Pine Cone Scale Sections

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rnabholz
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Pine Cone Scale Sections

#1 Post by rnabholz » Sat Feb 13, 2016 4:05 am

Sections of the scale of a pine cone.

All images are stacks of between 7 to 20 images.

AO One Ten, 20x and 40x objectives, darkfield mask, afocal using a Nexus 6 phone.
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Last edited by rnabholz on Sat Feb 13, 2016 4:10 am, edited 1 time in total.

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Re: Pine Cone Scale Sections

#2 Post by rnabholz » Sat Feb 13, 2016 4:07 am

More
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Re: Pine Cone Scale Sections

#3 Post by gekko » Sat Feb 13, 2016 3:05 pm

Another very nice set of sections with good detail (patiently composed of lots and lots of images-- over 100?). Hypothetical question (not a request for an answer :) ): suppose you use every other or every third section, would you notice a deterioration in the stacked image? I wanted to try this, but my stacks are almost never good enough, and my eyesight is just as bad, so any conclusion I would draw would be highly questionable. Excellent darkfield even with the 40x objective (dry condenser?)
Was it hard to cut sections through the pine scale?

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Re: Pine Cone Scale Sections

#4 Post by JimT » Sat Feb 13, 2016 3:35 pm

Really nice. Some of them look like Hubble images.

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Re: Pine Cone Scale Sections

#5 Post by rnabholz » Sat Feb 13, 2016 8:17 pm

gekko wrote:Another very nice set of sections with good detail (patiently composed of lots and lots of images-- over 100?). Hypothetical question (not a request for an answer :) ): suppose you use every other or every third section, would you notice a deterioration in the stacked image? I wanted to try this, but my stacks are almost never good enough, and my eyesight is just as bad, so any conclusion I would draw would be highly questionable. Excellent darkfield even with the 40x objective (dry condenser?)
Was it hard to cut sections through the pine scale?
Hey Gekko,

Thanks for your questions.

Regarding the darkfield configuration, yes the condenser was dry.

I soaked the scale in water for a couple of hours before attmpting to cut it which softened it up nicely, and then cut it with my freshly sharpened microtome knife - worked like a charm.

I am not sure about the effect of reducing the number of files, I always assumed that the more the merrier, so lets see if we can find out together.

Below are two copies of the same scene. The original stack of 24 images is seen first below as it appeared above in the thread. The second is the same scene, processed from the same files except using only every other one, for a total of 12. I processed it using the same techniques and values for correction and enhancements that I used on the original. (Curves and levels adjustments may vary ever so slightly, but not materially)

What do you think??
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Re: Pine Cone Scale Sections

#6 Post by rnabholz » Sat Feb 13, 2016 8:20 pm

JimT wrote:Really nice. Some of them look like Hubble images.
Thank you Jim, I agree about the space like quality, especially the second one above. The "stars" must have been material from the pine scale, perhaps sap. The effect was really cool live as the particles would "twinkle" as the rotated or moved slightly.

Thanks

Rod

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Re: Pine Cone Scale Sections

#7 Post by gekko » Sun Feb 14, 2016 1:45 pm

rnabholz wrote:
gekko wrote:Another very nice set of sections with good detail (patiently composed of lots and lots of images-- over 100?). Hypothetical question (not a request for an answer :) ): suppose you use every other or every third section, would you notice a deterioration in the stacked image? I wanted to try this, but my stacks are almost never good enough, and my eyesight is just as bad, so any conclusion I would draw would be highly questionable. Excellent darkfield even with the 40x objective (dry condenser?)
Was it hard to cut sections through the pine scale?
Hey Gekko,
Thanks for your questions.
Regarding the darkfield configuration, yes the condenser was dry.
I soaked the scale in water for a couple of hours before attmpting to cut it which softened it up nicely, and then cut it with my freshly sharpened microtome knife - worked like a charm.
I am not sure about the effect of reducing the number of files, I always assumed that the more the merrier, so lets see if we can find out together.
Below are two copies of the same scene. The original stack of 24 images is seen first below as it appeared above in the thread. The second is the same scene, processed from the same files except using only every other one, for a total of 12. I processed it using the same techniques and values for correction and enhancements that I used on the original. (Curves and levels adjustments may vary ever so slightly, but not materially)
What do you think??
Many thanks, Rod! I wasn't expecting that you answer my question so quickly! Well, the fact that I cannot see a difference doesn't mean at all that the difference is not very clearly visible to you or others, so what do you think? Many thanks!!!

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Re: Pine Cone Scale Sections

#8 Post by rnabholz » Sun Feb 14, 2016 3:31 pm

You are welcome, the file count question is one I wondered about as well.

I can see a small difference. If you look at the upper black area and look closely at the white dots of particles there, I think I can see more of them in the first image that uses all of the files. Not a huge difference, but there does seem to be more information presented.

Assuming the same thing is happening all across the image, you can make a case that there is an advantage to using more files.

