# 5Dsr MP-E 65 z-stacking experiments.



## Zeidora (Jul 13, 2015)

I've been playing around with the new 5D sr to see how it behaves in the macro area. I shot some small orchid flowers, total height 2 mm each, with MPE 65 mm at 1:1, 2:1, 3:1, 4:1, 5:1 and with TC 1.4xIII at 7:1. Each set at f/2.8 and f/4: all open for maximum resolution (here meant as the traditional separating two points in object), and 1 stop down for possible aberration correction and overall improved IQ. Between 60 and 175 frames were shot per set-up. Flashed manually with MT-24Ex at around 1/64-1/32 power, so very short exposure times. Flash heads mounted off lens to ensure consistent illumination angles. Stacking done on Cogynsis Stackshot with steps down to 19 µm. Stacking steps calculated as 70% of DoF with c = 0.03 mm in 8 x 10" print.

Processing on 6 core MacPro soup can with 32 GB RAM. Mac OS Yosemite does not reliably display thumbnails of CR2 file icons in finder. No idea why. RAW files were run through DxO (latest download). In previous DxO you could double click one image in directory, and all images in that directory would load, but this leads to many errors and hang-ups with 5Ds files and latest DxO version. Drag-drop images into the DxO interface is 100% reliable. It takes time to process >100 files, but can be done on MacPro. Activity monitor had all processors going full throttle for several minutes, with quite a bit of heat coming out of the vent. I would not advise doing that on a laptop.

In previous tests with 5D2 files, RAW file processing was quicker in DxO than in PS CS5.5 extended with batch processing. 

Stacking in Zerene with 300 MB 16 bit .tif files was flawless. For me the P-max algorithm works best.

With the huge file sizes and small pixels, the question arises at what magnification is nothing gained anymore in terms of better information? This point is reached at 4:1, where f/2.8 becomes effective f-stop f/14. This is a bit higher than what diffraction limited calculations arrive at (f/6.7–11, in most discussions). f/4 resulted in slightly softer images.
Comparing 5D2 images of same plant to the 5D sr, 5D2 still gains information at 5:1 (did not try 7:1), but still on fewer pixels. With 5D sr you can take a larger field of view at 4:1, print larger, and crop heavier, and get same information, mostly as expected.

Attached is a 4:1 f/2.8 image, 111 image stack: full image height down sampled to 1000 pixels, cropped some of the black side areas out. Again, flowers are 2 mm high, the bubbles are individual cells, about 20–40 µm in diameter, resolution limit seems to be at around 10 µm, which is about right for anything short of epi objective lenses on compound microscope. On dedicated stereomicroscope you can get down to around 4–6 µm (1.22 lamda/NA).

Enjoy!


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## tolusina (Jul 13, 2015)

Could you, would you please post a 100% crop?


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## Zeidora (Jul 13, 2015)

tolusina said:


> Could you, would you please post a 100% crop?



Here you go. For clarifications, shooting style neutral, no USM or any further processing done.


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## tolusina (Jul 13, 2015)

Zeidora said:


> Here you go. For clarifications, shooting style neutral, no USM or any further processing done.


Oh my yes, thanks!


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## Click (Jul 13, 2015)

Thanks for posting. Excellent work. 

Well done, Zeidora.


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## Zeidora (Jul 13, 2015)

Glad you like it. If anybody wonders why the bottom flowers are out of focus, that is done intentionally. Top focal plane is at top edge of flower, bottom edge at bottom of green stalk (rachis of inflorescence). You can get weird artifacts when you have near-overlapping elements in very different focal planes. And it is also visually confusing; I think one expects something to be out of focus, so if everything is sharp, then one looses a key for what is where in 3D.


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## Halfrack (Jul 14, 2015)

Very nice, an extreme level of detail.

I'm pretty sure that Canon isn't the one that'll make all the money off the 5DsR, it's Intel, Apple, SanDisk, Crucial, and Seagate/WD because even throwing a MacPro at it, the files are huge, complex and multiplying faster than rabbits.


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## Zeidora (Jul 14, 2015)

Halfrack said:


> Very nice, an extreme level of detail.
> 
> I'm pretty sure that Canon isn't the one that'll make all the money off the 5DsR, it's Intel, Apple, SanDisk, Crucial, and Seagate/WD because even throwing a MacPro at it, the files are huge, complex and multiplying faster than rabbits.



I've been doing z-stacking for a while, so not much new here. RAW files are 2.5 the size of 5d2. Smaller stacking steps due to larger f-stop are possibly counterbalance by lower max magnification (= fewer steps) that is need to get all the detail possible. The .tifs are just temp files anyway; I throw them away after I have the stacked product file. So I only keep the original RAWs plus the output Zerene .tif.
Example: 100 stack = 100x50 MB RAWs = 5 GB, 100 x 300 MB 16 bit tifs = 30 GB of temp files. Final output files is 1 300 MB 16 bit .tif: total storage space about 5.3 GB. With 5d2 it is 2.1 GB + 120 MB = 2.2 GB. 

