<div dir="ltr"><div>Hi there,<br><br><br></div><div>our experience with the K3 is compatible with what Craig describes: we collected 3 datasets of horse spleen (nasty) apo-ferritin @ 8e/pix/sec, 16e/pix/sec and 30e/pix/sec (30 was the maximum dose rate as we were told by Gatan people). The 8 and 16e datasets look ok, with nice C2D and 3D below 3A with like 50Kptc. However the dataset @30e was clearly sub-optimal, Relion3b could not even get us nice C2D. We adjust the frame size to have around 1e/A2/frame depending on pixel size. Our standard collection settings in the Polara are now: 16e/pix/sec, 4sec exposures (total dose 64e/pix) at apix 0.95A/pix and 100msec/frame total number of frames 40. With these settings we have very nice results with a variety of samples from different users ranging from big ribosomes to small membrane proteins. We use Leginon/Apion for automatic data collection.<br></div><div>Hope it helps.<br></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">El jue., 14 feb. 2019 a las 15:20, Garry P Morgan (<<a href="mailto:Garry.Morgan@colorado.edu">Garry.Morgan@colorado.edu</a>>) escribió:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">
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Craig,
<div>thanks! that gives me a good place to start…</div>
<div>i’ll let you know what we see here when pushing the FPS up above 40.</div>
<div>Garry</div>
<div><br>
<div><br>
<blockquote type="cite">
<div>On Feb 14, 2019, at 12:57 PM, Craig Yoshioka <<a href="mailto:yoshiokc@ohsu.edu" target="_blank">yoshiokc@ohsu.edu</a>> wrote:</div>
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<div>Hi Gary,</div>
<div><br>
</div>
We haven’t exhaustively tested yet, but we have been targeting around 15-20eps on the K3 and have been getting nice results. This general range was based on the 400 -> 1500 read rate increase.
<div><br>
</div>
<div>At very high-magnifications 15eps is quite a high flux, so sometimes we will go below 10eps just so that we don’t have to push very high FPS (40+) with < 1 sec exposures. We have seen some mild pattern noise occasionally creep into images when
trying to save at high frame rates. Not sure yet if this is just our K3, or if it also seen by others. So far it doesn’t seem to be a problem in practice, but feedback from others if they have seen this would be appreciated.
<div><br>
<div>Cheers,</div>
<div>-Craig</div>
<div><br>
</div>
<div><br>
<blockquote type="cite">
<div>On Feb 14, 2019, at 11:45 AM, Garry P Morgan <<a href="mailto:Garry.Morgan@Colorado.EDU" target="_blank">Garry.Morgan@Colorado.EDU</a>> wrote:</div>
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thanks everyone. <br>
<div><br>
<blockquote type="cite">
<div>On Feb 14, 2019, at 11:53 AM, Reyes, Francis <<a href="mailto:francis.reyes@thermofisher.com" target="_blank">francis.reyes@thermofisher.com</a>> wrote:</div>
<br class="gmail-m_-6092317298382020416Apple-interchange-newline">
<div><span style="font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none;float:none;display:inline">Yes
it is advisable to work in nanoprobe. At parallel illumination and a 50 um C2 you should have around a 1.8um illuminated area.</span>
<div style="font-family:Helvetica;font-size:12px;font-style:normal;font-variant-caps:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
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<br>
<div dir="ltr"><span style="background-color:rgba(255,255,255,0)">Francis Reyes, Ph.D<br>
Engineer III, Field Applications<br>
Materials & Structural Analysis<br>
<br>
Thermo Fisher Scientific<br>
<a dir="ltr">5350 NE Dawson Creek Drive</a></span>
<div><span style="background-color:rgba(255,255,255,0)"><a dir="ltr">Hillsboro,
OR 97124</a> </span>
<div><span style="background-color:rgba(255,255,255,0)">Phone - <a href="tel:+1%20(720)%20322-6150" dir="ltr" target="_blank">+1 (720) 322-6150</a></span></div>
<div><span style="background-color:rgba(255,255,255,0)"><a href="mailto:francis.reyes@thermofisher.com" dir="ltr" target="_blank">francis.reyes@thermofisher.com</a></span></div>
<div><span style="background-color:rgba(255,255,255,0)"><a href="http://thermofisher.com/" dir="ltr" target="_blank">thermofisher.com</a></span></div>
</div>
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<div dir="ltr"><br>
On Feb 14, 2019, at 12:36 PM, Garry P Morgan <<a href="mailto:Garry.Morgan@colorado.edu" target="_blank">Garry.Morgan@colorado.edu</a>> wrote:<br>
<br>
</div>
<blockquote type="cite">
<div dir="ltr"><span>CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you recognize the sender and know the content is safe.</span><br>
<span></span><br>
<span></span><br>
<span>Hello all,</span><br>
<span></span><br>
<span>i’m looking for some info on the optimal dose rate for acquiring K3 movie stacks. i would appreciate it if any of you could send me your optimum acquisition parameters for imaging cry-samples for SPA…ie, dose rate, best frame rate/number of
frames, etc.</span><br>
<span></span><br>
<span>i’m also trying to figure out how to setup my low dose so that we have parallel illumination for our record images (on an FEI Tecnai F20). the problem i see is that in microprobe the beam is spread much too wide to image/expose a single hole
at a time at our record mag. is it advisable to work in nano probe to get a smaller beam that is parallel? any advice on this would be appreciated.</span><br>
<span></span><br>
<span>thanks,</span><br>
<span>Garry</span><br>
<span></span><br>
<span></span><br>
<span></span><br>
<span>________________________________</span><br>
<span>Garry Morgan</span><br>
<span>Director — Boulder EM Services — Department of MCD Biology</span><br>
<span>Campus Box 347 — University of Colorado — Boulder, CO 80309</span><br>
<span>phone: 303-492-8402</span><br>
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</blockquote></div><br clear="all"><br>-- <br><div dir="ltr" class="gmail_signature"><div dir="ltr">Israel S Fernandez<div>Columbia University, New York City, NY, USA</div></div></div>