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

[Anchi Cheng]

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