Stacking with DOF < 1 um; What is the best solution?

[nielsgeode]

Recently, I purchased myself two new objectives: a Nikon M-Plan 60x NA 0.70 ELWD and a Olympus MS-Plan ULWD 80x NA 0.75.

I still have to get the objectivs and calculate the DOF, but I am pretty sure that it is less than one micrometer. This is beyond the limit of the Stackshot.

Some microscope stands have a fine-focus which can do much smaller steps and some companies (like Newport) make precision stages that can do steps of < 1 um as well.

My question is: what is the best and most compact solution for a setup that can be used for stacking with these objectives?

Any help and suggestions are greatly appreciated

Niels

[seta666]

If you are going to use a linear stage then a differential micrometer would be the wise option for such lenses.

By the way, the stack shot can make 0.5 um steps, not in a very accurate way but still can do it ;-)

Regards
Javier

[ChrisR]

Niels - little steps with a stackshot:
http://www.photomacrography.net/forum/v ... highlight=

[rjlittlefield]

Niels - little steps with a stackshot:
http://www.photomacrography.net/forum/v ... highlight=

That thread has a lot of good detail, but sometimes people find it confusing to start. The key ingredient is just to link a stepper motor to the fine focus knob and drive the stepper with a StackShot controller. See Fine focusing a microscope using StackShot for a quick intro.

I've edited the detailed thread to include this link also.

I still have to get the objectivs and calculate the DOF, but I am pretty sure that it is less than one micrometer.

True. DOF by 1/4 lambda calculates to 0.98 microns at NA 0.75.

--Rik

[nielsgeode]

Niels - little steps with a stackshot:
http://www.photomacrography.net/forum/v ... highlight=

That thread has a lot of good detail, but sometimes people find it confusing to start. The key ingredient is just to link a stepper motor to the fine focus knob and drive the stepper with a StackShot controller. See Fine focusing a microscope using StackShot for a quick intro.

I've edited the detailed thread to include this link also.

I still have to get the objectivs and calculate the DOF, but I am pretty sure that it is less than one micrometer.

True. DOF by 1/4 lambda calculates to 0.98 microns at NA 0.75.

--Rik

Nice thread, thanx a lot

Do you know of any microscope stands were you can easily remove the fine focus part from the rest of the stand or that have a lower (e.g. 100 um) travel distance per full rotation of the fine focusing knob? I'd like to use it in a horizontal setup, but the focusing block should not be (much) larger than the size of a single-axis translation stage. Although I can image that I can then just as well go for a Thorlabs (or similar) stage with a stepper motor attached. What do you think and do you know how they compare in accuracy?

Niels

[rjlittlefield]

Do you know of any microscope stands were you can easily remove the fine focus part from the rest of the stand or that have a lower (e.g. 100 um) travel distance per full rotation of the fine focusing knob?

See Nikon , Olympus, Meiji, Swift Focus Blocks; & Setups. It's a long thread (9 pages), with many options and issues discussed. The short story is that yes, one can get microscope focusing blocks as separate units, and some of them are 100 µm per rotation. I believe that Chris S.'s Bratcam now uses a 100 µm per rotation stage driven by a 400 full steps per rotation motor driven by a StackShot controller.

Although I can image that I can then just as well go for a Thorlabs (or similar) stage with a stepper motor attached. What do you think and do you know how they compare in accuracy?

I've never looked at the Thorlabs stages. In general, I would expect their precision to be established by the stepper and (for microsteps) by the drive electronics.

--Rik

[nielsgeode]

Do you know of any microscope stands were you can easily remove the fine focus part from the rest of the stand or that have a lower (e.g. 100 um) travel distance per full rotation of the fine focusing knob?

See Nikon , Olympus, Meiji, Swift Focus Blocks; & Setups. It's a long thread (9 pages), with many options and issues discussed. The short story is that yes, one can get microscope focusing blocks as separate units, and some of them are 100 µm per rotation. I believe that Chris S.'s Bratcam now uses a 100 µm per rotation stage driven by a 400 full steps per rotation motor driven by a StackShot controller.

