Which translation stage?

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NikonUser
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Post by NikonUser »

Rik wrote:
"An alternative approach that's worth considering is to do a DIY job and gear down the screw of your Proxxon table. If you can fit, say, a 5:1 planetary drive knob in place of that basic handwheel, then you're back to 0.2 mm per turn."

I think I uderstand what Rik was saying, so I increased the diameter of the Proxxon hand wheel from its original 34mm to 75mm. Now I can easily get the stage to move forward in 0.01mm increments; and I actually got it down to 0.005 mm increments.
Now the limiting factor seems to be choice of subject and stacking software.
Photos of a 4mm fly:
top photo: a crop from the full frame taken with MF 105mm reversed and fully extended on full bellows extension (22 cm)
center photo: a crop of the head from the full frame taken with a x10 Nikon microscope objective on 160mm extension; stack of 43 at 0.01 mm HF 4.1.
bottom photo: 800 x 800 pixels crop from the original stack (i.e., actual pixels); can see stacking problems at top of head and setae.
The Proxxon table appears to be an excellent product for image stacking especially considering its relatively low cost.

Image
Image
Image
NU.
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives

Harold Gough
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Post by Harold Gough »

I don't understand much of the technique but the first one is superb.

Are the others suffering slightly from the stack being shallower than some?

Harold
My images are a medium for sharing some of my experiences: they are not me.

Charles Krebs
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Post by Charles Krebs »

NikonUser,

It can be very "educational" (at least in the respect of understanding how/why it seems the software has "screwed up") to carefully look at the pertinent source files (at 1:1) for the regions where these "errors" occur. I do this regularly.

In many cases you will find that there is simply no source file where the area in question is shown in detail. For example, look at the upper picture... specifically the region where the front wing crosses the rear wing. There is a border that contains no rear wing detail running along the edge of the front wing. If you look at the source files I suspect there will be none where that detail can be found. As the front wing visually "expanded" as it went out of focus it blocked the view of that portion of the rear wing. This is a primary stacking difficulty. (Note that there is a difference between programs. TuFuse and CombineZ "pyramid" do a slightly better job with crossing hairs and certain edges like this one. But when there is no source file with the needed detail, then there is nothing any software can do).

Another common problem that occurs is when, due to the extreme shallow DOF, there is different detail that occurs at the same location in the picture at different depths. An example would be fine hairs that are seen in source images near the top of the stack that "disappear" in the final image because the program prefers to use the texture and detail that occurs in a surface behind it. (This is a case where the software could probably be improved, but it's a tricky problem).

If you haven't already, play around with different settings in HF. It can also be informative to run the stack in the mode where the screen updates as each image is processed (although it takes longer). This lets you see where and how some of these errors occur.

I still Like HF best overall, although as I said earlier, TuFuse and CombineZ seem to do slightly better in some respects with certain ("hairy" :wink: ) subjects. But when I look at the output 1:1 the results from HF seem to look more "naturally photographic", and it doesn't mess around with the contrast as much.

In most cases, retouching skills will be needed with any of this software if you really want to get "flawless" results.

rjlittlefield
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Post by rjlittlefield »

Nice work, NikonUser. :smt023

I see that Charlie has posted ahead of me. There's some overlap but also some differences in our comments.

1. In the top image, there is a large halo above and to the right of the foreground wing. The size of the halo shrinks toward the base of the wing, where the wings would be closer together, and I can't see any halo at all where the trailing edge of the wing aligns with the abdomen. This combination of effects suggests that the stacking movement was slightly skewed with respect to the optical axis. In this case, it looks like the fly essentially slid to the right and up as you focused backward, or vice versa if you focused forward. In either case, the bits of rear wing that should have lined up with the edge of the front wing were actually completely obscured by the front wing by the time the rear wing came into focus.

What Charlie describes is a similar problem caused by perspective effects, but I wouldn't expect that to be nearly so large with a stack shot using a 105mm lens.

You can tell the difference by loading the stack into software that treats the image sequence as a movie. Play the "movie" at several frames per second. If you see the subject just get bigger or smaller while staying centered, then you have the effect that Charlie described. If you see the subject drift left or right, you have misaligned axes.

2. The "partially transparent" spines visible in the third picture are a stacking error that is made by every software package I know of. What happens is that the software has no idea that those are opaque dark spines. It just shows whatever happens to have the most contrast at each pixel position. Edges of dark spines against a light background have very high contrast, so you end up seeing all the edges that the lens did. This problem gets worse at high magnifications because high-NA lenses are much more able to "look around" foreground features and see high contrast detail behind them. With some software, you can reduce bogus transparency at the cost of more halo, or vice versa, but I'm not aware of any software that does not have one or the other.

3. The last image (actual pixels crop) appears to show some alignment errors also. Those would be the apparent "steps" in certain bristles. Sometimes errors creep into the auto-alignment process when the software ends up trying to match details that don't really correspond. If your stacking gear is good about moving the subject in a straight line directly along the optical axis, then you might get a better result by turning off auto-alignment altogether.

--Rik

lauriek
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Post by lauriek »

With regards the 'crossing over detail at different levels' issue which I know we've all suffered from, I don't understand why this is hard to sort out, surely all it would take to fix would be a way to input which end of the stack is the front. Then the sw could simply use detail from the frontmost frame if it finds detail in two... I'm guessing there's a reason it's not that simple or it would have been sorted out by now, can anyone explain why that wouldn't work?

Does anyone else ever get tempted to 'snip' bits off of subjects half way through a stack to stop them obstructing details behind with a big OOF blob? ;)

Edited to add: Really nice work btw!

Edited again to add: I just re-read Rik's post an I think I understand the problem with the hairs, it's because there's no detail in the middle of the hair, the sw is looking for contrast steps...

