Sorus of rock cap fern (Polypodium virginianum)

[Chris S.]

First image with my newly-built macro rig. This is the underside of a leaf of the rock cap fern Polypodium virginianum.

For those who wonder what we're looking at: You may have noticed that fertile fern leaves have tiny, flat dots on their undersides. Those dots are called "sori" ("sorus" for singular), and a sorus--with the edges of three other sori--is shown here. Each sorus contains many fist-like "sporangia"--easily evident in this image--which in turn contain a multitude of individual spores that act as seeds for the plant. The spores in this image are mostly immature, but some sporangia have burst and scattered sand-like immature spores across the underside of the leaf surface. The rattle-snake-tail like segments on each sporangium function like springs, and are attached to a transparent capsule around the ripening spores. When the spores ripen, the transparent capsule dries out and splits. The springy stucture then catapults the spores around and about--some hopefully catching a breeze and falling away from the mother plant, spreading into a new area.

The sprout-like structure at the right of the main sorus is, apparently, a couple of ribs from the ruptured sporangium to the right of the strand. This is not easy to see in the Web version, but quite evident in a full-res image.

This is a 70-shot stack with a Nikon CF N Plan Apochromat 4x with an NA of .20. Movement increment 20 microns. Software Zerene Stacker. Camera Nikon D200.

As this was my first experiment with the instrument (spent a couple of happy months building it, followed by several frustrating months too busy with work to use it even once), it's far from perfect. Plan to reshoot with an increment of 10 microns, and slightly different contrast ratio. I'm aware of the evident visual artifacts, particularly around the margins of the sorus. Retouching didn't offer much benefit, as no individual image seems to have the necessary information that would eliminate the artifacts. Hence the desire to shoot with a tighter movement along Z axis--I suspect 20 microns was a bit much for this optic.

Comments and criticisms extremely welcome--I'm definitely in the learning phase.

Thanks!

--Chris S.

[rjlittlefield]

First image with my newly-built macro rig.

"First light", eh? It's very nice -- much better than most of my first shots.

This is the underside of a leaf of the rock cap fern Polypodium virginianum.

I remember being introduced to Polypodium sori in a college botany class. That was a long time ago. The image brings back nice memories.

I'm aware of the evident visual artifacts, particularly around the margins of the sorus.

They may be evident to you, but I can't find a thing that I could identify as an artifact. Are we talking about missing detail, some form of halo, or something else?

I see some cases, for example around 10 o'clock on the central sorus, where it looks like we're looking at the leaf surface through a bit of fog between two of the rattlesnake tails. If we're actually looking through clear space between the tails, then the fog is probably OOF foreground spread into a big blur, in which case I don't know any good way to get rid of it. That sort of contamination happens with the light itself, and it's the devil's own time to back out computationally.

This is a 70-shot stack with a Nikon CF N Plan Apochromat 4x with an NA of .20. Movement increment 20 microns.

Microscopyu lists DOF at 4X 0.10 as 55 microns. NA 0.20 would be about half that, so 20 microns does not seem excessive. Smaller steps are pretty harmless, of course, and would remove one source of uncertainty.

--Rik

Edit: fixed spelling.

[paulheijmink]

I still didn't know what to look at.

It must be Polypodium instead of Polygonum

Wonderful details.

Paul

[Chris S.]

It must be Polypodium instead of Polygonum
Paul

Right you are, Paul--I must be getting tired. After your reply, I edited the orginal post. The sorus is, of course, from a polypodium (as you said) and not a polygonum.

I guess it's time for me to catch some shut-eye--shouldn't be making mistakes like that. Thanks for keeping me straight!

Best regards,

--Chris

[Planapo]

Beautiful! From the documentation as well as the aesthetics! I like the composition.
One can nicely even recognize the cell crest on each sorus that, when drying out, will tear it open to release the spores !

--Betty

[ChrisR]

Brilliant. And the explanation adds enormously, thanks!

Have we seen your rig, Chris? My local hardware store is fresh out of goniometers so I'm not sure what to look out for

[Tardigrade37]

Beautiful image... For those interested in the process of the spores being flung, check out this YouTube video from Martin Microscope:
http://www.youtube.com/watch?v=-xF83pHEx6Q

[rjlittlefield]

It must be Polypodium instead of Polygonum

Thanks for the catch on the name. It was very late at night for me too. Normally I do Google search to confirm spellings even if I think they're correct, but in this case I was focused on the image and didn't do the search. Not catching the glitch is especially embarrassing to me because Polygonum is a genus of buckwheats that I encounter much more frequently than the ferns!

I still didn't know what to look at.

The pixel coordinates of my "10 o'clock" example are around x=270, y=170. There you can see two "rattlesnake tails" arranged in a wide V, one almost vertical, the other almost horizontal. In the center of the V there are several light blobs that look sort of like the ones we see all over the leaf, but in the V they are indistinct and have little contrast, like we are seeing them through a cloud of light tan fog.

This is a common effect when shooting through microscope objectives and other wide-aperture lenses. The cause is this: when the lens is focused on the closer detail, the background is seen in its natural colors but out of focus; when the lens is focused on the background detail, the foreground objects are out of focus and are seen as large fuzzy blobs. Where those blobs overlap the background, the background is never seen clearly; it is always either out of focus or contaminated by OOF foreground.

