December 12, 2017, 10:50:00 PM

Author Topic: 5Dsr MP-E 65 z-stacking experiments.  (Read 16320 times)

Zeidora

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Re: 5Dsr MP-E 65 z-stacking experiments.
« Reply #30 on: November 18, 2017, 01:53:40 PM »
@zeidora: I seem to understand form your comments that you are proper microscopist but I don't want to bother other readers with technical stuff.
I am interested in monitoring crystals during solid-state reactions. In some case these occur by varying temperature, in other cases by exposing crystals to humidity, pressure, or different gasses including HCl and ammonia, through the use of special cells that I have designed. Now for some reason people don't want me to use HCl in their ESEM  :-X.
Being more serious most of these reactions produce a change of the crystal colour that electron microscopy cannot reveal.

Anyway if as you shouw above the lens resolution is the limiting factor then the test results of DoX mark suggest that I'm better off with a 6D or 5D and an extender than the 5DS(r).

Yep, no HCl in "my" SEM either! I only brought up SEM because you mentioned TEM. Still waiting for the color SEM as well.

Re 5D vs 5Ds[R], does not matter really. Either you use the 5DsR and get low mag, high MP image, or you use 5D ... and get higher mag, lower MP image. The structure will be shown at the same pixel dimensions at the edge of resolution, once you factor in absolute pixel size.

I gather you have a cell with a window, and you want to image your crystals through that window. I still think long WD epi compound objective is your best bet; for NA<0.3 you can also use regular 0.17-coverslip-objectives [Maybe you have seen this British guy who does z-stack-stitch gigapixel images of insects using compound microscope lenses on dSLR.] You will have to take into account magnification change due to the cell window. Same problem as with underwater photography and the window on the lens ports. You will have to include a scale bar with your crystal, or separately photograph a micrometer in your cell for calibration.

Illumination may be another problem depending on the design of your cell. You did not mention whether you do trans or epi illumination. I tried epi using compound-epi-pathway, but for 3D structures it resulted in too flat illumination. Now use dual goosenecks diffused with ping-pong balls and am very happy with results. With trans you have all the polarization options. Cross polarization with epi is another option and may help eliminating specular highlights on crystal facets.
5D2 full spectrum, 5DsR, a bunch of Zeiss (some other) primes, for documentary natural history, macro, and micro.

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Re: 5Dsr MP-E 65 z-stacking experiments.
« Reply #30 on: November 18, 2017, 01:53:40 PM »

Zeidora

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Re: 5Dsr MP-E 65 z-stacking experiments.
« Reply #31 on: November 18, 2017, 02:02:47 PM »
@Zeidora are you still using AP to finish off your images?
Yep, using AP exclusively. By now I am sufficiently used to AP that PS seems strange when I have to use it at work. DxO optics Pro for RAW conversion. My OS 10.10 is not compatible for the new DxO (Grrr!) but upgrading OS would make a bunch of other software incompatible (Word, FileMaker, QuarkXPress). Will wait for a year till the new Mac Pro comes out and then face the music.
Now I just have to learn Coda to ditch Dreamweaver, and I am Adobe-free! :-)
5D2 full spectrum, 5DsR, a bunch of Zeiss (some other) primes, for documentary natural history, macro, and micro.

sanj

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Re: 5Dsr MP-E 65 z-stacking experiments.
« Reply #32 on: November 30, 2017, 10:18:56 AM »
Thank you for this post.

Alejandro

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Re: 5Dsr MP-E 65 z-stacking experiments.
« Reply #33 on: December 01, 2017, 02:58:46 PM »
@zeidora: I seem to understand form your comments that you are proper microscopist but I don't want to bother other readers with technical stuff.
I am interested in monitoring crystals during solid-state reactions. In some case these occur by varying temperature, in other cases by exposing crystals to humidity, pressure, or different gasses including HCl and ammonia, through the use of special cells that I have designed. Now for some reason people don't want me to use HCl in their ESEM  :-X.
Being more serious most of these reactions produce a change of the crystal colour that electron microscopy cannot reveal.

Anyway if as you shouw above the lens resolution is the limiting factor then the test results of DoX mark suggest that I'm better off with a 6D or 5D and an extender than the 5DS(r).

Yep, no HCl in "my" SEM either! I only brought up SEM because you mentioned TEM. Still waiting for the color SEM as well.

Re 5D vs 5Ds[R], does not matter really. Either you use the 5DsR and get low mag, high MP image, or you use 5D ... and get higher mag, lower MP image. The structure will be shown at the same pixel dimensions at the edge of resolution, once you factor in absolute pixel size.


So, you're saying that in between a 5DsR and a 5D2/5D are the limits, ¿any camera in between (6D, 6D2, 5D4) would just balance the quality/diffraction/magnification?

Zeidora

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Re: 5Dsr MP-E 65 z-stacking experiments.
« Reply #34 on: December 01, 2017, 04:47:28 PM »
@zeidora: I seem to understand form your comments that you are proper microscopist but I don't want to bother other readers with technical stuff.
I am interested in monitoring crystals during solid-state reactions. In some case these occur by varying temperature, in other cases by exposing crystals to humidity, pressure, or different gasses including HCl and ammonia, through the use of special cells that I have designed. Now for some reason people don't want me to use HCl in their ESEM  :-X.
Being more serious most of these reactions produce a change of the crystal colour that electron microscopy cannot reveal.

Anyway if as you shouw above the lens resolution is the limiting factor then the test results of DoX mark suggest that I'm better off with a 6D or 5D and an extender than the 5DS(r).

Yep, no HCl in "my" SEM either! I only brought up SEM because you mentioned TEM. Still waiting for the color SEM as well.

Re 5D vs 5Ds[R], does not matter really. Either you use the 5DsR and get low mag, high MP image, or you use 5D ... and get higher mag, lower MP image. The structure will be shown at the same pixel dimensions at the edge of resolution, once you factor in absolute pixel size.


So, you're saying that in between a 5DsR and a 5D2/5D are the limits, ¿any camera in between (6D, 6D2, 5D4) would just balance the quality/diffraction/magnification?

precisely. Optical resolution is measured of how small an object structure (two points/lines separated between x µm) can be recognized as distinct. There is the contrast issue added in and that is what MTF tells you. Keeping object contrast the same, then either you can use a low overall magnification (say 4:1) on a high MP body with small pixels (say 5DsR), or you can use an overall higher magnification (say 7:1) on a lower MP body with larger pixels (say 5D2) to reach the resolution limit of the lens. Going above 4:1 on a 5DsR is futile, because you only blur circles over many pixels, but not additional information. That is what the beginning of this thread was all about.

With crop bodies, the small pixel size vs small sensor size sort of cancel each other out.

The above is not fully mathematically exact, but gets us into the ball park, and we can avoid doing futile exercises (e.g., going to 10:1 with MPE65 & 2xTC on 5DsR).

That is why I suggested microscope objectives for your 0.5 mm crystals to begin with.
5D2 full spectrum, 5DsR, a bunch of Zeiss (some other) primes, for documentary natural history, macro, and micro.

canon rumors FORUM

Re: 5Dsr MP-E 65 z-stacking experiments.
« Reply #34 on: December 01, 2017, 04:47:28 PM »