Mr. Microscope Posted June 11, 2015 Author Share Posted June 11, 2015 I've never done it but a quick google search turned up this protocol:Hmm..That looks doable. I wonder if there's a protocol out there from EM? I'll have to do a little digging. Thanks Mr. Pants! Link to comment
Mr Pants Posted June 11, 2015 Share Posted June 11, 2015 I'd think that instead of aspirating into a pipette you could just collect it on a filter (probably coated with something to make them stick a bit) and go on to zero point drying and then gold coating. It should be pretty simple since the spicules are tough enough that you don't have to worry about preserving delicate structures like cell membranes. Link to comment
Mr. Microscope Posted June 11, 2015 Author Share Posted June 11, 2015 I'm thinking about something more along the lines of aldehydes and buffering to avoid osmotic and dehydration artifacts. Link to comment
Mr Pants Posted June 11, 2015 Share Posted June 11, 2015 I don't think you'll get a lot of dehydration artifacts from such hard structures. Maybe its my dino bias but I'd think if you wanted to be really careful you'd just do a series of ethanol dilutions with a rest in between each step. Slowly take the ethanol concentration up to 100%. That's all we need to do with dinos to keep the theca nice. Remember you are starting this process by dissolving the animal in acid or bleach. The stuff that remains is tough. How did you prep your coral samples? That actually might be very similar. Link to comment
Mr. Microscope Posted June 11, 2015 Author Share Posted June 11, 2015 The corals were just skeletons. My main concern was less with the dehydration (I usually do a graded series of ETOH anyway), but more with osmotic and preservation. I'm looking into ways to avoid such a harsh step as acid/bleach? It just seems so counter to everything I do in EM keep specimens pristine. Perhaps the spicules are roubust enough being calcareous or siliceous, although I don't know if spongin would come out in tact being collagen. I've found a few papers, though most of them refer to a, "classical method of dissociation." lol, I hate it when scientist just do things the way they've always been done without questioning whether or not there's a better way. Link to comment
Mr Pants Posted June 11, 2015 Share Posted June 11, 2015 Its just different when you are looking at hard parts. The dino theca and the sponge spicules are their skeleton. Its not really different than the coral in that respect. So you strip off all the soft stuff. Then that doesn't really need the delicate methods you are used to when looking at squishy things like mammal cells that seem to explode if you look at the wrong (stupid boring mammals) Link to comment
Mr. Microscope Posted June 11, 2015 Author Share Posted June 11, 2015 Its just different when you are looking at hard parts. The dino theca and the sponge spicules are their skeleton. Its not really different than the coral in that respect. So you strip off all the soft stuff. Then that doesn't really need the delicate methods you are used to when looking at squishy things like mammal cells that seem to explode if you look at the wrong (stupid boring mammals) Haha! Awesome. It's true. Even cytoskeleton seems to hold up to a lot. Sounds good. I think I have to go poking around under my rocks now (what do you bet I can't find any, lol). Thanks again for chiming in. Link to comment
amphipod Posted June 13, 2015 Share Posted June 13, 2015 Zombie Dino!You've got me wanting to find some sponges in my tank. I'm pretty sure I have a few. What is the protocol for dissolving a sponge? what would be kinda cool in my opinion is show a sample of sponge tissue, then show some spicules, before after sort of deal. Link to comment
FlowerMama Posted June 13, 2015 Share Posted June 13, 2015 Glad that my tank water is a source of great discussion for all of you. Don't understand what you're talking about but keep on researching!!!! I'm glad I was able to get help and I'm also proud of myself for having guessed right in the first place. Link to comment
Mr. Microscope Posted June 13, 2015 Author Share Posted June 13, 2015 what would be kinda cool in my opinion is show a sample of sponge tissue, then show some spicules, before after sort of deal. I wish I had the Keyence system so I could do the multi-focal image. Sponges have a lot of up and down topography. Glad that my tank water is a source of great discussion for all of you. Don't understand what you're talking about but keep on researching!!!! I'm glad I was able to get help and I'm also proud of myself for having guessed right in the first place. We're plotting a take over of your RFA tank! Yes, good guess. You did it just like any scientist. You observed, did your research, made a hypothesis, and in turn did an experiment (or outsourced it to me) to try to disprove it. Link to comment
CJJon Posted June 13, 2015 Share Posted June 13, 2015 I wish I had the Keyence system so I could do the multi-focal image. Sponges have a lot of up and down topography. Is that something that focus stacks a bunch of images like in macro photography? If you can export a series of images at various focal lengths, there are programs that will stack them. I use Helicon Focus 6. What image formats can you export? Link to comment
picoreef78 Posted June 13, 2015 Share Posted June 13, 2015 Awesome pictures guys, thank for sharing. Mr. M your thread always is a great read! Link to comment
Mr Pants Posted June 13, 2015 Share Posted June 13, 2015 \I use Helicon Focus 6. I've used Helicon Focus with a simple compound scope. It works beautifully. Link to comment
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