[3dem] Re: charging and astigmatism

[Xinghong Dai]

Hi.

I received a few responses regarding my question. Here is the summary.

1.       The image blurring I observed was most probably caused by electron
beam induced sample movement, instead of charging. What I observed, e.g.
higher chance of blurring occurred at  the edge of the hole, and also the
direction of blurring always being perpendicular to the curvature of the
carbon film edge, were pretty consistent with  observations and proposed
mechanism in Niko¡¯s paper http://www.ncbi.nlm.nih.gov/pubmed/22366277    

2.       I tested ctf determination of e1.mrc or e1, e2 and e3 combined (by
CTFFIND3), the results (particularly the defocus value) were pretty close.
That means a single image can also produce reliable ctf parameters for my
data. The three images combined may improve the accuracy, but I am not sure
this improvement will be crucial for my target resolution. However, 2 extra
exposures do slow down my data collection speed by >30%, so I abandoned it
after two days of trial. 

3.       I tried 1e/A2 pre-exposure today, seems that it indeed worked and
reduced the chance of blurring to some extent, so I am still using it. 

4.       ¡°Astigmatism is caused primarily by magnetic, not by electrostatic
fields. Charging causes specimen movement, not astigmatism.¡±

 

Thanks for all the advices and discussion!

 

Best regards,

 

Xinghong Dai
daixh at ucla.edu

Graduate Student
Dept of Microbiology, Immun. & Mol. Genetics

and Electron Imaging Center for Nanomachines (EICN), CNSI

University of California, Los Angeles
570 Westwood Plaza, CNSI 6350

Los Angeles, CA 90095

+1 (310) 447 4330 <tel:%28973%29%20972-4483%20x23160> 

 

·¢¼þÈË: Xinghong Dai [mailto:bestdz at gmail.com] 
·¢ËÍʱ¼ä: Sunday, August 05, 2012 6:11 AM
ÊÕ¼þÈË: 3dem at ncmir.ucsd.edu
Ö÷Ìâ: charging and astigmatism

 

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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