Although I can image that I can then just as well go for a Thorlabs (or similar) stage with a stepper motor attached. What do you think and do you know how they compare in accuracy?

I've never looked at the Thorlabs stages. In general, I would expect their precision to be established by the stepper and (for microsteps) by the drive electronics.

--Rik

I have an X-axis stage (do not know the brand) that moves 0.5 mm per full rotation. So, this means that at 200 microsteps (the Cognisys stepper motor) per rotation the smallest stepsize that you can get with this stage is 2.5 microns? Is there a way to obtain smaller steps with this stage or would the mechanics in the stage also itself limit it to steps > 1 micron?

[rjlittlefield]

I have an X-axis stage (do not know the brand) that moves 0.5 mm per full rotation. So, this means that at 200 microsteps (the Cognisys stepper motor) per rotation the smallest stepsize that you can get with this stage is 2.5 microns?

Not correct. The standard Cognisys system is 200 full steps per rotation, with 16 microsteps per full step, so 3200 microsteps per rotation. Microsteps are analog positions between full steps, accomplished by modulating the two phases of drive currents.

Is there a way to obtain smaller steps with this stage or would the mechanics in the stage also itself limit it to steps > 1 micron?

At 3200 microsteps per rotation, each microstep would be nominally .156 microns. The stage itself is undoubtedly a continuous drive device with a bit of slop. There won't be anything that limits its movements to some fixed value > 1 micron, but there also won't be any assurance that each of those microsteps ends up being quite the same, or even that the same drive sequence reaches exactly the same positions on two runs of the stack. If the drive screw and alignment tracks are smooth, clean, and snug it should be pretty close, but unfortunately I can't put a number of "pretty close".

--Rik

[nielsgeode]

I have an X-axis stage (do not know the brand) that moves 0.5 mm per full rotation. So, this means that at 200 microsteps (the Cognisys stepper motor) per rotation the smallest stepsize that you can get with this stage is 2.5 microns?

Not correct. The standard Cognisys system is 200 full steps per rotation, with 16 microsteps per full step, so 3200 microsteps per rotation. Microsteps are analog positions between full steps, accomplished by modulating the two phases of drive currents.

Is there a way to obtain smaller steps with this stage or would the mechanics in the stage also itself limit it to steps > 1 micron?

At 3200 microsteps per rotation, each microstep would be nominally .156 microns. The stage itself is undoubtedly a continuous drive device with a bit of slop. There won't be anything that limits its movements to some fixed value > 1 micron, but there also won't be any assurance that each of those microsteps ends up being quite the same, or even that the same drive sequence reaches exactly the same positions on two runs of the stack. If the drive screw and alignment tracks are smooth, clean, and snug it should be pretty close, but unfortunately I can't put a number of "pretty close".

--Rik

So this means that at 3200 microsteps per rotation and a distance of 200 micrometers (Olympus CH) a microstep would be 63 nm Somehow, I cannot believe that it is even possible at all to do a stack with steps of, for example 60 ± 30 nm.

Thorlabs has a simple micrometer stage: http://www.thorlabs.de/thorProduct.cfm?partNumber=MS1
It offers a travel distance of 250 micrometers per full rotation and a total travel distance of 1/4". With this device and a stepper motor, steps of 0.2 to 0.3 micrometers should be easily and accurately achieved. Am I correct?

[Chris S.]

Recently, I purchased myself two new objectives: a Nikon M-Plan 60x NA 0.70 ELWD and a Olympus MS-Plan ULWD 80x NA 0.75.

I still have to get the objectives and calculate the DOF, but I am pretty sure that it is less than one micrometer.

Niels, I have a Nikon 60/0.70 ELWD, and use 0.5 micron steps with it. Seta666 recently mentioned using 1 micron steps with his. Though I teased him about “being lazy,” I meant it in good fun; I don’t think his choice was unreasonable—it’s just not what I would likely do.

My question is: what is the best and most compact solution for a setup that can be used for stacking with these objectives?

“Best” and “most compact” are probably not the same. For “most compact,” I’d think of a motorized linear stage. For “best,” I’d think of a motorized microscope focus block. They pleasure of working with coaxial coarse and fine focus, on a microscope block, has to be experienced to be appreciated. While I have and use linear stages, I’d recommend you get a nice microscope focus block for your stacking movement.

