5Dsr MP-E 65 z-stacking experiments.

[Mt Spokane Photography]

I guess we will have to see what Neuro can produce with his conformal microscope

 

[Zeidora]

I guess we will have to see what Neuro can produce with his conformal microscope

Ah, dam you autocorrect! ;-) Confocal works with laser excited fluorescent markers, generally of translucent objects. I am unaware of epic-confocal microscopy. I guess you could extract the uppermost layer with computer processing of images, but that obviates the entire point of confocal.
Both SLR imaging and SEM are for surface structures, so completely different imaging approaches. For SLR, the object is generally completely untreated and un-stained. For SEM the specimen is often pre-treated (e.g., critical point drying of flowers), sometimes fixed/stained (e.g. with nasty osmium tetroxide, haven't done that in a long time), and generally also sputter coated with precious metals, mostly gold and/or palladium. I work quite a bit with uncoated material in variable pressure.

 

[Boromir883]

Zeidora,
-as a person with a degree in biotechnology (but working as quality Manager in food industry) i like reading about your working technics

 

[Zeidora]

Zeidora,
-as a person with a degree in biotechnology (but working as quality Manager in food industry) i like reading about your working technics

Thanks! Just in case you're interested, I wrote a couple of articles on imaging small flowers and z-stacking, and also a book chapter on scientific photography of mollusks. If interested, PM me and I send you links.

 

[malus]

sorry for reopening a old post.
I am considering using the mp-e to take high resolution images of small (0.1 to 1 mm) crystals and I am wondering if Moiere interference can be an issue with the 5dsr. Does anyone have experience with that? Is the 5ds a better option?

 

[Zeidora]

sorry for reopening a old post.
I am considering using the mp-e to take high resolution images of small (0.1 to 1 mm) crystals and I am wondering if Moiere interference can be an issue with the 5dsr. Does anyone have experience with that? Is the 5ds a better option?

If you want significantly <1 mm full frame where every pixel contains information (not glorified part of a blur circle), you want to go rather on a compound microscope with high NA epi objectives. Consider that on 5DsR with MP-E65 at f/2.8 maximum mag where you get info out on 5DsR is 4:1. Even at 5:1 you just get blur circles. 4:1 means field of view is 4x6 mm, so a 0.5 mm structure will be rather small. If the pixel dimensions are sufficient for your purposes, then that's fine, but otherwise go compound. If that is something you are interested, reply and I will provide details.

Hope that helps.

 

[malus]

I should have mentioned that I already have access to all sort of microscopic equipment: from magnifier lenses to state of the art TEMs.
For this project I need some space to access the crystals during the shots. Unfortunately compound microscopes do not have enough working distance for my need. The stereomicroscope I currently use has very bad glasses and ccd. A new one with some decent specs cost much more than a camera, which I could also use for other purposes.
I agree that it is a waste of money/time if I can't get some decent pictures.

 

[Zeidora]

I should have mentioned that I already have access to all sort of microscopic equipment: from magnifier lenses to state of the art TEMs.
For this project I need some space to access the crystals during the shots. Unfortunately compound microscopes do not have enough working distance for my need. The stereomicroscope I currently use has very bad glasses and ccd. A new one with some decent specs cost much more than a camera, which I could also use for other purposes.
I agree that it is a waste of money/time if I can't get some decent pictures.

How much do you need? is the problem bulk of specimen and limited stage travel, or are your specimens very long needles? There are long WD objectives, usually with a bit lower NA. Alternatively, use compound lens on photographic equipment. If you have older 160 mm tube length lenses, then use them on bellows. If you have more modern ICS objectives, then you need a relay lens. Then specimen bulk is of no concern.

If you also have access to VP or E SEM and chamber is sufficiently large, then you can go un-coated in VP or E, use long WD to get as much DOF as possible, and shoot at high probe current. 0.1 mm is pretty low mag on SEM, so you could go 500-1000 pA. You can even z-stack SEM images. Haven't done that myself, but know of people who have done that.

 

[zim]

Definitely a thread which deserves to bubble back to the surface, inspiring stuff, fantastic when such technical info is shared too.

@Zeidora are you still using AP to finish off your images?

 

[malus]

@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).

 

[Zeidora]

@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.

 

[Zeidora]

@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!

 

[sanj]

Thank you for this post.

 

[Alejandro]

@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]

@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.