I'd start by redoing the background corrections on your camera's software to see if this has an effect on the white spots. It's almost like someone used a carbon film with dense spots on it to create the background images, hence the white spots in the same locations on the images taken at the same magnifications. Just the beam (no sample) should be present when collecting background images.

B

When you move your specimen, do spots move with substrate? If they do move, you would know they are real and not a detector artefact.

Brent E Gowen , thank you, these are useful implementable suggestions!

I will have my next training in a week, so I'll ask if I can try this.

There is one detail that may also give some information, so we established hat the position does not change with magnification, but the intesity of the dots on other hand, does change with magnification!

So with higher magnification, the intensity of the dots decreases.. Could this indicate something about the lens? Or could this simply be a result of the grayscale autocorrect in the software.

Vladimir Dusevich , as I mentioned in the description, indeed the spots do not move with magnification nor sample movement. So I believe it may indeed come from a detector artefact! I hope to find out what the artefact is, so I can improve future images :)

1 Electromagnetic lenses cannot create the pattern you are seeing.

2 Contamination in the column would result in astigmatism; which is corrected in your images so this is not an issue.

3 Contamination of the column cannot create the pattern you are seeing.

4 "Intensity of the dots changes with magnification" As you change magnification, the number of electrons per square unit of area hitting your sample changes depending on how you are adjusting the beam for an image. This variation of the beam intensity, with a set background, could enhance or lessen the spots in the background as you are seeing.

Do you get these spots with a blank image (no sample present in the beam)? You should be able to find an empty square within your grid. Try taking a picture of a blank area to see the spots by themselves.

What kind of camera do you have (CCD, Direct Electron Detector,)?

As Brent E Gowen suggested you have an issue with background corrections. There are two types:

Dark reference, which set all pixels to zero read out at a fully blocked beam

Gain reference, which set all pixels to saturation read out at a fully illuminated CCD. (no specimen).

Dark reference is usually done when changing your exposure time, and

you probably can find the gain reference in your camera menu

Are you on a carbon/formvar grid, with negative staining? I see something similar when I am.

Flemming B. Grumsen , I also agree that your description of gain reference fits best with what I observe. Thanks a lot, this will help me a lot in knowing what to look for in improving the system!!

firstly, you should adjust the background of the camera (Bias / Gain ) from the user interface software, it is normally adjusted with no sample ( the sample holder need to be retracted for 1 cm) then from software it re-zero the camera screen. and the other option to adjust the beam intensity exposed to the camera screen in presence of the sample.

I hope it could help. Lotte Metz