Two small contributions of new member from southern bavaria

[michael_r]

Hi, this is my first contribution to this wonderful forum. My interest in photography lasts back for about four decades when (in school) i seriously tried to make good pictures in black and white with an own darkroom that fed up most of my pocket-money for years. Well i was honest enough to realize, that my pictures where medium class at best and so i decided not to become a professional photographer and went to university studying physics. I worked for the semiconductor industry for a couple of years and after that became professor about twenty years ago. I never stopped taking pictures, but my photographic activities had a real comeback after photokina 2008 when i bought one of those new full format dslrs and had a lot of fun to explore all these additional possibilities the digital workflow provides. From a book on multishot techniques i learned about photostacking and started my first experiments with helicon focus. The „twin rose“ (that i found in my garden last summer) was my first stacking result.



From the helicon focus page it was not far to krebsmicro.com, where i found an interesting article on the use of microscope objectives in photography. It was fascinating to see that this approach allows to produce pictures with a depth of field that required the use of a scanning electron microscope before. I wanted to be able to do this and skipped my plans of buying a long telephoto lens (would have been to heavy anyway) in favour of a stackshot, a zerene pro edition and Mitutoyo glass. And after some experiments with my setup i‘d like to report two ideas resp. observations.

My setup is rather conventional. I keep the camera fixed (eos 550d) and move the object with a stackshot. My present approach is to use the rear block of a sinar p2 as an xyz stage with additional possibility for two axis rotations on the stackshot (only rotation with respect to the optical axis is not possible). Such stages sell used typically for a little more than 200 € on ebay. I post a picture as a small contribution to the long list of ideas presented in this forum.



Another possibly interesting observation is that my old pentax 6x7 200mm lens works pretty well as a tube lens for my infinity microscope objectives (i tried a nikon epi plan 20x/0.35 slwd, a mitutoyo m plan apo 5x/0.14, a mitutoyo m plan apo 20x/0.42 and a mitutoyo m plan apo 50x/0.55 with this tube lens and think that the result is promising). With the results of the 200mm i also tried the 300 mm/4 as a tube lens for the mtty 20x. Here is a 100 % crop of a picture of the „standard butterfly“ taken with that configuration (i didn‘t take a „full stack“ as this was primarily for lens testing, and this is camera jpeg stacked with pmax and nothing else) - is it possible to improve on this resolution? With the 200mm and the 50x i got pictures with somewhat less resolution at a reduced field size. Well this is possibly due to weakness of my setup - the stackhot is a nice device but it is not designed for steps in the submicron range, required for that kind of stacking. There‘s still a way to go.


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[johnsankey]

The StackShot rail is totally reliable at 10 um steps, but has issues below that both with irregular steps and vibration. For magnification higher than about 10x, I suggest you'll get better results by auto-focusing a microscope rather than using a StackShot rail. I suspect vibration with your present system. You can buy their stepping motor separately to hook up to a microscope fine focus for use with your StackShot controller - several people have.

[michael_r]

Thanks for the advice. I already ordered a newport stage and an extra stepper motor from cognisys to work towards a more reliable translation stage for small increments. I do however not believe that vibration is a problem with my setup (the picture is probably a little misleading as i mounted the stackshot on a tripod for illustration) as i spent some effort on vibrational decoupling, and

1. The 100 % liveview test was run sucessfully

2. I use flash for lighting

Kind regards

Michael

[johnsankey]

Another option to simplify the optics and reduce the amount of glass in the optical path is to use the objective directly:
http://www.photomacrography.net/forum/v ... hp?t=12147
The correction of an infinity-corrected objective when used finite at ca. 200mm spacing from the sensor is very close to optimal. You may find that you get better results this way when your new stage arrives. I'm just waiting for the cone adaptor to arrive to use the method with 5x and 10x objectives.

[rjlittlefield]

Michael, welcome aboard! Your setup and images look good. Best resolution normally comes from the largest NA objective, so you'd probably be better off using that 50x/0.55 instead of pushing the 20/0.42 with a longer tube lens. Even at 200 mm, the effective aperture of the 20/0.42 is about f/24 (=20/(2*0.42)), and when you push it to 300 mm and 30X, it goes to f/36. You can see that effective f/36 on an APS-sized sensor will not be very sharp when viewed at actual pixels.

The sharpest images I've seen of unmounted butterfly scales come from a 100X/0.80 objective. Some of them are shown HERE, using a Nikon M Plan ELWD at 2 mm working distance. The corresponding Mitutoyo's have much more WD but smaller apertures. I have also seen one report showing good results from an Microscopes India lens, their LWD Long Working M PLAN 100x DRY Material science Microscope Objective. I do not have personal experience with any of these objectives.

The correction of an infinity-corrected objective when used finite at ca. 200mm spacing from the sensor is very close to optimal.

