A second attempt at a focus stack of a pollen

Here you can discuss topics such as focus stacking, stitching and other techniques that relate to the processing of micrographs.
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gekko
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A second attempt at a focus stack of a pollen

#1 Post by gekko » Thu Apr 28, 2016 11:18 pm

Thanks to all the helpful folks how gave me valuable advice on focus stacking, I had the temerity to try it again and post the restuls below. This time the result was somewhat better, I think. Not great, but not as horrible as the first time. Again, thanks to all the help. [objective: 40x; only 6 images, CombineZP].

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mrsonchus
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Re: A second attempt at a focus stack of a pollen

#2 Post by mrsonchus » Thu Apr 28, 2016 11:46 pm

Looks like an improvement to me, the pores in the exine are just coming into view - there are three I thnk, and the spines are a little clearer.
You'll get there with practice - pollen is a bit of a swine to stack, especially 'fresh'. Have you tried an OH wash to remove some of the 'pollenkit' - the yellowish covering of gluey resin a lot of pollens have - it plays havoc with optics....
John B

einman
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Re: A second attempt at a focus stack of a pollen

#3 Post by einman » Fri Apr 29, 2016 12:35 am

A 40x objective typically has a dof of about 1 micron. How sensitive is your fine focus? Most of my scopes are divided into 2 micron divisions on the fine focus. What is the depth of the pollen particles? Take the depth of the specimen divided by the DOF of your objective and that will give you the approximate number of images in your stack.

If the pollen particle has a depth of 10 microns you would need 10 images at 1 micron intervals for maximum detail using a 40X objective.


You could take 20 images say at 0.5 micron intervals but it would simply be overkill and not result in better detail.

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Re: A second attempt at a focus stack of a pollen

#4 Post by mrsonchus » Fri Apr 29, 2016 4:21 am

Hi gekko, is it possible to show us 2 images, one of focus 'at the top' of the 'ridges' that cover the grain (i.e. the first good focal-plane that appears as the grain is moved 'up & towards the objective'), and the other of the equatorial-plane where the spiny exine 'halo' is in focus - this may give us an idea of the actual condition of your pollen-grains and their readiness to be imaged well?

There are good images lurking within. :)

These pictures may help to see the planes that may work well, although these have lots of contrast via staining - your fresh grains are far harder to stack than images such as these, but I've 'arrowed' the planes of focus I would try to achieve gekko old chap....
ws_pollen_focus_planes.jpg
ws_pollen_focus_planes.jpg (65.24 KiB) Viewed 4039 times
John B

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Re: A second attempt at a focus stack of a pollen

#5 Post by rnabholz » Fri Apr 29, 2016 7:44 pm

Hey Gekko,

I agree with John, more detail visible in this one, and the spines are showing nicely.

Rod

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Re: A second attempt at a focus stack of a pollen

#6 Post by JimT » Fri Apr 29, 2016 9:03 pm

So, you are getting better at this but looks like pollen doesn't lend itself too well to stacking.

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gekko
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Re: A second attempt at a focus stack of a pollen

#7 Post by gekko » Fri Apr 29, 2016 10:44 pm

Many thanks to einman, John B, Rod, and JimT for your comments and help. I think I have collected plenty of helpful information to try and use next time I find that a stack is totally unavoidable. John, I think I've had it with stacking pollen, especially that they may not be in good shape to begin with, but thank you for your continued helpful suggestions. I may post a couple of non-stacked pollen images if they turn out to be semi-usable just so I won't have wasted the pollen grains :) .
Again, thank you all for taking the time to advise me.

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mrsonchus
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Re: A second attempt at a focus stack of a pollen

#8 Post by mrsonchus » Fri Apr 29, 2016 10:49 pm

gekko wrote:Many thanks to einman, John B, Rod, and JimT for your comments and help. I think I have collected plenty of helpful information to try and use next time I find that a stack is totally unavoidable. John, I think I've had it with stacking pollen, especially that they may not be in good shape to begin with, but thank you for your continued helpful suggestions. I may post a couple of non-stacked pollen images if they turn out to be semi-usable just so I won't have wasted the pollen grains :) .
Again, thank you all for taking the time to advise me.
Well gekko, you've had a good go at them, and it's been interesting - onward to other things! There's no shortage of tiny subjects waiting to be explored! :D :)

p.s. You were a little unlucky too, the heavily ribbed grains of many similar Compositae/Asteraceae are just about the hardest to get a good stack from I think, even the 'easier' lily-type pollens are very difficult.... :)
John B

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