Sharpness of microscopic photos at 10x?

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rjlittlefield
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Re: Sharpness of microscopic photos at 10x?

Post by rjlittlefield »

plantfan123 wrote:
Tue Nov 30, 2021 11:11 pm
With the lens focus method can you do movements of a few microns that you can do with the Stackshot that are so useful in microscopy? How precise is it?
Yes, this works fine, assuming that the lens uses internal focus so that the objective does not move as focus changes.

A good way of thinking about the situation is that the objective makes an object that is actually small and close, look like an object that is large and distant. Then the rear lens just focuses on the large distant object, and steps focus through that. So, if the rear lens has enough precision to focus-step its way through a real large object without focus banding (and they all do), then it also has enough precision to focus-step its way through the virtual large object that is created by the objective.

From the standpoint of precision, things actually get better at high magnification, because the magnification process stretches the large-and-distant virtual object along the depth axis. Unfortunately, there is a limit in how far the rear lens can shift focus away from infinity, before it starts seeing parts of the enlarged object where the image is degraded by pulling the objective away from its design point. As you saw, focusing the rear lens at 1:1 messed up your 10X objective pretty badly. With an objective of higher magnification and higher NA, that problem would be even worse.

See my thread on "AF motor focusing with a microscope objective" for some illustration. Note particularly the behavior with 50X NA 0.55, described near the end of the first page of the thread.

--Rik

plantfan123
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Joined: Tue Dec 08, 2020 12:39 pm

Re: Sharpness of microscopic photos at 10x?

Post by plantfan123 »

rjlittlefield wrote:
Tue Nov 30, 2021 11:45 pm
plantfan123 wrote:
Tue Nov 30, 2021 11:11 pm
With the lens focus method can you do movements of a few microns that you can do with the Stackshot that are so useful in microscopy? How precise is it?
Yes, this works fine, assuming that the lens uses internal focus so that the objective does not move as focus changes.

A good way of thinking about the situation is that the objective makes an object that is actually small and close, look like an object that is large and distant. Then the rear lens just focuses on the large distant object, and steps focus through that. So, if the rear lens has enough precision to focus-step its way through a real large object without focus banding (and they all do), then it also has enough precision to focus-step its way through the virtual large object that is created by the objective.

From the standpoint of precision, things actually get better at high magnification, because the magnification process stretches the large-and-distant virtual object along the depth axis. Unfortunately, there is a limit in how far the rear lens can shift focus away from infinity, before it starts seeing parts of the enlarged object where the image is degraded by pulling the objective away from its design point. As you saw, focusing the rear lens at 1:1 messed up your 10X objective pretty badly. With an objective of higher magnification and higher NA, that problem would be even worse.

See my thread on "AF motor focusing with a microscope objective" for some illustration. Note particularly the behavior with 50X NA 0.55, described near the end of the first page of the thread.

--Rik
Thanks for the lucid explanations! I am really enjoying learning about all of this. I don't have the set-up to try this now but look forward to experimenting with it.

Side question - has anyone seen an M25 - 52mm adapter 3D printer model? I see various microscopy-related 3D printer files online but can't find this one. The wait time for getting the adapters from China is just so long haha.

Scarodactyl
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Re: Sharpness of microscopic photos at 10x?

Post by Scarodactyl »

These are easy to make in fusion 360, though you have to manually edit an sql file to make custom threads. I find I need to add about 0.3mm to the dimensions of inner threads becsuse they print a bit tight.

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