Hello guys. Hope you are all good!!!
I have a doubt, and if you guys may answer me I will be grateful. So... I've been noticing from a while that everytime I analyze a biological sample something always intrigue me.
Usually biological samples the most interesting thing is the fine surface detail, so of course a lower KV is necessary, not only because the surface detail but that kind of sample needs a lower KV too. The only problem is that I can't get a good image, for example:
I can do a good image until 1000x, but beyond that a higher mag with lower KV is almost impossible. Take note that I've already set the parameters with lower WD, astigmatism, wobbler, spot size, probe current and etc.
And every time I use RDC for focus the image and etc, it "damage" the sample.
May someone explain me why this is happening? It is my equipment?
Thank you all for the attention!
What detection mode are you operating? Secondary electron? If so, try using a tilted stub with the sample's surface facing the detector. The problem with operating in low-kV is the inherent loss of resolution. Another tip-- operating in low-vacuum can mitigate charging/heating effects. It's a similar effect as coating the sample, as Kenneth Towe alluded to.
Sorry I forgot to tell @kenneth that I've coated the sample with gold.
Yes @james I used SED
What is your main problem? Poor resolution at low kV or specimen damage by a beam?
Some SEMs loose resolution much faster than others at lower kV, especially poorly aligned ones. You may want to check with metal specimen how your SEM behave at low kVs.
Some biological specimens are very sensitive to electron beam. Good fixation can help a bit. But anyway, you have to be very careful when focusing on such specimens. Do not use small area scanning, focus at your chosen magnification or at 2x magnification. If possible, try to focus not on area of interest, but on some nearby place.
Hey Vlad I did that I avoided using RDC, the problem is that the sample wasn't flat, so even if I focused out of the area of interest, when I go back to the area of interest it was out of focus.
I had both problems, lower resolution at low KV and damage by the beam specially using RDC. I already know that using low KV we loose resolution, but also has a better surface detail (and I needed a good surface detail), the problem is couldn't go beyond 1000x magnification
I used Accv 5KV and lower Probe current
Align your SEM properly (most modern SEMs should have no problems at magnifications up to x10k working at 5 kV.) If your SEM is unable (due to its specifics) to work properly at low kV, you are just out of luck.
To minimize damage try to acquire images at high frame rate utilizing integraion/averaging of images. Do not use RDC, learn to focus with full frame image at 2x magnification. And improve fixation of your specimens, sometimes it helps.
Sometimes beam damage is a real problem and you need to adjust your expectaions, be happy with results you can get.
- Badges
- Science topic
- Similar topics
- Computer Science
- Images