Whether that small difference is worth the extra capture time, processing time and storage space is another matter. Certainly it might be overkill for presentation in an online forum with 1024 pixel size limits, but if you intend to print in large sizes, would be something that I would invest the time and effort in.

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Re: Pine Cone Scale Sections

#9 Post by KurtM » Sun Feb 14, 2016 3:42 pm

Beautiful images! And interesting discussion - I have been experimenting with image stacks on diatoms, and have run a series or 3 image stacks and 6 image stacks, and seem to consistently prefer the 3 image stack results.

Rod, you're using Combine ZP aren't you?
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Re: Pine Cone Scale Sections

#10 Post by rnabholz » Sun Feb 14, 2016 5:34 pm

KurtM wrote:Beautiful images! And interesting discussion - I have been experimenting with image stacks on diatoms, and have run a series or 3 image stacks and 6 image stacks, and seem to consistently prefer the 3 image stack results.

Rod, you're using Combine ZP aren't you?
I am using Combine ZP.

I can't say that I have compared different stack counts before, so I don't have a preference that way. I can say that I do always run all the methods and do see a big difference in those, and not always in predictable ways. I don't think I could ever be comfortable just running one method a thinking that I got the best possible output.

I am also curious about everyone's process. When I intend to shoot a stack, I do a "dry run" to figure out where the top and bottom focus points are how many focus changes I need to cover that range, and then shoot it. I do generally find that I end up with a couple of extra frames which I attribute to being a bit conservative in the size of each focus adjustment causing me to need more changes to cover the range I decided on.

Obviously, depending on their size and depth, some subjects require many more frames than others, and I have shot images using as few as 3 and as many as 50 frames, both yielding good results.

How do all of you approach the process?

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Re: Pine Cone Scale Sections

#11 Post by mrsonchus » Sun Feb 14, 2016 8:20 pm

The biggest obstacle I encounter with the stacking of images of the essentially-transparent and at the very least translucent sections that I prepare is the problem of varying points of focus that exist at the same locations on a slide, that is to say multiple focusable 'layers' exist at each point, unlike with a solid and opaque object such as a fresh leaf say.

Pollen-grains are a good example, they have a variety of different focus-levels that will all give a potentially radically different view of the grain, leading to the need to standardize the focal plane used when assessing and sharing pollen images for identification or study etc. The most often used focal-plane used with pollen is the 'equatorial' plane that axists mid-way through the depth of the grain, another being the 'polar' plane at the very top of a grain as it is focused upon....

The main criterion I therefore try to satisfy is that I have only single points at any depth of a stacked images that have been used as 'in-focus' contributors to the stacked product. I rarely succeed here and almost always produce rather poor stacks from my slides I'm afraid. :cry:

I'd like a stacking algorithm to prevent the overlay of points in the Z-plane when constructing an image, of course it may be necessary to present a set of images to the process in Z-order, but that's no problem and quite a natural thing to do.

Personally I am usually less than happy with my stacking attempts I'm afraid. :)
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Re: Pine Cone Scale Sections

#12 Post by gekko » Mon Feb 15, 2016 12:42 am

John, I'm with you. I almost never succeed in getting statisfying stacks, hence I usually post single shots at the selected plane of focus. My understanding of focus stacks, is precisely that they do render the image with all levels of the object in focus as though your object is made of glass and everything is in focus at all levels and the result projected on the plane of the image (I would appreciate being corrected if, as is likely, I misunderstand the subject). Yet, as Rod said, if I use "all methods" in CombineZP, I get 6 different results, and, at least in my hands, more often than not, none of them is really satisfying (I have to hasten and say that this only applies to my use of focus stacks-- everyone else here posts exceptionally beautiful and excellent stacked images that I would be proud to be able to emulate), so in my case it is my incompetence at work. In contrast, in John's case I think that sections are a very different kind of animal, and one only needs to show one plane of focus, and even with that, use a fairly large aperture in order to limit the depth of field, which is the exact opposite of what focus stacking is meant to achieve. At least that is my understanding.

In terms of number and spacing of images for focus stacks, someone on the forum (sorry--I don't remember who) suggested the very rational idea of using the depth of field of the microscope to determine this. As an example, using a 40x/0.65 objective and a condenser set to about 80% of the objective's aperture resulting in roughly 0.6 NA for the microscope, the depth of field would be about 1.5 µm (see http://www.microscopyu.com/articles/for ... depth.html and notice that the ordinate scale is logarithmic). So one can focus on the top surface, and go down by increments of about 1.5 µm until the bottom surface is reached, however many images that comes to. For a 10x/0.25, the depth of field would be about 30 µm.

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Re: Pine Cone Scale Sections

#13 Post by KurtM » Mon Feb 15, 2016 1:25 am

Great discussion! I believe the results of stacking have a lot to do with the subject matter. I took several stabs at producing a decent stack of cotton pollen today, and can't say I'm thrilled with any of them. Some I deleted right away. Yes, I use All Methods too, seems like it'd be silly not to hedge your bet, given the opportunity.

