Motorized & Steromicroscope z-stack of Oberonia flower

[tropterrarium]

Here's something more interesting.
1) Oberonia inflorescence z-stacked using Cognysis StackShot, about 4:1 with Canon MPE 65 mm lens and Canon 5DmkII, Canon MT24EX twin macroflash, z-stacked with Zerene stacker based on a forty frame stack. f/5.6, steps 70 micrometers.

2) individual Oberonia flower, about 1 mm size, on Zeiss Discovery V12 with 0.63x planapo, Axiocam HRc, run through HeliconFocus, about 30 frames.

Resolution of latter is limited by low numerical aperture of system. For better resolution I am now doing scanning electron microscopy of critical point dried flowers. Loads of fun.

[cbuchman]

Beautiful pics - they looks like candy. for the techno-speak... I don't understand any of it I can just enjoy the outcome.

[tropterrarium]

Thanks Carrie, glad you like the pictures. Re the techno babble, suffice to say, those are very small flowers, the larger image has a field of view of about 8-9 mm tall, the smaller about 2-2.5 mm. This is serious macro, of some of the smallest orchid flowers. This particular species is even among the smaller Oberonias.

Depth of field is limited in macro. So I take multiple shots in successive focal planes, then combine the in-focus portions of each frame with a computer program, aka z-stacking. That way I get an image with greater depth of field, that would be un-attainable with regular photography.

The problem is even steps in z-stacking, which can be achieved with a motorized, computer controlled focusing rail. That is what the Cogynsis StackShot is all about.

For anybody interested, there should be an article on this sort of thing coming out in Orchid Digest sometime in spring.

[RosieC]

Wow, great flowers and such detailed pics.

Originally Posted by tropterrarium

Resolution of latter is limited by low numerical aperture of system. For better resolution I am now doing scanning electron microscopy of critical point dried flowers. Loads of fun.

Can you explain more. I thought this was at the atomic level and you wouldn't see flower structure or even cell structure with that. I used scanning electron microscopy to study the surface of silicon slices many many years ago. But it was in 1996 so I don't know the latest on these techniques.

[silken]

Very nice super macro shots. I used to have the Heliconfocus software but don't anymore. I do some photo stacking in Photoshop to achieve good depth of field but nothing real serious. It really helps tho.

[tropterrarium]

Originally Posted by RosieC

Wow, great flowers and such detailed pics.

Can you explain more. I thought this was at the atomic level and you wouldn't see flower structure or even cell structure with that. I used scanning electron microscopy to study the surface of silicon slices many many years ago. But it was in 1996 so I don't know the latest on these techniques.

Thanks.

SEM can easily be used for low mag shots. I attach an image of a different species done by SEM. Sorry for the ugly writing, but I still want to publish this image. The problem is, that the specimen needs to be in pretty high vacuum, so all water has to be removed without the specimen shriveling. That is done by Critical Point Drying, where the specimen is first preserved in 100% ethanol, then transferred to liquid carbon dioxide. The carbon dioxide is under pressure raised to above 31 deg C, where upon it goes "supercritical": the fluid has zero viscosity, and it is in a gas-fluid hybrid state. Then the gas is slowly released. So this procedure requires specific equipment.

I sputter coat the flowers with gold, then put them into a variable pressure SEM (Zeiss EVO 40XVP). Chamber pressure is relatively high for SEM, I like 30 Pa. The air helps with reducing charging on those highly branched structures, and produces more even illumination on the specimen. The column and the gun are isolated from the chamber by a lower 100 micron aperture, at which differential pumping is going on. The column is still under 10-4 to 10-5 Pa pressure, just with turbomolecular pump, not with ion gate.

I can do higher mag shots, too, say of pollinia, or details of cell surface structures on various parts of the flowers. Very interesting results.

Atomic resolution is very difficult to get with SEM. Our scope can't do that at all (I think specs are 4-5 nm resolution). In general, you would rather go to TEM or STEM, or atomic force microscopy. In silicates you can resolve crystals no problem, but not atoms or molecules.

Hope that feeds your curiosity.

[tropterrarium]

Originally Posted by silken

Very nice super macro shots. I used to have the Heliconfocus software but don't anymore. I do some photo stacking in Photoshop to achieve good depth of field but nothing real serious. It really helps tho.

Thanks!

In the latest phototechnique magazine issue, there is an article on z-stacking. The author also used CS6 with reasonable results. I tried it with CS5 on stereomicroscope images and it was worse than useless. Depending on the stack, either HF or ZS is better, so I run both and try things out.

[RosieC]

Originally Posted by tropterrarium

Thanks.

SEM can easily be used for low mag shots. I attach an image of a different species done by SEM. Sorry for the ugly writing, but I still want to publish this image. The problem is, that the specimen needs to be in pretty high vacuum, so all water has to be removed without the specimen shriveling. That is done by Critical Point Drying, where the specimen is first preserved in 100% ethanol, then transferred to liquid carbon dioxide. The carbon dioxide is under pressure raised to above 31 deg C, where upon it goes "supercritical": the fluid has zero viscosity, and it is in a gas-fluid hybrid state. Then the gas is slowly released. So this procedure requires specific equipment.

I sputter coat the flowers with gold, then put them into a variable pressure SEM (Zeiss EVO 40XVP). Chamber pressure is relatively high for SEM, I like 30 Pa. The air helps with reducing charging on those highly branched structures, and produces more even illumination on the specimen. The column and the gun are isolated from the chamber by a lower 100 micron aperture, at which differential pumping is going on. The column is still under 10-4 to 10-5 Pa pressure, just with turbomolecular pump, not with ion gate.

I can do higher mag shots, too, say of pollinia, or details of cell surface structures on various parts of the flowers. Very interesting results.

Atomic resolution is very difficult to get with SEM. Our scope can't do that at all (I think specs are 4-5 nm resolution). In general, you would rather go to TEM or STEM, or atomic force microscopy. In silicates you can resolve crystals no problem, but not atoms or molecules.

Hope that feeds your curiosity.

Thanks yes it does feed my curiosity. I used TEM as well in the same university project so I'm perhaps confusing which one gave us atomic level images. Really interesting about how you remove the water, of course I didn't have that problem with silicon structures, though I did have problems getting them clean enough to scan. TEM initially gave lots of pictures of the invisible to the eye debris because my supervisor thought you could just wash them in pure water in a fume cupboard (I wasn't convinced of that from the start). Ultra low vacuum techniques helped there though

Really interesting to see