rocking diatoms

nanometer · Post by **nanometer** » Mon Dec 25, 2017 11:17 pm

Thanks to Mike Tuell, I have finally tried Zerene. Amazing program. I went after a few diatoms with the Leitz 90x apo Pv and Heine condenser, and created a few rocking gifs. The Pv objectives have the phase ring at higher NA than the 160mm Floutar objectives which was helpful. Now I'm hoping to get new life out of my 40x Apo Pv 0.95

Lou Jost · Post by **Lou Jost** » Tue Dec 26, 2017 6:06 am

Wow, these are stunning, though I can't quite figure out the middle one. Very inspiring.

carlos.uruguay · Post by **carlos.uruguay** » Tue Dec 26, 2017 6:11 am

Super!!!

JH · Post by JH » Tue Dec 26, 2017 10:35 am

Easy to see the shape, nicely done!
Best regards
Jörgen Hellberg

rjlittlefield · Post by **rjlittlefield** » Tue Dec 26, 2017 11:19 am

Very nice!

Lou, I take the middle one to be every other wedge is forward/backward, so 3 wedges in front, 3 behind. The floating purple blobs in the middle could be some sort of imaging artifact, or they could really be physical blobs inside the specimen, perhaps being distorted by a rippled surface in front of them.

nanometer, what does direct observation tell you about the floating purple blobs?

--Rik

nanometer · Post by **nanometer** » Tue Dec 26, 2017 11:28 am

Thanks for looking guys. Rik, I would say that central region is more like your latter description rather than some imaging artifact. That's a funny one to look at while rocking for me. Sometimes, I see every other 3 high and then the next time, I see the highs and lows reversed. Sometimes I see two adjacent ones both high, and then it goes away.

I'm interested enough to do some more of this! It's amazing to see how well rocking gives you information compared to just a simple one-view stack. The latter can look like a bit of a mess, but then it all makes sense in rocking. Can't say enough great stuff about Zerene. When my trial period ends, I'll be buying the license.

Lou Jost · Post by **Lou Jost** » Tue Dec 26, 2017 11:34 am

Rik and nanometer, I see it now, but it grates against my expectation that a diatom shell is encasing a 3-dimensional body....

rjlittlefield · Post by **rjlittlefield** » Tue Dec 26, 2017 12:04 pm

Lou, the actual surface relief is not as much as these images make it appear to be. But yes, those diatoms have a definite up/down/up/down structure to their shell. See http://www.nhm.ac.uk/content/dam/nhmwww ... column.jpg (at http://www.nhm.ac.uk/our-science/our-wo ... earch.html) for an SEM image of a similar (not identical!) structure.

nanometer, have you tried these in stereo? I did a little trial with images 4 apart in your sequence, and for me it worked well.

--Rik

nanometer · Post by **nanometer** » Tue Dec 26, 2017 1:24 pm

Rik, I did try some stereo images and they turned out well, but I prefer rocking to crossing my eyes

Lou Jost · Post by **Lou Jost** » Tue Dec 26, 2017 1:36 pm

Me too, these rocking images are easy to look at compared to cross-eyed things.

nanometer · Post by **nanometer** » Tue Dec 26, 2017 3:07 pm

Rik,

I looked at the diatom again today, and I can tell you that that central structure is definitely a phase contrast artifact that changes with focus.

Steve

Smokedaddy · Post by **Smokedaddy** » Tue Dec 26, 2017 3:16 pm

Very nice.

rjlittlefield · Post by **rjlittlefield** » Tue Dec 26, 2017 6:23 pm

nanometer wrote:... central structure is definitely a phase contrast artifact that changes with focus.

Great, thanks for the info.

--Rik

rjlittlefield · Post by **rjlittlefield** » Tue Dec 26, 2017 6:36 pm

nanometer wrote:Sometimes, I see every other 3 high and then the next time, I see the highs and lows reversed. Sometimes I see two adjacent ones both high, and then it goes away.

There are lots of interesting ways for shape-from-motion to go weird.

With some effort, I can consciously shift between sharp patterns in front versus fuzzy patterns in front. That's OK, but then sometimes I get it one way on the left and the other way on the right, and then the apparent motion becomes a flexing/folding across the almost vertical line. First time it happened, I wondered "Say what?!"

None of those things happen to me with stereo. And of course stereo has the advantage that it does not require an electronic display.

But I agree that under a lot of circumstances, rocking works better. When I present to an audience, I always show the rocking form, usually implemented by forward/backward keystrokes in PowerPoint.

--Rik

nanometer · Post by **nanometer** » Tue Dec 26, 2017 7:00 pm

Rik,

Just for you, here's a stereo of a diatom very similar to the one in the article you linked above. This is DF with the 40x .95 APO. Not the cleanest, but the stereo image turned out nice--even the little glass tubes sticking out. I tried 40x phase, but the tubes didn't show up very well. I do see what you mean. The stereo image is just cleaner and there is no ambiguity as to what is high and what is low.