I am viewing fixed phytoplankton cells (on cover plate) under SEM. I gold coated the sample with 30mAmp for 240 seconds and mounted the cover plate. First time when I observed the plate, the cells did not "move" at all. However, when I came back a few hours later to observe the same plate again, I could start seeing the cells on the image screen shifting at 5 um, and the shifting problem got worse when I came back to observe it the next day... The image usually moved in one direction. This prevents me from getting a high resolution picture since I cannot focus the image well. What factors can cause image shift? How can I address the issue?
You can take into account the fact that some microorganisms selectively absorb gold. Perhaps the microorganisms remained alive.
Why do you need to keep your specimen in SEM for days?
What is the purpose of multiple observations of the same are?
If you see your specimen moving, it could be because:
Charging. Check if your specimen is well grounded. Sometimes after sputtering there are not enough gold on vertical edges of cover plates.
Soft mounting (like with carbon tape).
Stage instability, especially if you use specimen tilt. Avoid tilting.
SEM instability. Run SEM with a beam on for some time before you start taking pictures.
And, of course, nothing is alive on your plate.
The drifting in one direction is likely due to charging of your substrate.
If your cover plate is made of a dieletric material, as glass, you can have lots of charging depending on the accelerating voltage you use (more penetration depth) and grounding issues.
Try the following:
Keep in mind that small cover slips are the better as well. Let us know if you solved the issue.
Good luck,
If you keep the sample for days inside SEM, it will contaminate your machine . Because biological samples are volatile and affect your chamber vacuum while degradation . So do not keep samples in SEM for longer time and make sure that the grounding is proper .
Thank you so much for the replies. I just got recently trained with SEM, so reading all your comments really help me.
I am sorry about the confusion, but I did take the sample out every time after using the SEM. I did use the machine for hours a day to practice getting higher-resolution images, so perhaps I overworked the machine. After letting it rest for a day and changing the kV when imagining, the problem got resolved.
This normally happens at very high magnification and when the SEM is used for longer period. Once that happens I give the instrument a break and let is rest for a day or two. Just make sure that the sample holder is securely tightened on stub holder to prevent the specimen from shifting or avoid movements.
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