[3dem] Regarding Image Quality from FEI Tecnai G2 Polara

[Grassucci, Robert]

Hi Sayan
What is the vacuum in your projection chamber?  Generally contamination comes from the projection chamber assuming there is no leak previously.  Did the falcon camera just get installed?  That could be the source of your contamination.  Do you use film?  If it is not desiccated then that could be the source but if you have a falcon you probably do not use film.  Since the cryo box on the Polara has a relatively large hole in the bottom use of an objective aperture can help restrict the area that is contaminated on your grid.  Do you keep Valve B open and the MSC attached?  That could also be your vacuum leak.  Good luck.
Best,
Bob


From: Sayan Bhakta [mailto:19sayanbhakta89 at gmail.com]
Sent: Tuesday, September 06, 2016 1:35 PM
To: Grassucci, Robert
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Regarding Image Quality from FEI Tecnai G2 Polara

Hi Bob,
Thanks for your suggestion. I would surely convey the message to the FEI engineer.
Apart from this, we also have another issue and that is constant ice formation inside the column. The engineer who has come, kept a grid inside the column in the weekend and on Monday there was ice contamination formed onto the grid. After that when we inspect, we see continuous decrease in quality of images taken subsequently on the first day itself. This might be because of the fact that the ice starts growing from the day 1 itself. We don't see it on the screen because of the beam may be??? As suggested by many there must be some leakage in the column. But the vacuum of the system is constant at 6. May be that minor leakage is somewhere undetectable. If you share your view on this, It would be highly appreciated.
Sayan

On Tue, Sep 6, 2016 at 8:25 PM, Grassucci, Robert <rg2502 at cumc.columbia.edu<mailto:rg2502 at cumc.columbia.edu>> wrote:
Hi Sayan,
I believe what you are seeing is terracing or ring collapse.  This happens when the feg tip shape goes from a sharp 9high intensity) tip to a more blunt (dim) tip.  Sometimes you will even see it split and look like 2 sources.  This happens at our facility about once a month so we monitor the intensity very carefully.  The good news is that it generally recovers quickly and then remains stable for a long while.  FEI service engineers know about this phenomenon and have some things to do to make it less frequent.  One is to decrease the tip temperature and the other is to increase the extraction voltage.  You can also reshape the tip by bringing down the Ht and extraction and then bringing it back up  but this does not always work well.  If your tip is damaged then it just may need to be changed. Speak to your engineer so he can correct this.   I hope that helps.  Good luck.
Bob


From: 3dem [mailto:3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>] On Behalf Of Sayan Bhakta
Sent: Saturday, September 03, 2016 6:39 AM
To: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Subject: [3dem] Regarding Image Quality from FEI Tecnai G2 Polara

Hi,
We are facing many problems with our Cryo EM facility. I am giving some details of the machine and one of the main problems we are facing. It would be very helpful if people comment on this and help us to find the actual problem.
Machine:
FEI Tecnai G2 Polara
300Kv
Have been running for last two years.

Camera:
FEI Eagle 4k*4K

Problem:
On the first day when we are putting our sample into the Cryo EM, we are getting good images. From the second day the quality of the images deteriorates.
1.  If we look at the images taken on the first day and second day, the intensity values of the images decreased on the second day.
2.  From the FFT, we see nice thorn rings from the images taken on the first day. But for the images taken in the second day, the FFT is very bad. Most of them are having drifts.
3. Irrespective of the days, there are some images for which the 1D spectrum is having a cut in between. What I mean by that is there is spectrum upto certain distance in the 1D spectrum, then there is no waves, then again there are some waves.
4. Irrespective of the days, images are sometimes with blakish shadow on one side and with whitish shadow on other side. We need to do gain correction more frequently than usual- a number of times in a day.

For your information:
1. The vacuum is stable on the second day.
2. We are giving Cryo-cycle each weekend.
If anyone has ever faced the same problem or has any advice on this regard, it would be very helpful. We have been struggling very hard to standardise many issues with our microscope.
--

Sayan Bhakta

Research Scholar

Group of Dr. Jayati Sengupta

CSIR - Indian Institute of Chemical Biology

Kolkata, West Bengal

India



--

Sayan Bhakta

Research Scholar

Group of Dr. Jayati Sengupta

CSIR - Indian Institute of Chemical Biology

Kolkata, West Bengal

India
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