I believe that Chris S.'s Bratcam now uses a 100 µm per rotation stage driven by a 400 full steps per rotation motor driven by a StackShot controller.

Exactly so. And I’m very happy with it. I get 0.016 microns per microstep with the StackShot controller, or 0.25 micron full steps. (I usually work in full steps.)

Niels, I’ll send you a PM with a current eBay item you might want to try, even though I think it a bit overpriced (and the vendor declined my offer). I keep a pretty close watch on eBay focus blocks, and there are not too many good candidates at decent prices right now. Do be aware than any microscope block purchased second hand involves a gamble--something I know from experience, having purchased a number of them. It helps to ask the seller very specific questions about the focus block, and perhaps coach the seller if necessary on how to test a unit. But no matter how hard you try to assess a block before purchase, you will still sometimes receive a bad one, even with a seller apparently acting in good faith. Also, unless you’ve had a few focus blocks in hand, you may not immediately recognize if you received a good one.

Good luck!

--Chris

[seta666]

Niels, I have a Nikon 60/0.70 ELWD, and use 0.5 micron steps with it. Seta666 recently mentioned using 1 micron steps with his. Though I teased him about “being lazy,” I meant it in good fun; I don’t think his choice was unreasonable—it’s just not what I would likely do.

http://www.photomacrography.net/forum/v ... 6&start=14

Je, je; according to new Rik's DOF spreadsheet 1um step with 60/0.70 is still allowing a 10% overlap, 0,5 um steps give over 50% overlap

In fact a 0,5um step gives 40% overlap even with the 100/0.80 ELWD


Regarding focus block vs linear stage I thing both are more than enough for our needs, of couse if I had a microscope I would go the focus block route

Stackshot can do even stacks with the 100/0.80 ELWD (I have done it), you need to fool the controller so it can give 0,5 um steps. Just tell the stackshot it give 15,875mm per revolution and it will let you take those 0,5um steps (controller will read 5 um)

The weak link is the rail though, can not compare to a linear stage or focus block but Zerene will fix all or most missalignment


Regards

[Chris S.]

Niels, I have a Nikon 60/0.70 ELWD, and use 0.5 micron steps with it. Seta666 recently mentioned using 1 micron steps with his. Though I teased him about “being lazy,” I meant it in good fun; I don’t think his choice was unreasonable—it’s just not what I would likely do.

http://www.photomacrography.net/forum/v ... 6&start=14

Je, je; according to new Rik's DOF spreadsheet 1um step with 60/0.70 is still allowing a 10% overlap, 0,5 um steps give over 50% overlap

In fact a 0,5um step gives 40% overlap even with the 100/0.80 ELWD

For some reason, I'm currently barred from seeing Rik's spreadsheet; but I have my own, long-used, well-trusted spreadsheet. On my spreadsheet, I computed values separately for red and violet wavelengths, as they are at opposite ends of the spectrum. Empirically, I've found that increments that correspond to 50 percent overlap in violet result in the best stacks, so I routinely use these values.

But I also know that if I'm in a hurry (suppose a specimen is wilting), I will lose only a small amount of quality if I use an increment up to twice my violet, 50-percent overlap optimum.

Cheers,

--Chris

[nielsgeode]

Niels, I have a Nikon 60/0.70 ELWD, and use 0.5 micron steps with it. Seta666 recently mentioned using 1 micron steps with his. Though I teased him about “being lazy,” I meant it in good fun; I don’t think his choice was unreasonable—it’s just not what I would likely do.

http://www.photomacrography.net/forum/v ... 6&start=14

Je, je; according to new Rik's DOF spreadsheet 1um step with 60/0.70 is still allowing a 10% overlap, 0,5 um steps give over 50% overlap

In fact a 0,5um step gives 40% overlap even with the 100/0.80 ELWD

For some reason, I'm currently barred from seeing Rik's spreadsheet; but I have my own, long-used, well-trusted spreadsheet. On my spreadsheet, I computed values separately for red and violet wavelengths, as they are at opposite ends of the spectrum. Empirically, I've found that increments that correspond to 50 percent overlap in violet result in the best stacks, so I routinely use these values.