I got a couple of 32 GB Lexar 1066x CF cards, but those do fill up rather quickly (around 400+ shots). I will pick up at least one 128 GB card.

I already had a 3 TB RAID1 LaCie 2big via thunderbolt as my main data drive. The internal 500 GB MacPro disk is only for OS, apps and scratch disk. Deep storage is on a 8 TB LaCIe 4big RAID1, still with plenty of space.

One more expense that is coming is 1-2 NEC 32" 4k displays.

Seeing my images exhibited at the Smithsonian: priceless


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## Valvebounce (Jul 14, 2015)

Hi Zeidora. 
That is incredible detail, thanks for showing it to us. 

Cheers, Graham.


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## NancyP (Jul 14, 2015)

Wow! Thanks for the information. I am a mere newbie at macro in the 1:1 and higher range. 
What is this lovely flower? A terrestrial orchid? It reminds me a bit of some I see in Missouri.


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## Zeidora (Jul 14, 2015)

NancyP said:


> Wow! Thanks for the information. I am a mere newbie at macro in the 1:1 and higher range.
> What is this lovely flower? A terrestrial orchid? It reminds me a bit of some I see in Missouri.



Have fun jumping into >1:1 macro. Loads of fun!

The orchid is tropical epiphyte in the genus Oberonia. It just flowered the first time, and I try to figure out what it is. It is somewhat similar to O. aff. lunata, but is not quite it. Occurrence, not sure. Obtained it with provenance from Taiwan, but does not exist there. So could be anywhere from India through Solomon Islands, more or less. Only about 300 species in the genus ...


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## HighLowISO (Jul 23, 2015)

*Fantastic!*


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## Zeidora (Jul 24, 2015)

HighLowISO said:


> *Fantastic!*



Glad you like it!


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## Viggo (Jul 24, 2015)

Something else, very nicely done! Thanks for sharing!


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## Zeidora (Jul 25, 2015)

Viggo said:


> Something else, very nicely done! Thanks for sharing!


Thanks!

Re something else, I would guess that >95% of popular images have people in it. I think for me it's the inverse. I like to shoot plant & animal portraits. Pointing a camera at people is just too vulgar and intrusive. My main lens is the Zeiss Makroplanar 100 mm, next is the MP-E 65. The OM 80 mm bellows head lens from 1:2 to 2:1 was also wonderful to use. Stacking has opened a whole other level of possibilities.


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## NancyP (Jul 27, 2015)

Thanks for info, Zeidora


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## Boromir883 (Aug 11, 2015)

Thanks Zeidora. It`s impressiv how many details show up, when equipment is used at its max


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## Zeidora (Aug 11, 2015)

Thanks. I agree, seeing individual cells with "simple" SLR is cool. But there are limits to imaging SLR. To see cell surface sculpture I have to use the scanning electron microscope. Haven't done compound microscope imaging with epi-objectives, but most cell surface detail is too small for that anyway. And I like running the SEM


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## Boromir883 (Aug 11, 2015)

Zeidora said:


> Thanks. I agree, seeing individual cells with "simple" SLR is cool. But there are limits to imaging SLR. To see cell surface sculpture I have to use the scanning electron microscope. Haven't done compound microscope imaging with epi-objectives, but most cell surface detail is too small for that anyway. And I like running the SEM


I would like to have access to such a toy ;D


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## Zeidora (Aug 12, 2015)

Boromir883 said:


> Zeidora said:
> 
> 
> > Thanks. I agree, seeing individual cells with "simple" SLR is cool. But there are limits to imaging SLR. To see cell surface sculpture I have to use the scanning electron microscope. Haven't done compound microscope imaging with epi-objectives, but most cell surface detail is too small for that anyway. And I like running the SEM
> ...


Yep, one of those job perks. Not compainin' about that one!


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## Mt Spokane Photography (Aug 12, 2015)

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


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## Zeidora (Aug 12, 2015)

Mt Spokane Photography said:


> 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.


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## Boromir883 (Aug 12, 2015)

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


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## Zeidora (Aug 12, 2015)

Boromir883 said:


> 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.


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## malus (Nov 17, 2017)

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?


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## Zeidora (Nov 17, 2017)

malus said:


> 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.


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## malus (Nov 18, 2017)

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.


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## Zeidora (Nov 18, 2017)

malus said:


> 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.


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## zim (Nov 18, 2017)

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?


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## malus (Nov 18, 2017)

@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).


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## Zeidora (Nov 18, 2017)

malus said:


> @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.


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## Zeidora (Nov 18, 2017)

zim said:


> @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!


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## sanj (Nov 30, 2017)

Thank you for this post.


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## Alejandro (Dec 1, 2017)

Zeidora said:


> malus said:
> 
> 
> > @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.
> ...




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?


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## Zeidora (Dec 1, 2017)

Alejandro said:


> Zeidora said:
> 
> 
> > malus said:
> ...



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.


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