Technical quiz

[Pau]

It's no about the specimen, a Navicula diatom, but,
What's the technique used to take this image?

[g4lab]

Scanning Electron Microscopy

[Franz Neidl]

Maybe with a blue LED (455 nm) ?

Franz

[canonian]

It's not DIC, is it?

[Cactusdave]

I think it's an optical technique and it's certainly a beautiful image with outstanding clarity combined with high magnification. The possibility of blue end of the spectrum illumination for its greater resolving power combined with perhaps some form of oblique illumination and very small stacking steps? Or perhaps some kind of deconvolution technique such as Zeiss' Apotome or a laser confocal microscope? I'll be very interested to hear the answer.

[Craig Gerard]

I'm seeing a distinct right-angle.
The image has perceptual depth. 3D?

Craig

[René]

Darkfield, reversed?

[peter-h]

may be, this is made with a UV-led at 400nm or shorter. Here is one picture from me , made with a LED at 365nm and published in inverse gray scale.
http://www.mikroskopie-ph.de/Navicula-365UV-DMK.jpg

Peter

[Pau]

- Graham, I posted it because it seems SEM, but it's optical microscopy
- Franz, blue but no LED, only an old deep blue filter
- Canonian, no DIC, just oblique illumination
- CactusDave, Yes blue light and oblique, but no stack nor specialized techniques
- Craig, the relief impresion is form O.I.,
- René, Yes, reversed but not darkfield at this magnification
- Peter, YES! you did it before me, the only significative difference is blue vs UV (despite I saw your website, I don't understand german and I didn't see your navicula)

Thanks all for your participation, I think it's fun

This image is an unexpected result of my failed attempt to make an high resolution stack of this diatom. It's a single shot of a serie of 17. After spending an huge lot of time cloning out lots of dust spots, the stack was horrible but some frames like this one had a nice focused part, the image is Highly processed: cleaned, cropped, desatured, inverted and highly sharpened. Here you have the original frame (just levels adjusted from RAW)

Zeiss Neofluar 100 1,3 Phase 3, Zeiss KPL 8x and 0.25X photo adapter, Zeiss Standard microscope, a.a. 1.4 condenser decentered for O.I. , Canon EOS 20D

[rjlittlefield]

This image is an unexpected result of my failed attempt to make an high resolution stack of this diatom. It's a single shot of a serie of 17. After spending an huge lot of time cloning out lots of dust spots, the stack was horrible but some frames like this one had a nice focused part

Pau, I am curious what went wrong when you tried to stack this. Sometimes at high magnification things go OOF in strange ways that cause nasty artifacts, like bad alignment or dimples and rings on what should be smooth spheres. Looking at how areas go OOF in just this one frame, I don't see anything that I would expect to cause problems. But perhaps this frame looks well behaved only because everything is either focused or behind the focus plane, and the bad OOF behavior appears only for objects in front of focus.

Just to check, what happens if you process in Zerene Stacker using PMax, with scale adjustment turned off and maybe even Shift X and Shift Y turned off?

--Rik

[Charles Krebs]

Pau,

Just saw this... yes it was fun to guess.

After spending an huge lot of time cloning out lots of dust spots, the stack was horrible

Yes indeed, dust is just brutal with high mag objectives... dust that is no problem at all for "normal" photography. The camera I use on the microscope was in real need of cleaning (similar to what yours shows in the full shot, but with an additional one or two much bigger blobs ). Just the other day I took it off the microscope to photograph one of my grandsons playing soccer (with a 300mm f4 lens). Not a single speck of dust was visible in any of the shots. I see this all the time, but it's always amazing to realize how small a dust spec can be and still show up in these microscope shots.

the stack was horrible

Any idea why? Generally subjects like this stack pretty nicely. (At the very least for a few frames until the "back" side starts showing through). Did you try it with all "adjustments" turned off? You usually don't want them at these magnifications. (And there are also no dust "tracks" to deal with then as well)

Edit:
Awwww..... I see Rik got ahead of me again!

[Pau]

Rik & Charles,

Many many thanks for your interest and advice

I delayed my response working again in the image ( I can't count how many hours I spent in total ) and the result is a lot beter but stil far from perfect.

Pau, I am curious what went wrong when you tried to stack this. Sometimes at high magnification things go OOF in strange ways that cause nasty artifacts, like bad alignment or dimples and rings on what should be smooth spheres. Looking at how areas go OOF in just this one frame, I don't see anything that I would expect to cause problems. But perhaps this frame looks well behaved only because everything is either focused or behind the focus plane, and the bad OOF behavior appears only for objects in front of focus.

Just to check, what happens if you process in Zerene Stacker using PMax, with scale adjustment turned off and maybe even Shift X and Shift Y turned off?--Rik

The problems were all you say plus bad alignement of some of my dust corrections (I did clone the dust in each image, a big mistake)
I use Combine ZP and this software has a malsane tendence to find detail were there isn't any, rsulting in artifacts. My Zerene trial vesion expired, I will soon buy it for this kind of work)

Yes indeed, dust is just brutal with high mag objectives... dust that is no problem at all for "normal" photography. The camera I use on the microscope was in real need of cleaning (similar to what yours shows in the full shot, but with an additional one or two much bigger blobs ). ...
....

the stack was horrible

Any idea why? Generally subjects like this stack pretty nicely. (At the very least for a few frames until the "back" side starts showing through). Did you try it with all "adjustments" turned off? You usually don't want them at these magnifications. (And there are also no dust "tracks" to deal with then as well)

I'm allways surprised -and pleased- with the cleanness of your photomicrographies, and I now also appreciate your skills removing dust. Some advice (or mini tutorial) on this topic would be extremelly useful. I only clean my sensor with canned air (at some distance to avoid condensation). Would a wet cleaning manage this microscopic dust particles?

I have more problems stacking high magnification images than sharp macro ones. Turning off all alignemets was necesary here (lots of dust and oblique illumination that fool the software) but in some cases it's needed due to small bad calibration of the paralelism between the stage and camera or small movements of my too sensitive stage due to the shutter vibration.
Here you have the final result of the stack. The processing of the final image (here sack of 14 shots) was similar to the first posted one:

[rjlittlefield]

Pau, thank you for this follow-up. The final image looks good to me. Those hours were productive even though I'm sure they were frustrating.

What usually happens to me is that the second or third time goes much faster than the first, as I have learned which techniques work well and which ones do not. (I have many memories of ones that do not!) With luck, you have now figured out a way to process this stack that will work well on the next one also.

One thing I should mention for other readers. This last image is rotated 180 degrees from the blue image shown earlier in the thread. I was very puzzled for a while, wondering why this last image showed obvious features that I could not see a hint of in the earlier image. When I realized about the rotation, things made more sense!

--Rik

[Pau]

Thanks Rik, I did rotate it because the sense of depth is a bit more natural with the light coming from up

[rjlittlefield]

Ah, good point! The blue one had inverted brightness compared to the stacked image, so the blue one already looked like the light was coming from above. But when I swap bright and dark, I see that it does look odd without the rotation.

--Rik