Zeiss Axioskop2plus epi for z-stacking?

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dlg
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Zeiss Axioskop2plus epi for z-stacking?

Post by dlg »

I currently take images of small (1-3 mm) flowers with Canon MPE-65 and stack shot (resolution limit around 10 µm), or on a Zeiss Discovery V20 mot with planapo 1.5x (resolution limit around 4–6 µm). I've been toying with the idea of using my compound for z-stacking (resolution to 1.6 µm with NA 0.4) but was hamstrung by the lack of motorized focus. Found
http://www.photomacrography.net/forum/v ... sc&start=0
[totally genius!], so that can be overcome.

Some of the issues and what I think about it below (feedback welcome!):

- Chromatic correction. Apparently Zeiss uses tube lenses for partial color correction, so I am more or less stuck with Zeiss lenses. Looking at LD Plan-Neofluar 20x/0.4 Corr M27 with 7.9 mm working distance. A bit more expensive than Mitutoyo, but all works well together. There is a Zeiss apo version with 5.6 mm WD, but at $20K a bit over the top.

I have a set of 5-10-20-40corr ICS/0.17 planapos for histology slides, but working distance is short and they are designed for use with cover slips. At low mag end, WD should be fine; does anybody know how much of a difference /0 vs /0.17 makes?

- Steps: Still have to calculate DOF and steps, but no problem. I have optovars, so may have to experiment a bit for the three magnification settings. Not even sure whether the optovars affect step size or not.
Not sure whether there is data on how much stage travel is generated by one revolution of fine focus. Worst case scenario, some experimentation.
I can control Stackshot and Camera through Zerene, and use fixed step-sizes, most likely with dummy values that produce the right result. Given the limited number of mags = step sizes, a simple table takes care of that.

- Illumination: I have an epi-illumination pathway on the scope, though never used it. Should a flash (have Canon Speedlight 580EX) be placed where traditional epi lamp house goes and use fibre optics as external modeling/focusing lights? Or is it better to use ring diffuser around objective and object, flash from outside, and use epi-pathway with traditional lamp house as focusing light?

Thanks for any insights!

Chris S.
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Re: Zeiss Axioskop2plus epi for z-stacking?

Post by Chris S. »

dlg wrote:I have a set of 5-10-20-40corr ICS/0.17 planapos for histology slides, but working distance is short and they are designed for use with cover slips. At low mag end, WD should be fine; does anybody know how much of a difference /0 vs /0.17 makes?
This has been discussed a few times. Generally, for an NA of 0.30 or less, the lack of a cover slide doesn't matter. For higher NA's, there is a problem, which very rapidly escalates. See Rik Littlefield's post here.
Steps: Still have to calculate DOF and steps, but no problem. I have optovars, so may have to experiment a bit for the three magnification settings. Not even sure whether the optovars affect step size or not.
This prompted a brief, interesting read on what an Optovar is. My bet is that an Optovar won't affect DOF, as DOF is essentially determined by NA. This said, experimentation is always in order, as calculations only get you in the neighborhood. A nice place to start is, again, something written by Rik: DOF Estimates For Macro/Micro (depth of field, step sizes), in particular table 2-C. I suggest you shoot a few stacks with smaller increments, and use Zerene Stacker's "Stack every N'th frame" feature to determine what looks best to you.
Not sure whether there is data on how much stage travel is generated by one revolution of fine focus. Worst case scenario, some experimentation.
As you've no-doubt envisioned, the experiment is quick and painless. When I measure an unknown focus block, I zero my digital caliper between two appropriate points, then crank the fine focus block 100 revolutions, and adjust the caliper for the same two points. Distance on the caliper's readout, divided by 100, is the distance per single revolution. And most sources of error are reduced by two orders of magnitude by measuring across a cumulative 100 revolutions.

But my bet is that you have long ago thought of this. :D

--Chris

Pau
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Post by Pau »

Not sure whether there is data on how much stage travel is generated by one revolution of fine focus
Most microscopes have one or both fine focus knobs graduated. If so the numbers will refer to micrometers for sure. I've seen axios with 1 division = 1 micrometer but I ignore if it is the general rule. All microscopes I have seen have divisons each 5 micron (like my Zeiss Standard), 2 micron or 1 micron

About flash, maybe this post could suggest you some ideas: http://www.photomacrography.net/forum/v ... hp?t=26185

For epi illumination almost for sure you will need to implement cross polarization to avoid unwanted reflections and glare. Zeiss has cross polarization cubes to fit their fluorescence epi illuminators in place of the fluorescence cube.
Pau

Charles Krebs
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Post by Charles Krebs »

- Illumination: I have an epi-illumination pathway on the scope, though never used it. Should a flash (have Canon Speedlight 580EX) be placed where traditional epi lamp house goes and use fibre optics as external modeling/focusing lights? Or is it better to use ring diffuser around objective and object, flash from outside, and use epi-pathway with traditional lamp house as focusing light?
I would give this some thought. With a working distance of 7.9mm you will have some decent options for external lighting.

