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<p>Thank you Chris for the paper, and Robert your explanation is pretty clear to me.</p>
<p><br>
</p>
<p>The grid we used is UltrAuFoil 1.2/1.3, The object Aperture is 100, defocus value is 2-4. </p>
<p><br>
</p>
<p>I think it should have less problem if people are handling high electron-dense complexes since naturally they don't need to put high defocus value. It is easy for them to align the particles once they can have enough contrast. For the protein I handled is
elongated and in one direction is pretty thin. So We usually put more defocus to see the particles clearly. Now I feel that maybe the effect is oppsite for the gold grids. </p>
<p><br>
</p>
<p>I will pay attention the points we discuss here for the next Krios acquisition.</p>
<p><br>
</p>
<p>Thank you all.</p>
<p><br>
</p>
<p>Xiaodi</p>
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<div id="x_divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" color="#000000" style="font-size:11pt"><b>From:</b> 3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Christopher Russo <crusso@mrc-lmb.cam.ac.uk><br>
<b>Sent:</b> Friday, June 10, 2016 4:48:43 AM<br>
<b>To:</b> Robert McLeod<br>
<b>Cc:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] Image processing from the gold grids</font>
<div> </div>
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<font size="2"><span style="font-size:10pt;">
<div class="PlainText">Dear Robert, <br>
Your observation about the diffracted beams in the images is correct. In practice these are not a problem and can actually be used to quickly determine focus. We typically use a 70 micrometer objective aperture and defocus of 1-3 micrometers for routine
data collection on a Krios. This and many other details of the properties of all gold supports that may be helpful are in a recent paper we published:<br>
<br>
<a href="http://dx.doi.org/10.1016/j.jsb.2015.11.006">http://dx.doi.org/10.1016/j.jsb.2015.11.006</a><br>
<br>
which is available open access.<br>
<br>
Kind regards, <br>
Chris <br>
<br>
<br>
Christopher J Russo, PhD<br>
MRC-LMB, Francis Crick Ave, Cambridge CB2 0QH, UK<br>
Phone +44 1223 26 7535 <a href="http://www.mrc-lmb.cam.ac.uk/crusso/">http://www.mrc-lmb.cam.ac.uk/crusso/</a><br>
<br>
> On 10 Jun 2016, at 09:42, Robert McLeod <robert.mcleod@UNIBAS.CH> wrote:<br>
> <br>
> Hi All,<br>
> <br>
> I've only taken a brief look at the Ultrafoil grids but one problem I did notice was the appearance of diffraction contrast from the Au in the middle of empty holes. Is that what's being discussed here? Basically due to the large amount of defocus used in
the cryo-TEM you have a detector plane that is in-between the image and diffraction planes in the instrument. If the edges of the beam touch the Au support (as they probably should), you can see dark-field images of the crystallites in the center of the hole.
<br>
> <br>
> At 300 keV, the [111] Au spacing has 2*theta_Bragg = 8.4 mrad, and then the next ring at [211] is 11.9 mrad. I.e.<br>
> <br>
> 2*theta_bragg( Au[111] ) = 8.4 mrad = 2.35 Angstrom<br>
> 2*theta_bragg( Au[211] ) = 11.9 mrad = 1.67 Angstrom<br>
> <br>
> To compare the objective apertures on our Titan correspond to<br>
> <br>
> OA(30 um) = 4.3 mrad = 4.6 Angstrom (too small)<br>
> OA(70 um) = 9.7 mrad = 2.03 Angstrom (about right)<br>
> OA(100 um) = 13.6 mrad = 1.45 Angstrom (too big, let's in higher order spots)<br>
> <br>
> So the 70 um aperture is the best choice but it still lets the first Au spacing through. A 58 um aperture would be about perfect to completely cut-off all the crystalline gold signal (and limit your structure resolution to a maximum of 2.5 Angstrom).
<br>
> <br>
> I don't know nor want to derive the exact formula for how camera length scales with defocus but for parallel illumination it should scale linearly with defocus (and also be a function of wavelength). I.e.
