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

Thu Feb 14 19:38:37 PST 2019

Here's how pixel size changes  at given C2 values as a function of changing
Z on the arctica.

a 1% change in pixel size may not matter much for apoferritin, but it
probably will for ribosomes and viruses.

[image: TalosParallelicity_spot4.png]

On Thu, Feb 14, 2019 at 7:44 PM Craig Yoshioka <yoshiokc at ohsu.edu> wrote:

> 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>
> 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> 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>
> 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> 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> 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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