[3dem] charging and astigmatism

[Xinghong Dai]

Hi.

I got a question about charging and astigmatism of cryoEM imaging: Of
course, they are two different kinds of distortion of the final images, but
will charging also introduce some extra astigmatism to the WHOLE image?
Since there is an extra electron field introduced by the charging area. 

The reason I concern about this is that, recently, we are considering about
a cryoEM imaging procedure that, after taking one exposure image (e1.mrc for
reconstruction), we will take two more extra exposures (e2.mrc & e3.mrc) at
the same place with exactly the same settings (particularly same defocus).
We will combine e1, e2 and e3 for ctf determination, which has much better
S/N ratio than just one CCD frame, and we hope this will help us to
determine the ctf parameters more accurately. Usually one 4kx4k CCD image
produces very noisy power spectrum that shows only one or two thon rings,
especially for large particles in relatively thick ice.   However, during
the imaging process, we noticed that, sometimes the first exposure image e1
shows some charging effect, but e2 and e3 always have very nice thon rings.
So, if the charging will also introduce extra astigmatism, then the ctf
parameters (especially the astigmatism) determined by averaging e1, e2 and
e3, may not be applicable to e1. Any comment?

 

Another thing is, if the exposure area is at the edge of the hole so that
there is carbon film covered in the electron beam, then there is a much
higher chance to get charging images.  I was told that a pre-exposure at a
does about 1e/A2 may help to reduce charging. For example, if the does rate
is 25e/A2*s, we can take a pre-exposure for 40ms, then after a few seconds,
take the exposure image for 1000ms. Does anyone have experience about this? 

 

Any comment is appreciated!

Best regards,

 

Xinghong Dai
Graduate Student
University of California, Los Angeles



 

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