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

[Grassucci, Robert]

Hi Sayan,
Anchi's suggestion that it may camera related triggered a thought that it might be a camera cooling problem.  If the camera temperature fluctuates too much then it can greatly affect the bias of the camera.  Check your water temperature and flow rate.  Just another one of the many suggestions you can throw at the FEI engineer.
Best,
Bob


-----Original Message-----
From: 3dem [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of Anchi Cheng
Sent: Tuesday, September 06, 2016 2:22 PM
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] Regarding Image Quality from FEI Tecnai G2 Polara

Hi, Sayan,

Some of your problems seem more camera related than microscope.

My reasoning are the followings:

> 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.
> 
As Bob pointed out, the biggest change of intensity caused by tip terracing is a relatively short event (hours), and it recovers afterward.  You should monitor the intensity and room temperature nevertheless.  With single camera, and with the suspicion of problem with it, I would recommend put the screen down with a fixed condition (spot size, beam spread) in empty area (vacuum) several times a day if you want to isolate the problem to tip.

I could not tell if this observation is of every session that last two days or just from one single session.
If it happens every time you try a long run, it is unlikely a tip terracing mainly because the odds of your session in sync with the tip terracing is small.  You should take into consideration differences in operation as you prolong your scope session.  For example, gun and column alignment stabilize over time.  If you always realign at the beginning of the session and tend to adjust a lot, the scope may settle to a different value by second day.

> 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.
> 

This observation points to camera problem.  Other than the objective aperture and cryo box aperture, the scope has no way to introduce a cut above LM mode.  Again, check directly on the scope in diffraction mode with something that scatter enough (carbon, thicker ice, or grating replica) first to rule that out.  In addition, you can take an image at a very different magnification but the same lens series.  If you get the same cut in your 1D spectrum, it comes from the camera.

> 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.
This is a typical camera bias problem. Eagle and other multi-port camera stetches sensors together.
The base value (bias) of these sensors may not be the same.  Therefore, a correction is needed in its software.  If you see becomes more frequent than spec and becoming unbearable, you should have it serviced.  I do note that unless it becomes so severe that it takes it out of the camera dynamic range, your data would be o.k.  I point this out because I know an Eagle camera with this problem and was never fixed quite right but still gave usable data….

Anchi Cheng
Research Scientist
National Resource for Automated Molecular Microscopy Simons Electron Microscopy Center New York Structural Biology Center _______________________________________________
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