But I also know that if I'm in a hurry (suppose a specimen is wilting), I will lose only a small amount of quality if I use an increment up to twice my violet, 50-percent overlap optimum.

Cheers,

--Chris

Thanx a lot for you reply (and your PM!). Would you mind to send me te spreadsheet so I can see how you calculated the DOF values?

My first idea is/was to mount the stackshot on a stable aluminum plate with shock absorbers on it (horizontal setup). Then, I'd like to have a fixed object-positioning (goniometer) in front of the stackshot. Whenever, I need steps of less than 1 micrometer, I would just use the stackshot only for course focusing and a small stage (or focusing block) to do the actual stacking. With this idea in mind, One or two milimeters of travel distance is already more than enough, because I can still use my stackshot for course movements.

Thorlabs sells a brand new small stage for only about $200: http://www.thorlabs.com/newgrouppage9.c ... oup_id=761

It will certainly be accurate enough when a Cognisys stepper motor is attached and a travel distance of more than 6 mm is a lot when using over 50x magnification.

What would be the advantage of a focusing block compared to a micrometer stage in this (type of) setup? I don't like the "risk" of spending $200 (or more) on a focusing block and getting one that is old, intensively used and not in good shape.

How much do these things approximately weigh? Because I'm dealing mainly with crystal (specimens) that differ a lot in size I need a setup where the specimen can be "moved" quite a large distance to get the crystals in front of the lens. Also, I need full six-axis rotation since the orientation of these crystals is completely dfiferent with each new specimen. My current idea is to machine some support to fix the Novoflex Cross-Q in a vertical position and then "attach" some piece of custom made aluminum to the Arca-clamp of the Cross-Q to obtain a horizontal setup for my specimens. On this custom made piece of aluminum I can then fix the micrometer stage and some goniometer-like device to allow for (some) rotation and tilting of the crystals.

[rjlittlefield]

Stackshot can do even stacks with the 100/0.80 ELWD (I have done it), you need to fool the controller so it can give 0,5 um steps. Just tell the stackshot it give 15,875mm per revolution and it will let you take those 0,5um steps (controller will read 5 um)

Alternatively, set the controller to work in "steps" (which are actually microsteps) and set a value of 1. No fooling needed.

--Rik

[Craig Gerard]

What would be the advantage of a focusing block compared to a micrometer stage in this (type of) setup?

The answer to that question would depend on whether you intend automating either a translation stage or a 'focus block'. Manual translation of 1 micron with specific 'focus blocks' is very easy to accomplish, so too 0.5 micron. The same exercise with a linear translation stage is a rather difficult matter when you are working with such fine increments; 0.5 and 1 micron incremental adjustments would only come into play at magnifications > 40X.

Based on your proposed setup, the StackShot (which you already own) would appear to be the most appropriate candidate for the focus stacking role. The addition of a linear translation stage could serve as a manual, fine positioner; but I'd go with a larger model than the referenced Thorlabs stage. I find some form of manual adjustment capability to be fundamentally essential. Setting a 'start' and 'end' point for a stack is the last thing I do; I like to have a good look at and around a subject from various angles before commencing to focus stack. Such exploration is undertaken using a combination of positioning devices at both the camera and/or subject end.

Which StackShot rail do you have, the standard or extended version?

I'm dealing mainly with crystal (specimens) that differ a lot in size I need a setup where the specimen can be "moved" quite a large distance to get the crystals in front of the lens.

There are a number of viable options for such a scenario if we are considering a horizontal setup. One such option, for camera positioning, would be to incorporate a long (12" to 24") manual positioning rail such as the Newport PRL + PRC combo or a length of aluminium extrusion with a lockable, 'gliding' platform. A manual linear translation stage could be attached to such a platform or to the Newport PRC; an automated 'fine' focus device could be attached to the linear translation stage. Such an arrangement would provide manual 'course' positioning, manual 'fine' positioning, and automated 'ultra fine' translation.





Craig