How to prepare your samples

Samples should be prepared prior to coming to the CAM. Fixed samples or live cells can be imaged, but all live cells must be BSL-1. Reach out to Melinda with any questions.

Due to the wide range of possible materials, we don't have a standard protocol to follow for preparing SEM samples. Consult with Amy or Abby prior to bringing your sample. See below for some general guidelines and tips to help you plan your experiement.

Sample Preparation Guidelines for SEM

1. Wet, sticky, and gooey materials are generally unsuitable for SEM imaging, including all liquids. Residues from tapes and stickers can contaminate samples and degrade the vacuum/ imaging conditions. 
2. Many biological or wet/hydrated samples can be imaged successfully following specific processing procedures. The CAM is well-equipped to assist with these, please ask.
3. Mounting materials. Only mounting materials provided by the Center for Advanced Microscopy are allowed. All other materials are specifically prohibited.  The only adhesives allowed are properly dried carbon cement, properly dried rapid cure epoxy cement and the special carbon tabs (not carbon tape)  For the best results, use the minimum amount of adhesive possible.
   • Adhesive drying times. 
     Carbon cement - 4 hours at room temperature
     Rapid cure epoxy - 12 hours at room temperature.
   • Never use carbon cement to mount samples like glass cover slips, silicon wafers, etc. It will not dry.  Use only the rapid cure epoxy on samples like these.
   • Although the special carbon tabs are approved for use, they may not provide the best results at high resolution and may take up to 40 minutes to stabilize in high vacuum conditions.
4. Only stubs provided by the Center for Advanced Microscopy are to be used. These stubs have been ultrasonically cleaned. The CAM SEMs use cylindrical mounts rather than pin stubs. We will provide 12.5 mm or 25 mm diameter mounts. Pin stubs can be accomodated with an adapter but they are not provided.
5. For the best resolution, the smaller the sample the better the results. All samples will release residual materials in a vacuum (outgas), and is particularly a concern with the ultrahigh vacuum of the 7500F. Also, thermal expansion and contraction is more of a problem with larger samples.
6. Gloves must be used at all times when touching samples, stubs, and holders - anything that will go into the vacuum. The cleaner the conditions, the better the results.
7. Sample height. Best results are acheived when all samples on a given stub are approximately the same height. 
8. Store your samples in a vacuum dessicator for the best results. Samples older than 5 years will be discarded unless specific accomodations are made with CAM staff.
9. Samples with magnetic materials present special problems for the 7500F SEM. Consult CAM staff for assistance before placing these samples in the 7500F.

How to bring samples to the lab for biological TEM

For tissues:
1x1 mm²  samples fixed in 2.5% glutaraldehye+2.0% paraformaldehyde in 0.1 M cacodylate buffer

For cells:

100 ul pellet size (around 6 million cells fixed in 2.5% glutaraldehyde

Example protocols

fixation of leaves

Reach out to Alicia with any questions.

Consult with Andy prior to bringing your sample. For particulate samples, we do have grids in-house that can be used.