You can get a better understanding of the problem by considering a side view of the lens and subject. When the lens is focused on the background, a portion of its entrance cone is blocked by nearby foreground objects. Light from those foreground objects is mixed uniformly with light from the background. It is like an extreme form of flare that occurs close to edges in a depth map.

The problem is especially bad when the background is darker and foreground is lighter, as in this case. When the foreground is darker, then its OOF contribution darkens the background but does not degrade the contrast as much.

check out this YouTube video

Very nice -- thanks for the link. It is interesting in the video that after the spores are flung out, the structures curl back into almost their original shapes. I would have assumed that they would stay open.

--Rik

[Chris S.]

Thanks, everyone, for the compliments, corrections, and discussion. I really appreciate it.

Rik and Paul, my goof between Polypodium and Polygonum is embarrassing—a bit like calling an old friend "Anderson" instead of "Henderson." Doh! For both of these genera, I've photographed most of the species that occur here in northeastern Ohio, and usually can tell a fern from a buckwheat—at least on a good day.

Rik, I especially appreciate your checking the image over as you did. I was aware of the problem you describe, though could not have explained it with anything approaching your clarity. I figured the effect probably accounted for some of the haze around the sorus, but not all. Better informed now, I likely won't worry about re-shooting this image.

Tardigrade37, what a link! Very nifty video. Unfortunately, it's put thoughts into my head about building a macro video rig with full movements. . . .

Chris and Betty, thank you! No, Chris, I haven't yet posted pictures of my rig, but will. I have a couple of major work commitments to finish up first, but after that, will show goniometers and all.

--Chris

[Planapo]

Homebrew mounting/movement rig with six-axis movement: Steel plate base, microscope focus block for X-axis on camera mount; other five axes covered on subject mount with microscope focusing block, translation stage, rotation stage, and two goniometers.

I'm sure there are a lot like me who are curious to sneak a peek on this one.

Beautiful image... For those interested in the process of the spores being flung, check out this YouTube video from Martin Microscope:
http://www.youtube.com/watch?v=-xF83pHEx6Q

Very nice video! There's so much great stuff out on the i-net, which otherwise would go unnoticed, so thanks for linking, Tardigrade37.

BTW, Martin Microscopes have chosen a lovely tune as "soundtrack" for this video haven't they?! For those who enjoyed the music too, I'd like to provide an ID : It's the ending of Johann Sebastian Bach's famous Air from the Orchestral Suite No. 3 in D major, BWV (Bachwerkeverzeichnis) 1068. I had never heard it played on the guitar before, but this sounded good!

--Betty

[AndrewC]

I'm interested to see the difference between a rotation stage and a goniometer. Do you mean the different planes in which the rotation occurs or something more subtle ?

Andrew

[Harold Gough]

This is a common effect when shooting through microscope objectives and other wide-aperture lenses. The cause is this: when the lens is focused on the closer detail, the background is seen in its natural colors but out of focus; when the lens is focused on the background detail, the foreground objects are out of focus and are seen as large fuzzy blobs. Where those blobs overlap the background, the background is never seen clearly; it is always either out of focus or contaminated by OOF foreground.

Or when directly observing fairly thick specimens, such as Collembola, at high magnification through a microscope, probably more so with phase contrast. Within limits, our brains select what we perceive

Harold

[Chris S.]

I'm interested to see the difference between a rotation stage and a goniometer. Do you mean the different planes in which the rotation occurs or something more subtle ?

Andrew

What I'm about to say will make much more sense after I post images, which I can't do now, but will eventually (though it will likely be a couple of weeks before I get the chance). That said, here goes. Please bear in mind that this is for a horizontal rig.

On this rig, the rotation stage and goniometers are used to position the subject along any of the three axes of rotation. The rotation stage is mounted to spin in the same axis as a phonograph record player. It can be rough positioned by hand, then fine-tuned with a micrometer drive.

On top of the rotation stage, I have a matched pair of goniometers. These tilt in the remaing two axes of rotation--like a very small table that tilts from side-to-side and front-to-back. These are micrometer driven.

The rotation stage and pair of goniometers rotate about a single point in space, which is 25mm (1") above the surface of the uppermost goniometer. This keeps the specimen in one place while being rotatable in all three axes.

The benefit of using goniometers on top of the rotation stage is that unlike some other approaches to three-axis rotation, this leaves the space around the specimen mostly free (only the area below is blocked). This makes it easy to arrange lights and manipulate the specimen.

Thorlabs has good documentation on the goniometer portion. It's a Thorlabs GNL, which consists of a GNL10 mounted upon a GNL18. These two devices are made to work together. I mounted them on top of a Newport rotation stage. To do that, I had a very simple adapter plate made.

Again, sorry for all the words and no pix. Will remedy that in another post at a later date.

Andrew, thanks for your interest.

--Chris

[AndrewC]

The words make sense, thanks. I'm trying to contrive something similar myself but don't have the nice Thorlabs goniometers or similar. It's relatively easy to get two axis to rotate about a centre point, the 3rd axis is driving me nuts and has exceeded my fabrication skills - so far !

Andrew

[Cyclops]

Very cool, a little fern nursery! (I love ferns!)