[3dem] VPP phase development curve

[Radostin Danev]

Hi Jian,

The "good" phase shift range is between 0.2 and 0.8 pi, which translates to
35 to 145 deg. Please check our recent paper:
https://elifesciences.org/content/6/e23006

To reduce the beam current and keep the dose on the sample the same you have
to shrink the beam. The practical limitation is, if your beam is too tight
around the camera, you'll start seeing Fresnel fringes coming in from the
corners of the image.

Cheers,

Rado


-----Original Message-----
From: jamon [mailto:jamonshi at gmail.com] 
Sent: 24 February 2017 09:54
To: Radostin Danev <danev at biochem.mpg.de>; 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] VPP phase development curve

Thanks Rado for detail info.

Since the phase shift will never stay put, what typical range of phase shift
would give good data for high resolution reconstruction, say
80-100 degree?  Or it can be much broader?

I do notice sometime the output of CTFFIND4 and GCTF are not correct. 
Normally I will go back to change the parameter to retrial.

My heat is at the maximum setting now. My beam current is 0.5nA, do you mean
EXPAND the beam to reduce the beam current?

Thanks and Regards,
Jian
On 02/24/2017 04:01 PM, Radostin Danev wrote:
> Hi Jian,
>
> If you see rapid jumps in the phase shift curve that usually means 
> that some of the CTF fits failed. Did you look at the diagnostic 
> output? Do the CTFs look reasonable? Try limiting the defocus search 
> range to something like
> 3000 - 7000 A, assuming you collected with 500 nm target defocus. 
> Also, set the low resolution limit for the fits to 20 A.
> In principle there is no way to stabilize the phase shift. It will 
> gradually increase with the dose on the VPP. There are two ways to 
> slow down the phase shift development: increase the phase plate 
> temperature and/or reduce the beam current. The maximum recommended 
> heater settings are (somebody from FEI please correct me if I'm wrong) 
> 4.2 V and 25 mA. The current is the more critical setting, please do not
exceed 25 mA.
> To reduce the beam current you could try to shrink the beam. We 
> typically use beam diameters ~1.2 um with beam currents 0.100 - 0.200 
> nA. The dose on the phase plate per image is = (beam current in nA) x
(exposure time in s).
> The dose you mentioned is the dose on the sample and that is not 
> representative of the total dose on the phase plate.
>
> Cheers,
>
> Rado
>
>
> -----Original Message-----
> From: 3dem [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of jamon
> Sent: 24 February 2017 03:27
> To: 3dem at ncmir.ucsd.edu
> Subject: [3dem] VPP phase development curve
>
> Dear Lister,
>
> Does anyone calibrated the VPP phase development curve during data 
> collection, using ctffind4.1.x or gCTF? I ran some test on Krios and 
> notice the phase changes rapidly from 0 degree to 90 degree with less 
> than <500e/A2, then reach 120 degree at 1000e/A2. Later the phase 
> shift moves much slower from 120 degree to 140 degree within 5000e/A2. 
> It suggest that the usable dose window is very narrow. Is there a way 
> to control phase shift stable at 90 degree instead of 120 degree? Your 
> suggestions are appreciated in advance.
>
> Thanks and Regards,
>
> Jian
>
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