[3dem] Image processing from the gold grids
Alushin, Gregory (NIH/NHLBI) [E]
gregory.alushin at nih.gov
Thu Jun 9 11:25:17 PDT 2016
Following up on Bob's comment, if you do have gold occupying a significant portion of your image (e.g. the edge of the hole), it can give you problems with image normalization and CTF estimation with standard practices (programs like CTFFIND). We once collected a dataset where we had enough images of this type that I wrote a small program to try and salvage them. It finds the mean and stdev of the pixel values in a central 1024 region of the image (which is assumed to be free of gold), then "removes" the gold by replacing pixels more than a certain stdev threshhold with random noise from a distribution that matches the statistics of the central 1024 region. We were able to get OK CTF estimates from these images and they were good for picking particles. Obviously you would want to actually extract particles from the original, unmodified images.
If you (or anyone else on this list) would find this useful, I'll put up a download link.
On Thu, Jun 9, 2016 at 9:08 AM, Grassucci, Robert <rg2502 at cumc.columbia.edu<https://mail.nih.gov/owa/redir.aspx?REF=E7wSDmAL0YTVvEXRbevKHDT8SAFDhzC6wavn_J3wO7uKtiBNk5DTCAFtYWlsdG86cmcyNTAyQGN1bWMuY29sdW1iaWEuZWR1>> wrote:
What you may be seeing is normalization/display issue if the gold is a significant portion of your image. Of course I am assuming that your ice is the same thickness in both types of grids. In our hands the holey gold grids do seem to require a slightly longer blot time so that could also be the problem. We have also noticed some gold particles on both the commercial and home-made holey gold grids but as long as it does not significantly alter the overall standard deviation it is not a problem . I hope that helps you.
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Subject: [3dem] Image processing from the gold grids
We have one dataset collected from the gold grids. We feel that the particle contrast from the gold grids is weaker comparing with the one from the regular carbon grids. We do find some gold particles in the images. So we wonder is it possible that these gold particles affect the image normalization/contrast? And is any prefilter or re-adjust the normalization of the images needed before we process the dataset? The last question is comparing with the carbon grids, do the gold grids prefer longer blotting time in order to get the thin ice?
This is the first time we collected the data from the gold grids. So any suggestion is welcome. Thank you in advanced.
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Gregory M. Alushin, Ph.D.
Early Independent Scientist
Laboratory of Macromolecular Interactions
Cell Biology and Physiology Center, NHLBI
Building 50, Room 4140
E: alushing at mail.nih.gov
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