John, that trick will work OK for low-NA objectives like the 5X and 10X that you mention. But it would be a disaster for larger NA objectives like the 50x/0.55. The sensitivity to "tube length variation" goes as NA to the fourth, so an objective at NA 0.55 is roughly 23 times more picky than one at 0.25, and over 900 times more picky than one at 0.10. Even at 10X and NA 0.25, there's noticeable degradation from omitting the tube lens and running finite. Microscope manufacturers have a reason for including those tube lenses.

--Rik

[michael_r]

Rik, thanks for the comments. I'm aware that the picture posted suffers from diffraction. The crop comes from a sensor area about 4 to 5 mm wide and is displayed on my screen about 27 cm wide, so magnification from sensor to screen is something like 60x. Combined with the 30x magnification from object to sensor total magnification of the displayed image is something like 1800x: too much for a tack sharp image with na 0.42.

The choice of an objective almost inevitably requires some sort of compromise. I choose the Mitutoyo because of long WD sacrificing some NA and therefore useful magnification. I hope to find out if there is someone in this forum who got better results with another tube lens?

Michael

P.S.: I highly appreciate your recent discussion of magnification and pixel size (really great hands-on experiments), which is somewhat related to this point. If i need three pixels to resolve on the sensor what my objective can resolve in the object plane, and if my objective can resolve 0.7 um in the object plane, the maximum useful magnification should be some 14 - 15 um divided by 0.7 um, i.e. about 20x if pixel size is somewhat below 5 um.

[rjlittlefield]

I hope to find out if there is someone in this forum who got better results with another tube lens?

Chris S. has reported getting better results with Mitutoyo's own tube lens, versus a particular Nikon and especially with polarizer added in the infinite space between objective and tube lens. See http://www.photomacrography.net/forum/v ... hp?t=14494. Unfortunately Chris is incommunicado right now (until March 19, said the autoreply).

And I believe that Charles Krebs has reported slightly better performance from a true Nikon tube lens with Nikon objectives, versus using a telephoto as tube lens. I don't have the reference for that handy, and as I recall the effect was pretty small.

General consensus is that any long lens that works well by itself at infinity focus will also work well as a tube lens, assuming that the pupils are sized and placed so that vignetting does not occur. The standard explanation for that good behavior is the small effective aperture. Even 10X NA 0.25 means that the tube lens is only working at f/20. In the early days of playing with infinites on telephotos, we were concerned about degradation due to the aperture placement causing unusual bundles of rays to be accepted for off-axis points. (See HERE and the surrounding thread for discussion of the issue.) But experience indicates that that effect is actually not a big problem.

--Rik

Edit: typo and formatting

[nernelly]

Michael, my experiences in this magnification range are rather limited, but I do know that iridescent scales can be quite troublesome at higher magnifications (probably, because it gets very hard to avoid specular reflections). So, if you are still optimizing your setup you might consider using an easier subject while you're in the testing phase (e.g. non iridescent scales).

Best regards, Steffen

[Craig Gerard]

Michael,

From my understanding you are controlling the StackShot via the Zerene Stacker Pro interface? If not, be sure to turn 'High Precision Mode' ON via the StackShot controller when moving the rail in steps smaller than 10μm. The stepper motor makes a high-pitch noise when 'high precison mode' is 'ON' (most annoying if you have sonar ears). Also have a look at the Options>Preferences settings in Zerene; there are times when many of the default paremeters can be safely 'tweaked', that's why Rik made them...'tweakable'.


Chris S. is off dancing with wolves, so I'll grab a quote from the post Rik mentioned and add some additional information.

Something you might or might not care about: A big difference I found between the 200mm Nikon micro and the Mitutoyo tube lens is that with the micro, image quality deteriorated substantially when I added a polarizer. This was likely because the addition placed the objective a bit farther from the decollimating lens, sending more light through outer, less corrected portions of the decollimating lens. The Mitutoyo tube lens does not exhibit this degradation, which is very important in my use. I suspect that your 200 AI will be better in this regard than the 200 micro. But if you have any interest in using a polarizer or placing an iris behind your objective, you might want to check this.

The "placing an iris behind your objective" is an important consideration, as too are other inclusions such as a polariser which led me to use a dedicated tube lens.

The dedicated Nikon tube lens (Part# MXA20696 '2nd objective Lens') is excellent when using either Nikon or Mitutoyo ∞ objectives. Charlie did mention in an earlier thread the Nikon tube lense is slightly telephoto, so although it is rated as a 200mm lens, an appropriate distance from the image plane is 150mm.

http://www.edmundoptics.com/images/catalog/7257.gif

The Mitutoyo tube lens MT-1 is considerably more expensive and more particular about its placement in the optics chain. More information is available via the 'Technical Images' tab on the 'Accessory Tube Lenses' page on Edmund's website.
http://www.edmundoptics.com/products/di ... uctid=2230


Craig

[rjlittlefield]

The Mitutoyo tube lens MT-1 is considerably more expensive and more particular about its placement in the optics chain. More information is available via the 'Technical Images' tab on the 'Accessory Tube Lenses' page on Edmund's website.