<br>
> <br>
> R = (2*theta_bragg) * L( defocus, wavelength )<br>
> <br>
> It would be easy to measure empirically. Other things that would help: less defocus, good objective aperture centering, bigger Ultrafoil holes (although this may reduce the ability of the Au to reduce the charge), and a condenser aperture and illumination
fit to the hole size. <br>
> <br>
> Robert<br>
> <br>
> --<br>
> Robert McLeod, Ph.D.<br>
> Center for Cellular Imaging and Nano Analytics (C-CINA)<br>
> Biozentrum der Universität Basel<br>
> Mattenstrasse 26, 4058 Basel<br>
> Office: +41.061.387.3225<br>
> robert.mcleod@unibas.ch<mailto:robert.mcleod@unibas.ch><br>
> robert.mcleod@bsse.ethz.ch<mailto:robert.mcleod@ethz.ch><br>
> robbmcleod@gmail.com<mailto:robbmcleod@gmail.com><br>
> ________________________________<br>
> From: 3dem [3dem-bounces@ncmir.ucsd.edu] on behalf of Alushin, Gregory (NIH/NHLBI) [E] [gregory.alushin@nih.gov]<br>
> Sent: Thursday, June 09, 2016 8:59 PM<br>
> To: Reza Khayat; Xiaodi Yu; 3dem@ncmir.ucsd.edu<br>
> Subject: Re: [3dem] Image processing from the gold grids<br>
> <br>
> Hi Xiaodi and Sharon-<br>
> <br>
> Here is the script:<br>
> <br>
> <a href="https://www.dropbox.com/s/l05l48xcz04wrxk/degold.py?dl=0<redir.aspx?REF=GDlUNOnHX4pFU3xf9ybQQzCdpFV6pX94w-dr5AagOeiTvp0x85DTCAFodHRwczovL3d3dy5kcm9wYm94LmNvbS9zL2wwNWw0OHhjejA0d3J4ay9kZWdvbGQucHk_ZGw9MA">
https://www.dropbox.com/s/l05l48xcz04wrxk/degold.py?dl=0<redir.aspx?REF=GDlUNOnHX4pFU3xf9ybQQzCdpFV6pX94w-dr5AagOeiTvp0x85DTCAFodHRwczovL3d3dy5kcm9wYm94LmNvbS9zL2wwNWw0OHhjejA0d3J4ay9kZWdvbGQucHk_ZGw9MA</a>..><br>
> <br>
> It uses the EMAN2 image handling library, scipy, and numpy (hopefully you have these installed), and should run very fast even for fairly large images. Let me know if you have any problems.<br>
> <br>
> Also, I did not try CTFFIND4 at the time. If it can handle gold-containing images as mentioned, it is almost ceratinly a better option than using this program. It could nevertheless be helpful just for looking at the images without funky contrast.<br>
> <br>
> Cheers,<br>
> -Greg<br>
> <br>
> <br>
> Gregory M. Alushin, Ph.D.<br>
> Early Independent Scientist<br>
> Laboratory of Macromolecular Interactions<br>
> Cell Biology and Physiology Center, NHLBI<br>
> Building 50, Room 4140<br>
> T: (301)-496-6530<br>
> E: alushing@mail.nih.gov<br>
> <br>
> ________________________________<br>
> From: Reza Khayat [rkhayat@ccny.cuny.edu]<br>
> Sent: Thursday, June 09, 2016 2:54 PM<br>
> To: Xiaodi Yu; Alushin, Gregory (NIH/NHLBI) [E]; 3dem@ncmir.ucsd.edu<br>
> Subject: RE: [3dem] Image processing from the gold grids<br>
> <br>
> Hi,<br>
> <br>
> The latest version of CTFFIND 4 can deal with the mentioned CTF problems –at least in my hands it does.<br>
> <br>
> Best wishes,<br>
> Reza<br>
> <br>
> Reza Khayat, PhD<br>
> Assistant Professor<br>
> Department of Chemistry<br>
> City College of New York<br>
> 85 Saint Nicholas Terrace, CDI 2.318<br>
> New York, NY 10031<br>