What is the primary usage of the resulting photographs? Epi illumination (through the microscope) can look pretty "clinical". It is very direct and stark (talking about epi-brightfield now), and comes with it's own set of problems such as the glare and reflections Pau mentioned (which makes it very impractical to use a separate light source for modeling/focus, since you really need to be observing the exact effect your imaging light has in order to make good adjustments). Epi-darkfield is very useful at times, but with 3D subjects it will often yield the circular highlights similar to those seen with a ring-flash (and of course requires brightfield/darkfield objectives and components).

With 7.9mm to work with you can obtain much more aesthetically pleasing lighting by using external lights together with additional diffusers and reflectors. (Cut up ping-pong balls are very useful . :wink:, and very often a simple circular ring of Lee or Rosco diffusion material is all you need).

Is there a particular reason you want to use flash? Nothing wrong with that, and it is certainly recommended if you are trying to avoid troublesome equipment vibrations or "freeze" an active subject. But with a static subject and a speedlight (like the 580EX) you will have the problem of getting accurate modeling as you adjust your lighting.

dlg
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Post by dlg »

Thanks to all three of you to the various indications.

the NA 0.3 for coverslips is very helpful. 4th power function is fully appreciated. The 5x/0.16 ICS/0.17 planapo has a WD of 12 mm, so should work just fine. the 10x/0.3 ICS/0.17 planapo has a WD of 2 mm, so too tight. There is an EC Neofluar 10x/0.25 ICS/0 with WD of 9.3 mm. The 10x/0.4 ICS/0 planapo has a WD of 5 mm, which seems a bit tight.

RE optovars, I also sort of though that it should not have an effect, but proof is always in the pudding. I have done n-th sample routine in Zerene. Checked whether stacking steps of 0.2xDOF as used by Zeiss Zen Blue software makes a difference to the usual 0.7xDOF. Answer: no. only at >1xDOF you start to see image problems.

Re finefocus interval, digital calipers certainly will get me into ball park. If they are actually calibrated to something sensible, I can also check with Zeiss sales rep.

Re flash, I like it for added crisp-ness of images. Thanks for pointers re epi illumination. I had been wondering about too flat illumination, which you confirm. The round highlights are a further issue. I am familiar with them from SLR macrophotography, so prefer dual head macro flash (Canon MT24Ex). I position them with Wimberley plamps with epoxy glued flash-shoes (see https://www.aos.org/Default.aspx?id=355 for set-up on my old DV12). With SLR, I usually check lighting with test firing in upper third of stacking area, then adjust. I have a rotating petrographic stage, so that will make things quite a bit easier.

I noticed Krebs' penchant for ping-pong balls in other posts already. I have an LED dual gooseneck with focusing lens and also matching pol filter, so can try the various approaches. With flash, you have to be careful re fluorescent color shifts due to diffusor material. Photocopier paper with UV brighteners imparts a strong blue cast. Nowadays with custom color balance, that is not too much of an issue anymore. Lee material should not have that problem.

Re purpose, those are flower pictures for a taxonomic monograph. So should be highly accurate representations of what the flower looks like, nothing arty, just the facts. Z-stacking will give a bit of hyper-realism, but that is OK. 3D aspect is important, another reason to rather not use flat epi-illumination, but a bit more sculpturing light from oblique sources.

Thanks again for all your wisdom! The stepper motor has been ordered. Some fun for thanksgiving.

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Post by rjlittlefield »

For capturing/representing 3D structure, consider whether stereo might be helpful. Zerene Stacker can construct good stereo from a single stack, when shot at high magnification and wide NA as in your application. See http://www.zerenesystems.com/cms/stacke ... eticstereo for starters.

--Rik

dlg
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Post by dlg »

rjlittlefield wrote:For capturing/representing 3D structure, consider whether stereo might be helpful. Zerene Stacker can construct good stereo from a single stack, when shot at high magnification and wide NA as in your application. See http://www.zerenesystems.com/cms/stacke ... eticstereo for starters.

