the centre portion should be sharp, if I'm not mistaken.
redaipaul1 wrote:@Pau thank you for your replay.
Please see below some pictures with my current set-up
Looking forward for your feedback.
Solid foundation. Some recommendations:
- A vertical setup might be better for you. It was a major improvement for me
- You're using LEDs. What's the exposure time? Your setup will see lots of vibrations
- I don't see adequate diffusion. Even with no vibrations, the images might not be that great. Undiffused VS diffused, using container wrapped with tracing paper:
Image
- You need a way to support the tube, can't leave it floating like that
https://www.wemacro.com/?product_cat=le ... es&paged=2
Wemacro sell tubes and clamps, these work well. Just buy a cheap long arca-swiss rail. This is what mine looks like (I use thorlabs parts):
Image
- Use mirror up and EFSC in liveview, refer to camera manual
- Make your base heavier. You can do so by bundling bottles of water to the table or better, exchange a huge load of pennies (or equivalent smallest currency) at your bank! A heavy slab of marble or granite works well too.
- I would mount the camera directly to the motorised rail to lower the centre of gravity. You can use the pan-tilt head when you need it, maybe for lower mag.

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Post by rjlittlefield »

Macro_Cosmos wrote:
Pau wrote: I disagree, as JKT says at 0.25 NA this is not important (often this kind of objectives are labelled 0.25/- meaning that cover glass doesn't matter)
Standard cover glass mismatch only matters at more than 0.40 NA
I see, noted. Is there any literature to look into?
See discussion and links at https://www.photomacrography.net/forum/ ... 514#198514 . Also Figure 3 at https://www.microscopyu.com/microscopy- ... correction .
Even if it's a high NA objective used improperly without the cover glass, the centre portion should be sharp, if I'm not mistaken.
The center will go "milky" also, with sufficiently large NA and no cover slip. It is a matter of spherical aberration, where different rings of the lens focus at different places. If you focus the outside of the lens, then you will still resolve the finest detail corresponding to the NA, but then all parts of the lens closer to center will be out-of-focus and contributing haze. I think the PSF under these conditions will still have a narrow central peak corresponding to the full NA, but only at low amplitude superimposed on a broad pedestal caused by defocus of the other rings. Note the drop in "Maximum Intensity in an Image of a Point Object", the previously mentioned Figure 3.

--Rik

redaipaul1
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Post by redaipaul1 »

Thank you all for pointing me in the right direction, the information that I recieved is gold mine.

I will take in consideration all of the above and I will keep you posted with imporovemets when available.
Redai Paul

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Post by Deanimator »

Macro_Cosmos wrote:- I don't see adequate diffusion. Even with no vibrations, the images might not be that great.
This is definitely a HUGE issue.

Until I got a good handle on diffusion, I got mediocre results at best, even at relatively low magnifications.

It takes a lot of trial and error, especially for a beginner like me, but when you get the diffusion dialed in properly, the results are amazing.

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Post by JKT »

On second thought I'm no longer so sure that the cover glass correction doesn't matter. I don't seem to be getting really sharp pictures of slides with Mitutoyo 10x, but it seems to work fine without cover glass.

Could it be that the limit when it matters is from earlier times and not valid for current high resolution photography? I'd love to see a comparison by someone with a verified good Mitutoyo.

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Post by Macro_Cosmos »

JKT wrote:On second thought I'm no longer so sure that the cover glass correction doesn't matter. I don't seem to be getting really sharp pictures of slides with Mitutoyo 10x, but it seems to work fine without cover glass.

Could it be that the limit when it matters is from earlier times and not valid for current high resolution photography? I'd love to see a comparison by someone with a verified good Mitutoyo.
I'm happy to get a quick comparison up, however my slides are premade and due to the virus, I can't get any blank slides. Is there a method that doesn't involve destroying my current slides? I suppose I can compare a thin coverglass VS thick microscope slide (ie, flip the thing over).

My mit 10x was purchased new from verified supplier.
Z6, 24MP BSI, with coverslip: https://live.staticflickr.com/65535/497 ... bce2_o.jpg
Image

Removing it will definitely destroy the slide, I'm not doing that.

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