[3dem] K3 optimal dose rate & F20 parallel illumination

[Israel Fernandez]

Hi there,


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.
Hope it helps.

El jue., 14 feb. 2019 a las 15:20, Garry P Morgan (<
Garry.Morgan at colorado.edu>) escribió:

> Craig,
> thanks!  that gives me a good place to start…
> i’ll let you know what we see here when pushing the FPS up above 40.
> Garry
>
>
> On Feb 14, 2019, at 12:57 PM, Craig Yoshioka <yoshiokc at ohsu.edu> wrote:
>
> Hi Gary,
>
> 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.
>
> 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.
>
> Cheers,
> -Craig
>
>
> On Feb 14, 2019, at 11:45 AM, Garry P Morgan <Garry.Morgan at Colorado.EDU>
> wrote:
>
> thanks everyone.
>
> On Feb 14, 2019, at 11:53 AM, Reyes, Francis <
> francis.reyes at thermofisher.com> wrote:
>
> 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.
>
>
> Francis Reyes, Ph.D
> Engineer III, Field Applications
> Materials & Structural Analysis
>
> Thermo Fisher Scientific
> 5350 NE Dawson Creek Drive
> Hillsboro, OR 97124
> Phone - +1 (720) 322-6150 <+1%20(720)%20322-6150>
> francis.reyes at thermofisher.com
> thermofisher.com
>
> On Feb 14, 2019, at 12:36 PM, Garry P Morgan <Garry.Morgan at colorado.edu>
> wrote:
>
> 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.
>
>
> Hello all,
>
> 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.
>
> 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.
>
> thanks,
> Garry
>
>
>
> ________________________________
> Garry Morgan
> Director  —  Boulder EM Services —  Department of MCD Biology
> Campus Box 347  —  University of Colorado  —  Boulder, CO 80309
> phone:  303-492-8402
>
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-- 
Israel S Fernandez
Columbia University, New York City, NY, USA
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