--Rik
Sorry for being mis-leading, but I did not mean 3D stereo pairs, but 3D clues due to shadow/highlights due to lighting. I've never been able to see 3D images anyway, so that is completely lost on me.

Chris S.
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Post by Chris S. »

dlg wrote:Re finefocus interval, digital calipers certainly will get me into ball park. If they are actually calibrated to something sensible, I can also check with Zeiss sales rep.
If my experience is any guide, an inexpensive digital caliper can do more than get you into the ballpark—it can give you a measurement more trustworthy than the manufacturer’s specifications.

My digital caliper is an inexpensive (roughly $25) unit from Igaging. For precision, it reports to 1/100 millimeter--aka 10 microns. For repeatability, iGaging specifies about 13 microns—but I don’t know if they mean “plus or minus 13 microns” or “within a total range of 13 microns.”

Consider a case where we take the former, more conservative, interpretation, and round up to 30 microns, in keeping with the readout precision of the caliper.

I have quite a few microscope stages on hand. The finest increment of any of these is 100 microns of travel per turn of the fine focus knob. If I measure the total travel after 100 turns of the knob, I’d expect 10,000 microns (10mm). Across 10,000 microns of travel, a potential measurement error of 30 microns is trivial.

I’ve seen a couple of focus blocks that repeatably measured 10 percent less travel than specified by the manufacturer (Olympus, IIRC). Since my range of error is a tiny fraction of that, I’m inclined to trust my measurements, rather than manufacturer’s specs.

Measuring 100 turns of the fine focus knob is easier than it sounds. I put a fine strip of masking tape on the knob, and begin the measurement with the tape strip facing straight up. Then I crank the knob with one finger for 100 revolutions, ending with the tape—again—straight up. Takes only a couple of minutes.

--Chris

dlg
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Post by dlg »

Chris: thanks for the detailed explanations. Interesting note re error in manufacturers' data. That certainly calls for some ground truthing.

One thing I still have to figure out, but not that hard either, is what the nominal interval defined in the Zerene-Stack-Shot interface will generate one turn of the stacker motor. The tape method will tell me that as well.

Not sure whether Zerene-Stack-Shot can be re-calibrated for different gears from the stack-shot rail. Another bridge to jump off after I get there. Worst case scenario, I have to input dummy values. No big deal.

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Post by rjlittlefield »

dlg wrote:One thing I still have to figure out, but not that hard either, is what the nominal interval defined in the Zerene-Stack-Shot interface will generate one turn of the stacker motor. The tape method will tell me that as well.

Not sure whether Zerene-Stack-Shot can be re-calibrated for different gears from the stack-shot rail. Another bridge to jump off after I get there. Worst case scenario, I have to input dummy values.
There should be no need for dummy values.

On the Configuration tab of the ZS StackShot controller panel, there are parameters for "Millimeters per rotation of stepper motor" and "Microsteps per rotation".

Quoting the example given at http://www.zerenesystems.com/cms/stacke ... r_controls: if a 0.9° stepper motor were used to drive a microscope focus block at 100 microns per rotation, then the proper settings would be 0.100 millimeters per rotation, 6400 microsteps per rotation, and backlash distance 0.

Similar parameters are available in StackShot's own controller menus.

--Rik

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Post by ChrisR »

For multiple turns of fine focus knobs, micrometer handles etc, I clip on a plastic spring clamp or clothes peg. 4 turns per second is quick and easy to count.

WD of 4mm is possoble, affected considerably by the breadth of the rest of the metalwork around the lens. Some workers here use a 40x NA 0.65 "BD" objective which has a WD of 1mm, but once you've removed the outer shell there's just the very small tip to work around.
I have found that I have to diffuse even more than I ever thought necessary when doing that, otherwise anything like a highlight gives major problems.


Oh, and cross-eye stereo - just learn it! It's worth perseverance. The hard part is controlling the uncrossing, having overdone it. When you can do that slowly enough to approach the correct (small) amount of cross gradually, the 3D image "snaps" into view and "locks".

dlg
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Post by dlg »

Rik: thanks for the info on rotation calibration. Obviously, I had not RTFM.

Chris R: have to see whether I have enough clearance for a clip, or may have to move scope to the edge of the table.

Re WD, so I will give my existing 10x/0.3 with 2 mm WD a try.

Re diffusion, thanks for emphasizing that. will keep it in mind.

Re stereo, great that it works for you. It's not sufficiently important for me, and I don't think I an justify using all that printing space. There are a couple of other things I will rather put time into.

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