The Mitutoyo is definitely more expensive, and more information is published about its designed placement. But I don't recall anyone showing that it's actually more particular than the Nikon about where it's placed. Do you have a reference for that?

But [leaving out the tube lens] would be a disaster for larger NA objectives like the 50x/0.55.

Since I had a suitable subject already mounted up, I thought I'd take a few minutes and illustrate "disaster". The first two panels are using the Nikon CF Plan 50X NA 0.55 inf/0 EPI ELWD (8.7 mm) on empty extension and on an ancient Vivitar 200 mm telephoto that I've previously found works well. Both are PMax, untouched except for resizing to 50%.



The third panel is the result of taking the first panel and applying a heavy dose of unsharp masking, Photoshop's 145% at 4.4 pixels as shown here. I think it's interesting that most of the ill effects of the spherical aberration that's introduced by improper conjugates can be removed by such a simple digital filter.

--Rik

[Craig Gerard]

The Mitutoyo is definitely more expensive, and more information is published about its designed placement. But I don't recall anyone showing that it's actually more particular than the Nikon about where it's placed. Do you have a reference for that?

Rik, short answer regarding a specific reference or actual experience is 'no'; just an assumption on my part, although the various tube lens diagrams on Edmunds do indicate some differences. I've not had the opportunity to play with a regular Mitutoyo tube lens (although there was one of some description incorporated into this piece). Mitutoyo tube lenses rarely appear on the secondhand market, whilst Mitutoyo objectives are not so rare.

I do recall a somewhat related discussion:
http://www.photomacrography.net/forum/v ... hp?p=77168
and an extension to that discussion:
http://www.photomacrography.net/forum/v ... hp?t=12267

Regarding the use of inifinity-corrected objectives orphaned from a tube lens, we need to remember (at least I think we do) that the tube lens or 2nd objective lens is actually part of the objective, though separate, it's part of the overall equation; a somewhat similar relationship exists between cover glass and high NA (biological) objectives.

Michael,

When you have a project open in Zerene Stacker do you see much variation in the position of the subject when scrolling through the individual frames before and after alignment (with 'Show as Adjusted' checked)?
Do you need to crop much around the edges of the end result?


Craig

[rjlittlefield]

With the 200mm and the 50x i got pictures with somewhat less resolution at a reduced field size. Well this is possibly due to weakness of my setup - the stackhot is a nice device but it is not designed for steps in the submicron range, required for that kind of stacking.

True, but the limitations are not as severe as sometimes thought.

For my own StackShot unit, I have measured and written as follows (at http://www.photomacrography.net/forum/v ... 1767#71767)

If you set units to "steps" (actually microsteps), then it is possible to set an interval of one microstep = 0.496 µm. However, if you do that [then without High Precision] you'll discover that the resulting physical movements are not uniform, with physical steps occasionally being up to about 10X larger than the average step.

The recent modifications [to enable High Precision] do not involve changes to the rail itself. Rather they are modifications to the controller that allow it to make the rail move with much more uniform microsteps. The movements are still not perfectly uniform, but they are now good enough to limit the maximum physical step to something like 0.0013 mm = 1.3 µm, at least in the unit that I have. In the Noctua pronuba stack, I used 2 microsteps per focus interval, which (based on measurements made separately) resulted in actual focus intervals varying from roughly 0.6 to 1.9 µm.

Consulting the DOF calculator at http://www.microscopyu.com/tutorials/ja ... index.html, we see that 2 µm DOF is sufficient to cover everything up to the 50X NA 0.55 objective. Setting 1 µm to get a maximum of 2 µm is not optimal, of course, but it does get the job done. You can easily check whether there was a problem with focus step by comparing adjacent frames at 100% in Zerene Stacker.

--Rik

[Craig Gerard]

Here's the spooky bit.

Edmund Optics list the depth of focus at 0.9μm for the Mitutoyo M Plan Apo 50X 0.55 and the Resolving Power at 0.5μm.

Using those figures I could easily be inclined to shoot at 0.5μm when focus stacking with the aforementioned objective.

What is the relevance of the two terms depth of field and depth of focus and what is their relationship to oneanother?

Could someone put this into a bite size nutshell?

http://en.wikipedia.org/wiki/Depth_of_focus



Craig

[johnsankey]

Microscopists are much more generous in their acceptance of blurry images than most photographers are - all they have to do is identify things whereas we want them to look sharp. DoF very much depends on who is judging it. However, DoFocus and DoField are essentially the same concept.

Edmund's DoF of 0.9um for the 50x/0.55 corresponds to an 800px wide image, lower resolution than we photographers normally consider sharp.

[ChrisR]

the relevance of the two terms depth of field and depth of focus

For 40 odd years I've only noticed the former to mean subject side, and the latter, at the film/sensor.
More recently there seems to be looser application , as suggested in the Wikipedia entry.
All too often when there's a calculation, an outdated figure for Circle of Confusion lurks behind. . Perhaps when the next Canon comes out with 48Mpixels a few more equations will be revisited?