NetNotes
HMDS was not available and we only had 98% (greater or equal). Nonetheless, I prepared a sample and everything seemed to be fine. Drying was ok and there was no problem with the vacuum. However, I observed a slight discoloration of the gold target during sputter coating—the non-sputtered gold surface turned grayish. Have you experienced a similar phenomenon and can you explain it to me? Is it a consequence of bad drying and/or the wrong quality of HMDS? Edith Stabentheiner
edith.stabentheiner@
uni-graz.at Wed May 11 We use 98% pure HMDS and have never noticed discoloration of
our gold target. I would like to open out the discussion. Should I look for greater than HMDS of greater than 98% purity for dehydration of cultured human cell lines? Dave Patton
david.patton@
uwe.ac.uk Wed May 11 We use 98–99% HMDS and have not noticed any discoloration
of our sputter coater targets. It may be that you did not completely remove the ethanol, or did not completely dry the sample from HMDS. How did the pump-down go in the sputter coater? Did you have trouble getting to vacuum at first? Did you do a few purges with argon before coating in order to completely flush out all the air and any adsorbed water vapor, etc., from the chamber? Phil Oshel
oshel1pe@cmich.edu Wed May 11 I believe the discoloration was the result of incomplete drying
of your sample, but could also be due to sample outgassing. You may need to extend your dehydration steps. What I recommend, also, is to place samples in a 45°C vacuum oven aſter HMDS and pull a vacuum on the sample overnight to insure the sample is completely dry if you are working with a thick or difficult sample. A graduate student in my lab is working with shark skin, which contains a lot of connective tissue. She was unable to coat 6 samples at a time in my sputter coater. Tere was too much sample outgassing with her samples even with the overnight baking in the oven. With the 6 samples in the chamber, our gold-palladium target discolored, also, and her samples never coated properly, although a plasma formed in the chamber. She asked me to try another run with the coater, and I had the same results as she did with another set of samples. I noticed that I needed almost no argon gas to produce a plasma. At first, I thought I was having a problem with my gas feed and disassembled my needle valve and cleaned it, but that was not the case. Te same phenomenon occurred with the next set of samples. Tese were samples that had been processed, oven dried and been in a bell jar over desiccant for weeks. Tis led me to conclude that the samples were outgassing. Extended baking at 45°C did not cure the problem. When we reduced the sample number to 3 in the chamber, all three samples coat fine. Try placing fewer samples in the chamber with each coating run as well. Ed Haller ehaller@
health.usf.edu Wed May 11
Specimen Preparation: perfect loop I had the chance to acquire a Perfect Loop recently, thinking that
I can increase the quality of the collected sections. Unfortunately, it was not that much helpful (at least in my hands). I lose plenty of good section when picking them up with the “perfect” loop. Te grids that I routinely use are slot grids coated with Formvar, and it is very difficult to transfer from the loop to the grid. I bought tabbed grids in order to handle them better, but then I had to cut the tab, so I had to buy cutter tweezers. All in all, a tool that didn’t deserve the investment. However, before I leave it in some box and forget it for years, I’d like to hear if there is anybody out there that has used it or still uses it and if there are any special tricks that I can apply. In the meantime, I will still call it the “Imperfect Loop.” Josif Mircheski
jmircheski@us.es Fri Jun 10 Here is a how I use it: aſter cutting the ultra sections, I separate them (by ones, twos, threes etc.) and then lower the loop on top
64
of them, making sure that the rim of the loop does not touch the sections. Ten, I raise the loop, trying to minimize the water droplet taken by the loop by raising the loop near the edge of the boat. Ten, while holding the grid with tweezers (grid is single slot, Formvar coated), I touch the loop on the grid surface, trying to match the orientation of the section with the orientation of the slot so that it sticks to the film only, but not on the metal. Te tricky part is there— the presence of the tweezers’ tips prevents the loop and the grid from sticking perfectly and easily transfers the section. Tis is the point where I lose most of the sections. I used the tabbed slot grids in order to remove the tip away from the contact area, but then I still have problems while picking up the sections and transferring them with the loop at the grid. Also, the tabs need to be removed, so I have to put three more steps in the handling of the grid—I lose grids there, too. So, I guess this explanation can help somebody to tell me what I do wrong, or where I can improve. And, I have been trying various modes, for months now. Josif Mircheski
jmircheski@us.es Fri Jun 10 I also use the perfect-loop for naked grids and it works perfectly.
It is most useful when ribbons are not achieved and sections are all individual. I tried it with coated grids for a short while and had trouble like you described. I did not hold the grid but had it sitting on a piece of filter paper. I got the best results by first filling the open loop with as many sections as would fit. Tat way, when they moved with the water being wicked out, there were still sections covering the slot of the grid. Getting the final bit of water out between the grid and the loop was where I had the most trouble. I finally gave up as it was easier to take the time to trim better and make ribbons of sections. I pick them up the old fashioned way of dipping the grid into the water of the boat, hooking the top of the ribbon over the edge of the grid and liſting the grid up at a fairly steep angle so that the rest of the ribbon lays nicely across the slot of the grid. Newly coated grids or ones that have been glow discharged are needed to do this for older ones tend to be hydrophobic and the sections will “run away.” Patricia Stranen Connelly
connellyps@nhlbi.nih.gov Fri Jun 10 Te best technique I’ve tried in getting sections onto a coated
slotted grid with consistent results is to use homemade support films (e.g., Formvar or Butvar) and a domino rack (EMS# 70620). Te domino rack is a great investment. I use it every time I transfer serial sections on coated slotted grids. Aſter casting the film on the water surface, the domino rack is carefully used to pick up the film. Te film on the rack is allowed to dry. Sections are picked up with the slotted grid. (Te grid serves now as your loop.) Te slotted grid containing the sections suspended in water is carefully placed on the film on the rack and allowed to dry. Tis technique is carefully described in Biological Specimen Preparation for Correlative Light and Electron Microscopy by David Moran and Carter Rowley. Aſter reading your post, I wanted to start looking for my commercial perfect loop. It’s been missing for a while but I don’t think I need it for now. Claire Haueter
chaueter@bcm.edu Fri Jun 10
Immunocytochemistry: immunogold for SEM Does anyone know of literature references or vendor technical
notes that mentions labeling of surface antigens on cells in tissue explants? I am looking for large probes (~100 nm) for SEM studies. Vickie Kimler
vakimler@med.wayne.edu Wed May 25 I would recommend using nanogold labeled probes followed
by gold enhancement. Te Nanoprobes kit worked great for us— see: Meagher, C.K., H. Liu, C.P. Moore, and T.E. Phillips. 2005. Conjunctival M cells selectively bind and translocate Maackia amurensis leukoagglutinin. Experimental Eye Research 80(4):545– 553. Tomas E. Phillips
phillipst@missouri.edu Wed May 25
www.microscopy-today.com • 2011 September
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84