Shooting slides with D800E over Nikon Eclipse E200 questions

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ckatosmith
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Shooting slides with D800E over Nikon Eclipse E200 questions

Post by ckatosmith »

OK, I finally have StackShot working on my Nikon Eclipse E200 microscope, which have infinity plan acromat objectives.

The setup is Nikon D800E / bellows / Raynox DCR-150 over the microscope (binocular head removed).

At 40X, the DOF is so shallow that I seem to only get/need 3 steps (1 uM each) to stack. Does that sound right? Seems like I would 'need' a deeper stack. These are prepared educational slides with cover slips.

At 10X I don't seem to need many steps either, although I recall a much higher count stack when doing off microscope stacks with the objective basically attached to a 'tube lens' and the setup was on a StackShot rail, also even just using my Sony 4K video cam over the microscope.

At 4X, I can't even get things focused... it's like I need the stage to come up higher, though it has reached it's limit. But again, I have no trouble focusing when the setup is not through the microscope; D800E/tube lens (I use my Micro Nikkor 70-180mm set at 180mm) /objective attached to lens on a regular StackShot Rail.

Same objectives, same camera. But I realize the objectives are a different distance from the sensor when they are on the microscope nosepiece versus close to/right on the 'tube lens' when shooting on the rail system.
Could that have something to do with it? Comments/help welcome.

Thanks.

Pau
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Re: Shooting slides with D800E over Nikon Eclipse E200 quest

Post by Pau »

ckatosmith wrote: At 4X, I can't even get things focused... it's like I need the stage to come up higher, though it has reached it's limit. But again, I have no trouble focusing when the setup is not through the microscope; D800E/tube lens (I use my Micro Nikkor 70-180mm set at 180mm) /objective attached to lens on a regular StackShot Rail.
Something must be wrong, this happens when the microscope tube lenght is not well matched with the objectives correction. Being an infinite corrected system I guess that the most probable issue is the tube lens not focussed to infinite. Outside the microscope, focus with the bellows/Raynox to a distant subject to assure that it is really focussed to infinite. Some pictures of your setup also could help.

About the deep of the stack, biological prepared slide subjects usualy range from 5 to 30 micron. Another important point is the condenser aperture: if it is too closed you get more DOF and contrast but less resolution compared with the right aperture, this can alter your need of stack. I do not know your skils in microscopy, if you want we can recommend some easy and excellent readings.
Pau

ckatosmith
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Post by ckatosmith »

Thanks Pau,

My skills in microscopy I would say are nominal. I did, for example, experiment with the condenser aperture some and realized closing the aperture down would make things look sharper, increase DOF (just like squinting an eye, or using a smaller aperture in photography), but I didn't know it was at the cost of resolution.

Does it follow, then, that stacking 1um slices using a wider condenser aperture will yield more detail than stacking 1um slices of a smaller aperture because of a loss of resolution?

How does one set a fixed lens (the Raynox DCR-150) to infinity when it is just a piece of glass on bellows?

Thanks for the note that slide subjects usually range from 5 to 30 microns. I was hoping someone would give that range information.

Will try and post pictures of setup when I return from a short trip.

I may have been confusing when I stated using the 70-180mm Nikkor lens at 180mm and set to infinity... that 'tube lens' was with the objective screwed onto the lens (M25 adapter) and used OFF the microscope setup... on a vertical StackShot Rail. Since OVER the microscope (removed binocular eyepiece section) the objective is fixed in the nose piece, I can't see how to close the gap from the objective to the 180mm lens (which showed a tiny field of view), so I felt my only option OVER the microscope was to use the 'bellows/DCR-150' route because of the larger field of view (practically covers the D800E frame at 4X (but can't focus), and full field coverage with 10X and 40X.

I used CamRanger to focus at 100% so my eyesight should not be a confounding variable.

Sure, open to a good intro to microscopy. My introduction to the microscope was just one semester of Pathology where the focus was slide content, not any mechanics or understanding of how to bring details out or how to work with different lighting methods.

I have Enrico Savazzi's book on my wish list but it looks really daunting. Is there a good 'bridge' book, or should I just jump in and get that?

Pau
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Post by Pau »

(quotes in blue)

My skills in microscopy I would say are nominal. I did, for example, experiment with the condenser aperture some and realized closing the aperture down would make things look sharper, increase DOF (just like squinting an eye, or using a smaller aperture in photography), but I didn't know it was at the cost of resolution.
In general photography it also is (at least theorically) at cost of resolution, but only relevant in real life at very small apertures as you know, in microscopy we are closer to the physics limits...

Does it follow, then, that stacking 1um slices using a wider condenser aperture will yield more detail than stacking 1um slices of a smaller aperture because of a loss of resolution?
Yes, but to have actual resolution you need to have enough contrast, in BF microscopy you have to play with the condenser to play with this tradeof

How does one set a fixed lens (the Raynox DCR-150) to infinity when it is just a piece of glass on bellows?
Just playing with the distance between the lens and the sensor with the bellows extension, like with any other simple camera lens. We are refering to the Raynox placed at the end of bellows, not with it placed on another lens.

Thanks for the note that slide subjects usually range from 5 to 30 microns. I was hoping someone would give that range information.
May be thinner or thicker but this may be an useful reference

I may have been confusing when I stated using the 70-180mm Nikkor lens at 180mm and set to infinity... that 'tube lens' was with the objective screwed onto the lens (M25 adapter) and used OFF the microscope setup... on a vertical StackShot Rail. Since OVER the microscope (removed binocular eyepiece section) the objective is fixed in the nose piece, I can't see how to close the gap from the objective to the 180mm lens (which showed a tiny field of view), so I felt my only option OVER the microscope was to use the 'bellows/DCR-150' route because of the larger field of view (practically covers the D800E frame at 4X (but can't focus), and full field coverage with 10X and 40X.
Yes, that's the idea, it's about the same with true microscope tube lenses, they don't need to be very close to the objective, otherwise the infinite microscope desing would have no sense. Be aware that your Nikon zoom is a complex lens with the entrance aperture very recessed from the front lens.

Sure, open to a good intro to microscopy...
... Is there a good 'bridge' book, or should I just jump in and get that?

I always recommend this Zeiss booklet:
https://www.micro-shop.zeiss.com/index. ... 1-9901-000
(until recently it was free downoadable, I don't know if now it's still free but under register and log in)
The use of the condenser to set Kölher es very well explained.
Pau

Pau
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Post by Pau »

a link actually working for download it:
http://www.well.ox.ac.uk/cytogenetics/d ... inning.pdf
Pau

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