Charles Krebs
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Post by Charles Krebs »

Laurie,

There is a piece of software that has some provision for giving greater emphasis of certain parts of the stack (i.e. top or bottom). Last I tried it I still preferred HF.

http://www.saphicon.com/extended-depth-field.htm

NikonUser
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Post by NikonUser »

Thanks Charles and Rik. I'll approach this 1 idea at a time:
1st
Charles wrote "As the front wing visually "expanded" as it went out of focus it blocked the view of that portion of the rear wing. "
This is exactly what happened. The distance between the, in focus, front wing to the, in focus, rear wing was 3 cm. In the last image of the stack the front wing is a large OOF area.
2nd
Rik wrote "You can tell the difference by loading the stack into software that treats the image sequence as a movie. Play the "movie" at several frames per second. If you see the subject just get bigger or smaller while staying centered, then you have the effect that Charlie described. If you see the subject drift left or right, you have misaligned axes."
I looked at the sequence of images in Adobe Bridge, not as a movie but as a very quick switch between frames. I could see no displacement of the fly between frames; the fly appears to stay centered and just increase in size .

The original stack was of 31 images at 0.1mm and this gave both wings in focus (top image); when I re-ran the stack with just the 1st 22 images the rear wing is OOF, the halo around the front wing is still there but is much less noticeable because of the OOF rear wing.

When photographing this type of subject I normally just have the front wing in focus. In flies with strongly contrasting wing veins the wing detail gets confusing when both sets of veins are in focus. This fly has weak veins and a wing pattern that doesn't overlap so I put both wings in focus. However you can see a shadow of the lower 'dot' of the rear wing showing through as a shadow on the front wing - poor judgement on my part.
NU.
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives

rjlittlefield
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Post by rjlittlefield »

NU, thanks for the further information. I still can't make the numbers work out to explain such a large halo just from perspective effects, but if you're confident there's no shift, perspective must be the problem.

BTW, I'm presuming the "3 cm" in your posting was a typo. 31 images at .1 mm would be 3 mm, which also makes good sense with a 4 mm fly.

In the unlikely event that perspective issues become really troublesome, one cure lies in telecentric optics. Drop me a note when/if the time comes, and I can say more about that.

--Rik

NikonUser
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Post by NikonUser »

rjlittlefield wrote: I still can't make the numbers work out to explain such a large halo just from perspective effects, but if you're confident there's no shift, perspective must be the problem.
--Rik
Yes, of course, 3mm.
Does the following help to confirm perspective effects rather than an alignment problem?
(I take images in this sequence, 1st is the front of the subject, I then move the subject towards the lens for subsequent shots.)
I opened the first and last images in Photoshop, pasted the last image into the 1st and made this last image 50% opacity and then flattened the image. Appears to me that they line up perfectly, note that the halo around the front wing is much less in this image.
Image
NU.
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives

rjlittlefield
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Post by rjlittlefield »

NikonUser wrote:
rjlittlefield wrote: I still can't make the numbers work out to explain such a large halo just from perspective effects, but if you're confident there's no shift, perspective must be the problem.
--Rik
Does the following help to confirm perspective effects rather than an alignment problem?
(I take images in this sequence, 1st is the front of the subject, I then move the subject towards the lens for subsequent shots.)
I opened the first and last images in Photoshop, pasted the last image into the 1st and made this last image 50% opacity and then flattened the image. Appears to me that they line up perfectly, note that the halo around the front wing is much less in this image.
Great test, and fascinating results!

I pulled your earlier HF image (from 9 posts above here) and your Photoshop'd image back into Photoshop so I could see them together and click between them. (To make viewing easier, I also did a very minor rescale and shift to get the two results registered against each other, presumably due to slightly different cropping in preparing them for posting.) Then I sat for a while, clicking back and forth and trying to make sense out of what I'm seeing.

This is a challenging problem.

I agree, the front and rear images appear to line up perfectly with the HF result, and there's a lot less halo in this image.

What this tells me is that none of the previous explanations are correct. Despite what I wrote, the halo is not coming from either shift or perspective.

I had been assuming, and I think Charlie had as well, that the haloed-out portions of rear wing were completely obscured by OOF front wing.

Obviously that's not true, since in your Photoshop merge we can see rear-wing detail clear to the edge of the front wing. No doubt the contrast of the rear wing detail is reduced because of the OOF front wing, but there's still plenty left to work with.

So the question becomes, why did the software choose not to show that rear-wing detail, in the vicinity of the front wing?

With Helicon Focus, big halo will sometimes happen if the "Radius" or "Smoothing" parameters are increased a lot beyond their default values of 8 and 4. But then I would expect to see halo in other places too, like where the base of the front wing occludes those bristles on top of the thorax.

So I'm confused. I can't recall seeing this combination of effects before, and I don't understand what's going on. That makes it a learning opportunity for me!

One other thing I notice is that the Photoshop merge seems to show a lot more fine detail than the HF result does. Take a look at the tips of the wings. In the Photoshop image, the fine serrations on the leading edges are sharply resolved, and there's a lot of fine-scale texture in the membrane, both in the clear regions and in the darker spots. In the HF image, the serrations are blurred and there's much less texture visible in the membrane. These differences may be hard to see in separated images, but they become obvious if you do the layer-and-click thing in Photoshop.

Maybe the difference in visible detail is due to JPEG compression. I notice that both files are about the same size. That would allow a higher JPEG quality in the Photoshop merge, since most of that image is OOF. But conceivably it's due instead to whatever process is generating the halo, and it would be nice to know that's not the case.

So, more questions...

What method and what parameter settings are you using?

And if you look at the actual pixels on your end, is the HF result losing detail compared to the Photoshop merge, or more importantly, compared to the original images?

--Rik

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