> <a href="http://www.khayatlab.org/<UrlBlockedError.aspx">http://www.khayatlab.org/<UrlBlockedError.aspx</a>><br>
> 212-650-6070<br>
> <br>
> From: 3dem [<a href="mailto:3dem-bounces@ncmir.ucsd.edu">mailto:3dem-bounces@ncmir.ucsd.edu</a>] On Behalf Of Xiaodi Yu<br>
> Sent: Thursday, June 09, 2016 2:48 PM<br>
> To: Alushin, Gregory (NIH/NHLBI) [E] <gregory.alushin@nih.gov>; 3dem@ncmir.ucsd.edu<br>
> Subject: Re: [3dem] Image processing from the gold grids<br>
> <br>
> <br>
> Hi Greg Alushin:<br>
> <br>
> <br>
> <br>
> That would be very useful for us. Do you mind to share the link of the program? I would like to have a test for my images.<br>
> <br>
> <br>
> <br>
> Thanks a lot.<br>
> <br>
> <br>
> <br>
> Xiaodi<br>
> <br>
> ________________________________<br>
> From: 3dem <3dem-bounces@ncmir.ucsd.edu<UrlBlockedError.aspx>> on behalf of Alushin, Gregory (NIH/NHLBI) [E] <gregory.alushin@nih.gov<UrlBlockedError.aspx>><br>
> Sent: Thursday, June 9, 2016 11:25:17 AM<br>
> To: 3dem@ncmir.ucsd.edu<UrlBlockedError.aspx><br>
> Subject: Re: [3dem] Image processing from the gold grids<br>
> <br>
> Hi Xiaodi-<br>
> <br>
> 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.<br>
> <br>
> If you (or anyone else on this list) would find this useful, I'll put up a download link.<br>
> <br>
> Good luck!<br>
> -Greg Alushin<br>
> <br>
> On Thu, Jun 9, 2016 at 9:08 AM, Grassucci, Robert <rg2502@cumc.columbia.edu<UrlBlockedError.aspx>> wrote:<br>
> Dear Xiaodi,<br>
> 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.<br>
> Best,<br>
> Bob<br>
> <br>
> <br>
> From: 3dem [<a href="mailto:3dem-bounces@ncmir.ucsd.edu<UrlBlockedError.aspx">mailto:3dem-bounces@ncmir.ucsd.edu<UrlBlockedError.aspx</a>>] On Behalf Of Xiaodi Yu<br>
> Sent: Wednesday, June 08, 2016 11:27 PM<br>
> To: 3dem@ncmir.ucsd.edu<UrlBlockedError.aspx><br>
> Subject: [3dem] Image processing from the gold grids<br>
> <br>
> <br>
> <br>
> <br>
> Dear All:<br>
> <br>
> <br>
> <br>
> 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?<br>
> <br>
> <br>
> <br>
> This is the first time we collected the data from the gold grids. So any suggestion is welcome. Thank you in advanced.<br>
> <br>
> <br>
> <br>
> Best ,<br>
> <br>
> <br>
> <br>
> Xiaodi<br>
> <br>
> _______________________________________________<br>
> 3dem mailing list<br>
> 3dem@ncmir.ucsd.edu<UrlBlockedError.aspx><br>
> <a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem<UrlBlockedError.aspx">
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem<UrlBlockedError.aspx</a>><br>
> <br>
> <br>
> <br>
> Gregory M. Alushin, Ph.D.<br>
> Early Independent Scientist<br>
> Laboratory of Macromolecular Interactions<br>
> Cell Biology and Physiology Center, NHLBI<br>
> Building 50, Room 4140<br>
> T: (301)-496-6530<br>
> E: alushing@mail.nih.gov<UrlBlockedError.aspx><br>
> <br>
> _______________________________________________<br>
> 3dem mailing list<br>
> 3dem@ncmir.ucsd.edu<br>
> <a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a><br>
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