Here's my method, such as it is: I do dry runs too; first get a good idea of where top and bottom occur, in this case from the tip of the polar spines to the equatorial spines. Then practice running through the stack-image-gathering sequence several times first to decide how few increments I can get away with (again, depends on the specimen); I figure there's a balance to strike between fewest images to minimize gremlins, and many slices to maximize resolution.

On these pollen grains, I tried doing 8 image stacks, and 16 image stacks, and the 16'ers came out so awful I deleted them right away. Had I known there was to be this discussion, I'd've kept 'em and showed you. But lacking that possibility, I'll go ahead and post what I consider to be the least offensive of the bunch. Again, I wasn't happy with any of them, and didn't intend to post any of today's pictures. This image certainly doesn't do the eyepiece view justice, where the spines all 'round make 'er look like the world's most dangerous beach ball! And it ain't a bad slide either, made last spring using glycerin jelly. I'm very enthusiastic about pollen, and need to learn to make better slides.

Anyway ... once I establish what I want to do and practice doing it a few times, then it's a matter of watching each step on the monitor since I operate my camera by laptop through remote software and USB cable, but mostly moving the fine focus knob by feel in between start and stop. Hope this makes sense...?
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Re: Pine Cone Scale Sections

#14 Post by gekko » Mon Feb 15, 2016 2:10 am

Thank you, Kurt: that is very interesting. In my view your and Rod's and others' pragmatic approach is best, as the actual math used to design the stacking algorithms is bound to be complicated and way beyond my understanding, and I think for a really effective use of those programs, one needs to understand at least the basics of those algorithms (but maybe I'm wrong, as is often the case), at least to effectively set the many parameters that they provide, which I never use-- I just use the default values, and that likely is the cause of the poor results I get.

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Re: Pine Cone Scale Sections

#15 Post by rnabholz » Mon Feb 15, 2016 2:05 pm

Tov the degree that I have had success using stacking it needs to be entirely credited to the software's author, as I use it in complete autopilot mode.

The only step that I would consider outside that statement is that on large stacks I do use the more thorough alignment routine.

I do agree with Kurt and Gekko that some subjects do lend themselves better to stacking, and would as that magnification and lighting also have their effect.

John's incredibly thin sections, as gekko suggests may not be the easiest for the software to handle. High contrast lighting and high magnifications create halos that I also believe confuse the algorithms. Perhaps that explains difficulties stacking DF, DIC, Phase, etc.

I would like to hear from those using other stacking programs to see if they face the same challenges. While I like "free" as much as the next guy, I would be willing to pay for a product that is demonstrably better.

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Re: Pine Cone Scale Sections

#16 Post by gekko » Mon Feb 15, 2016 2:21 pm

rnabholz wrote:While I like "free" as much as the next guy, I would be willing to pay for a product that is demonstrably better.
I would suggest that you download a free trial copy of Zerene Stacker ( http://zerenesystems.com/cms/stacker ) and try it out. It has an excellent reputation. If you find it suitable, you can then buy it.

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Re: Pine Cone Scale Sections

#17 Post by KurtM » Mon Feb 15, 2016 4:28 pm

rnabholz wrote:While I like "free" as much as the next guy, I would be willing to pay for a product that is demonstrably better.
Yeah, this is something I need to answer for myself too, one of these days. But I'm in no mood to think about spending money on anything right at the moment.
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Re: Pine Cone Scale Sections

#18 Post by gekko » Mon Feb 15, 2016 8:48 pm

I have recently taken multiple images for focus stacks of several testate amoeba shells, but I never get results even 1/10th as good as, for example, 75RR's recent absolutely superb images. And single images are not useful there. Perhaps if I knew how to set the program's parmeters I would get acceptable images (but I'm just too impatient to even try to learn how to use the program: obviously not the program's fault, but purely mine).

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Re: Pine Cone Scale Sections

#19 Post by KurtM » Tue Feb 16, 2016 1:12 am

Well, if that's true, at least you're not the only impatient microscopist cluttering up the home labs of the world. I just made several stacks of a really cool testate amoeba today and don't like any of them. And just as you say, single images are pretty useless. Maybe we need to get 75RR to give a seminar (webinar).
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Re: Pine Cone Scale Sections

#20 Post by gekko » Tue Feb 16, 2016 1:17 am

KurtM wrote:Maybe we need to get 75RR to give a seminar (webinar).
I'll vote for that :)

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Re: Pine Cone Scale Sections

#21 Post by zzffnn » Tue Feb 16, 2016 1:52 am

I 3rd that. I am impressed with his high magnification stack of that amoeba test.

But 75RR uses Photoshop for stacking though, if I remember correctly, which may work differently from CombineZP.

I opened a new thread for discussing stacking:
viewtopic.php?f=27&t=2500

And I have also PM'ed 75RR and asked for his comments.
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