Suggestions for flow visualization?

[Michelangelo von Dassow]

Hello,

I'm really excited to have found and joined Slimoco!

 

I'm trying to test a hypothesis about how the outer diameter of the Physarum veins is controlled. To do this, I need good visualization of the boundary between flowing and non-flowing cytoplasm. I'd appreciate any suggestions people might have.

 

My attempts so far have not been especially successful. In the attached false-color image, the red channel is moving material (differences between video frames), and the blue channel is the average of everything (still and moving) for a few video frames. The boundary is not very distinct on the right, and only distinguishable with a fair bit of processing.

 

I tried feeding the plasmodium sumi ink because the black carbon particles are very visible in bright field. However, it didn't take up the ink. Then, inspired by Anna Sagatov's and Soichiro Tsuda's movies, I tried smooshing the plasmodium with dried sumi ink (dried after a wash to get rid of solvents). It worked pretty well for the few thin, short veins that grew out just after smooshing the plasmodium with the dried ink. I need a long vein for my experiment. Unfortunately, the plasmodium grew away, and left all the particles behind.

 

Any other ideas about how I can either convince it to eat sumi ink/carbon particles, or improve the imaging without added particles? Has anyone tried coating particles with something (maybe protein or mushed oat) to get them to eat them?

Thanks!

 

--Mickey

[Nazwa Zarrella]

Hello Mickey, Please have a look at this short video of what I believe may be slime mold cytoplasmic streaming.

                    I haven't been able to get any professionals interested in looking into it so I can't say what it is.

                   But just maybe you will know what you're looking at and it could tell you something.

                   Of course I don't expect you to tell me what it is, but maybe if it's clearly NOT slime mold you could give

                  me a hint and I'll bark up a different tree. If you do watch this thank you for watching and good luck

                  with all of your endeavors!  Best Regards-Nazwa_  [e-mail]      http://www.youtube.com/watch?v=AH3miMVVhpk&list=UUg3FCcpEX_SRqJ...

[Michelangelo von Dassow]

I'm afraid I'm not sure what you are asking. Are you asking whether your video is of a slime mold? Or do you know for sure it is a slime mold of some kind, and are asking whether it is cytoplasmic streaming?

If the former, you'd need to provide more information, e.g. where was the material found and info about its habitat (e.g. substrate type: wood, leaves, moss...), pictures of what it looked like as a whole, the scale (spatial and temporal) in the video, lighting and microscopy information, preparation methods, etc.). Even still, I've only worked a bit with Physarum, not other slime molds, but it does not look like what I've seen of Physarum. The flow seems unidirectional (hence the need for timescale if it is time lapse), the color is not strongly yellow (hence the need for lighting information), and the texture does not look like the cytoplasm of Physarum.

Sorry I can't be more help.

Good luck.

--Mickey

[Michelangelo von Dassow]

I wanted to follow up on this because I'm still looking for a good low-tech way to visualize the flow within the veins or measure the thickness of the wall of the veins (I had to put slime molds aside for a few months as I focused on my main research project). Any thoughts?

If you'd like to see what I've tried so far, I've posted a page with a bit more description of what I was working on here: http://igor.wikidot.com/projects:self-organizing-cells

[ian]

I've been feeding them fluorescent microscopy dyes soaked onto oats - they'll eat them if they have no other choice. Neutral red has worked the best so far and it was easy to see under the 'scope. I'm going to try some lipophillic dyes over the next couple of weeks which would directly label the cell membrane structures making the boundaries far clearer.

[Michelangelo von Dassow]

Please let me know how this works! If you get some pictures, I'd love to see them.

The experiments I'd been trying to do would be most easily done on an inverted 'scope (because I want to the plasmodium growing across differences in elevation), and -- unfortunately -- the inverted 'scope I have access to at the moment doesn't have fluorescence. However, neutral red might work well enough with transmitted light. If that fails, I can find a way to set it up on an upright 'scope.

[ian]

The streaming's waaaay too fast to catch on confocal so I'm going to have to try it on the widefield once I've done messing around with TIRF.

I've just got hold of DiI for staining the amoebae without actually poisoning them - pricey but as it only stains the membranes it'd be ideal for the job. I've Sudan III but it's toxic, though not very quickly.

There is an old fashioned method of doing fluorescence without a dichroic equipped microscope - a triangle of glass mounted on the surface of a slide with a drop of immersion oil or glycerol -

http://www.viewsfromscience.com/documents/webpages/led_fluorescence...

I've never tried setting it up but I reckon the glass knives I make for EM would make perfect prisms for the job.

Have you tried polarisation techniques? - they might actually give you the contrast you need.

[Michelangelo von Dassow]

What a nice idea about using LEDs for fluorescence. That should be quite doable. I did try polarization, but didn't get much contrast.

Out of curiosity, what are you doing with TIRF? 

[ian]

Mostly teaching other people how to use it. Today was six hours of trying to get it to work through the plant cell wall. I did stain up some physarum amoebae to try it on them but I'm guessing I had the centrifuge up a little too high as all I had was a slide of limp, drifting pink tatters. I'll try again with them tomorrow. I'm wondering if I'd see much from the plasmodium in TIRF - especially at the traveling front.

[ian]

DiI works quite well under Epi - movie coming soon, you do get a clear distinction between the layers but the physarum gets fed up of intense illumination and moves out of the way over a ten minute time course. 

I tried TIRF and got a nice view of vesicles moving around but the very close focus on such an active organism that really doesn't like being hit with 50mw of laser means the view doesn't last for long

[Michelangelo von Dassow]

I look forward to seeing your movies!

[ian]

Just put the vid up A little contrast enhancement would bring it out more and this was on a manual filter set otherwise I reckon a water soluble dye alongside the lipid soluble DiI would bring out the differences very nicely.

[Michelangelo von Dassow]

Looks great!