Mapping Grid Squares

Goal:

Select grid squares of interest.
Setup microscope for medium magnification montages, with low dose and high contrast, to find features of interest.
Auto-acquire montages.

Shift and rotate the grid map

If the currently loaded grid is not the one that was collected last (meaning the current grid has been reloaded), there will be a shift and rotation between the acquired map and the grid which needs to be fixed.

Serial EM:

Navigator window, increase the current registration to 2.
Take a Record image.
Load the grid map.
Open Navigator menu, Align with Rotation dialog, set the angle to rotate to 0, search range 10, press Align to Map (TAKES A WHILE and SerialEM can freeze for some time!).
Toggle between the aligned image and map (‘Home’ and ‘Delete’ keys) to check the alignment.
If the alignment is good, press Apply Image Shift on Scope and Transform.

Setup optics for grid square mapping

SerialEM:

Load (double clock) imaging state "Grid square map" (EFTEM 2250, spot 7, MicroProbe, Ill. Area 35.3 um).

Titan UI:

Dial in -100 micron defocus.
We should see this in the bottom of the UI:

SerialEM:

For Eucentric Height usage, set Trial mode to bin 4 Linear mode, 0.1 sec, check the exposure time so Trial images are not overexposed (Camera & Macro Controls, Setup).
Record camera parameters should already been set by the imaging state, check if you see what you need to see, otherwise change optics accordingly.

Insert and center objective aperture

Titan UI:

Insert 70 micron objective aperture by clicking the Objective button, yellow means inserted.
FluCam viewer, Insert Screen.
Go into Diffraction mode, right hand panel, Diffraction.
Use the highest camera length, D 8.5 m.
Click in the FluCam viewer and scroll the mouse wheel up until you see the aperture around the beam. You may need to make the beam a bit smaller with Intensity knob on the left hand panel to see this pattern better.

NOTE: there should be some carbon in the beam, otherwise you do not see the white disk at all.

Click Adjust (next to Objective) in the Aperture control panel, use multi function buttons to center.
De-select Adjust in Aperture control panel.
Go out of Diffraction.
Load (double click) imaging state again, "Grid square map" (EFTEM 2250, spot 7, MicroProbe, Ill. Area 35.3 um).
FluCam viewer Retract Screen.

Fix shift between grid map and SA map

There will always be a shift difference between the grid map and the current settings which needs to be fixed.

SerialEM:

Add a point on some clearly recognizable feature in the grid map.
Navigator window, select the point, go to XYZ.
Take a Trial image.
Put the green marker on the actual feature you selected in the grid map (left click in SA map image).
Navigator menu, shift to marker.
Save the Navigator!

Select grid squares

SerialEM:

Select all grid squares to be mapped.

Broken squares are Problematic.
The centers of the squares need to have some carbon for the eucentric height procedure.

Make all points Acquisition points A.

For the first position, select New File at Item
Montage
3 x 4 covers a 200 mesh square. Smaller is faster, watch the total area size.
Name it "MMM_01.mrc".
In the Navigator window select "New File at Item" for all Acquisitions points, numbering will auto increase.

Run

SerialEM:

Navigator menu, Acquire at Points.
Select Rough Eucentricity.
Select Acquire map image.
Select Close column valves at end.
Select Send email at end.

This takes a while!!!

Save the Navigator file when things are done!!!