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

Thu Feb 14 16:44:10 PST 2019

yes, but the point is the beam might be huge when perfectly parallel in uP, but if you resize it to something like 1.2um, it might actually be closer to parallel than if you resized the perfectly parallel beam in nP at 1.8um down to 1.2um.

On Feb 14, 2019, at 4:40 PM, Mariena Silvestry Ramos <ms3289 at cornell.edu<mailto:ms3289 at cornell.edu>> wrote:

Same in our experience, Gabriel. For 1.2/1.3 grids, even at 50um C2, the beam illuminated probably 5/6 holes so that would fry everything around the ROI. When we consulted your notes, it was the exact same behaviour. Thanks for that paper. It rocks!

Sent from my Game Boy

On Feb 14, 2019, at 7:32 PM, Gabriel Lander <glander at gmail.com<mailto:glander at gmail.com>> wrote:

Just set the appropriate beam size for parallel illumination in nP at the start of your session, save the setting, collect for a day (or a week if you have that luxury), and solve your < 2Å resolution structure. The beam size shouldn't change and is very stable in our experience. On our Arctica in microprobe the beam is massive, even with a 50uM C2 aperture, which would limit the number of images you can acquire in an area using image shift.

On Feb 14, 2019, at 4:16 PM, Israel Fernandez <israel.elotro at gmail.com<mailto:israel.elotro at gmail.com>> wrote:

hi again,

We operate the Polara and the F20 in microprobe, with small C2 apertures [50um for the F20, and 30um for the Polara, if i remember well...] and we have the feeling is pretty close to parallel. Win Hagen reported in twitter a under 2A reconstruction of apo-fe in microprobe AND FalconII in linear mode...so...

On Thu, Feb 14, 2019, 18:56 Craig Yoshioka <yoshiokc at ohsu.edu<mailto:yoshiokc at ohsu.edu>> wrote:
Oh, I have one comment about nP vs uP.  Yes, you can get a smaller parallel beam in nP -but- it is also way more sensitive to changes in beam size in nP.  As a result, we have been able to get below 3Å on an Arctica in microprobe, even when the beam size was not set to be perfectly parallel- it remains fairly close over a pretty large range.  This is a way more convenient configuration for a lot of use.

On Feb 14, 2019, at 11:57 AM, Craig Yoshioka <yoshiokc at ohsu.edu<mailto: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<mailto:Garry.Morgan at Colorado.EDU>> wrote:

thanks everyone.

On Feb 14, 2019, at 11:53 AM, Reyes, Francis <francis.reyes at thermofisher.com<mailto: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<tel:+1%20(720)%20322-6150>
francis.reyes at thermofisher.com<mailto:francis.reyes at thermofisher.com>
thermofisher.com<http://thermofisher.com/>

On Feb 14, 2019, at 12:36 PM, Garry P Morgan <Garry.Morgan at colorado.edu<mailto: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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