High and Low Resolutions Alignments                zhy, jpg,vdb2010

                           

Direct Alignments for Tomography and lower-resolution Single Particle work

FEI engineers or the supervisor will set most of the Titan alignments. As these alignments are very stable, the user only needs to adjust beam tilt pivot points (pp) and rotational center before collecting a tomography tilt series or lower resolution single particle data (worse than 10 angstroms).

1. Align Beam Tilt Pivot Points

a) locate a test region on the sample

1. insert the screen

2. find an area of carbon film that can be imaged with high dose

3. set sample at eucentric height

b) remove objective aperture.

c) set the magnification to data collection mag. or higher (i.e. 96,000 or 120,000)

d) press eucentric focus button to set objective lens current approximately to focus

e) condense the beam to a spot or a small disk.

f) go to Direct Alignment control panel

1. click on beam tilt pp x

2. minimize beam movement (wobble)using Multi Function Knobs (MFs )

3. usually only MF X needs adjusted, not Y).

4. repeat this for beam tilt pp y.

2. Set Beam Shift (still in Direct Alignment) (when running FEI tomo, adjust tomo beam shift instead)

a) expand the beam with Intensity knob

b) use MF knobs to center the expanded beam (NOT user beam shift, or will change back later when leave Direct Alignment page)

3. Set Rotational center (still in Direct Alignment)

a) click on rotation center on the Direct Alignment control panel

b) use MF knobs to minimize any sideways movement of features in the image (when running FEI tomo, adjust tomo rotation center instead of rotation center)

4. click on Done on the Direct Alignment work page

  1. this leaves Direct Alignment and saves the settings
  2. it also releases the MF knobs so the alignments don’t get changed accidentally

         

After direct alignment:

5. Center Objective Apeture

  1. Insert the 100 um objective aperture
  2. press Diffraction on the control pads to see if the aperture is centered.        
  3. If not centered, center it use the Apertures work panel (OCX) and MFs
  4. press Diffraction again to get back to imaging mode                 

6. Check Objective Astigmatism  

1. retract the screen

2. click Preview on the CCD/Camera control panel to get an unbinned live image of carbon film on the CCD in Digital Micrograph

3.generate a live FFT to check objective lens astigmasm

4.use a stronger beam and larger under focus If Thon rings are difficult to see

5.Use Stigmator control panel and MF knobs to fix the objective lens stigmasm first at larger under focus, then fine tune it at closer to focus. The CTF central circle and Thon rings will be absolutely round when stigmatism is correctly adjusted.

6.check objective astigmatism often, especially after moving from one grid square to another

Coma-free alignment for high (atomic) resolution Single Particle work

Coma-free alignment of the Titan is used for high-resolution data collection. It may be more precise than rotation center alignment and make a difference for atomic-resolution imaging. Rotational center and coma-free alignment are two different, independent methods to correct the same parameter, the tilt angle of the electron beam-- to make it parallel to the optical axis of the objective lens.  It is also necessary when selecting beam intensity to pick a beam size that is in the PARALLEL RANGE for that mag. and spot size.  Using a beam NOT in the parallel range means that some of the beam will not be IN PHASE.  It is crucial for high resolution work that the beam be in phase to prevent artifacts which will limit resolution.    

Rotational center alignment does this by wobbling the objective lens current and adjusting to minimize side-ways movement of image features. The coma-free alignment procedure wobbles the beam tilt between pre-defined positive and negative angles, producing 2 images. The goal of coma-free alignment is to make the two images have the same apparent defocus while the beam tilt wobbles.

It is only necessary to do one rotational center alignment or the other, not both.

1. Align Coma-free Pivot Points

a) locate a test region on the sample

1. insert the screen

2. find an area of carbon film that can be imaged with high dose

3. set sample at eucentric height

b) remove objective aperture.

c) set the magnification to high mag. 250,000x

d) increase beam intensity and burn off ice so only carbon film is left.

d) press eucentric focus button to set objective lens current approximately to focus

e) condense the beam to a spot or a small disk.

f) go to Direct Alignment work page

1. click on coma-free pp x

2. minimize beam movement (wobble)using MFs

3. usually only MF X needs adjusted, not Y).

4. repeat this for coma-free pp y.

g) click on Done on Direct Alignment OCX to get out of Direct Alignment

1. this leaves Direct Alignment and saves the settings

2. it also releases the MF knobs so the alignments don’t get changed accidentally

h) expand the beam on the screen with the Intensity knob.

6. Check Objective Astigmatism

a) retract the screen

b) click Preview on the CCD/Camera OCX to get an unbinned live image of carbon film on the CCD in Digital Micrograph

c) generate a live FFT to check objective lens stigmasm

d) use a stronger beam and larger under focus If Thon rings are difficult to see

e) Use Stigmator OCX to correct the objective lens astigmasm first at a larger under focus, then fine tune it at closer to focus. The CTF central circle and Thon rings will be absolutely round when stigmatism is correctly adjusted.

7. Correct Coma- Free Alignment

a) keep the live imaging (“Preview”) and live FFT on in Digital Micrograph

b) adjust the Focus knob to get close to focus.

c) click on coma-free alignment x on the Direct Alignment OCX

1. The live images and live FFT will wobble between two images taken with a positive beam tilt and a negative beam tilt.

2. Use the MF knob X to make the “apparent” defocus in the two images identical.

(Cont. page 7)

a. Adjust MF X to make the Thon rings in the two FFT images have the same size

b. It may be helpful to make the difference between the two images more obvious by first making the alignment worse by adjusting the MF X knobs a few turns randomly so that one live FFT has more Thon rings than the other one.

c. only MF X knob needs to be adjusted, the MF Y is not linked to anything for this alignment

3. The two FFT images may seem to be astigmatic, as the Thon rings are oval and their orientations are different. This is a result of the tilted beam going through the objective lens (not necessarily due to true stigmasm).

4. These oval-shaped Thon rings and different orientations can be ignored as long as the size of the ovals is the same.

5. if only one image is present, and the other is black- the objective apeture is blocking the beam at one of the tilts. Remove it and try again.

d) repeat the above procedure for coma-free alignment Y.

e) click Done button to get out of Direct Alignment (so alignment is saved).

8. Check Objective Apeture Centering

a) insert the screen (to avoid strong diffraction beam hitting the CCD);

b) Insert the 100 um objective aperture;

c) press Diffraction on the control pads

d) check if the aperture is centered. If not centered, center it use the Apertures OCX and MF knobs. e) press Diffraction again to get back to imaging mode.

9. Check Objective Astigmatism

a) retract the screen;

b) click Preview on the CCD/Camera OCX and live FFTin Digital Micrograph

1. get an unbinned live image of carbon film on the CCD in Digital Micrograph

2. generate a live FFT to check objective lens stigmasm one more time

3. correct as before as necessary