Setup positions

Goal:

Find targets for acquisition.
Take your time and make the right choices here!
Possible strategy: select everything that you think is ok, then revisit all a few times to be sure.
Setup targets to acquire tilt series: we expect to be in properly setup Low Dose mode.

Find positions

NOTE: when setting up positions, the imaging conditions are saved and can not be changed later, so make sure dose, dose rate, exposure time, beam size, frames etc. are setup properly!!!

SerialEM:

Close all open files.
Navigator window, Add Points.
Navigator window, double click in any grid square map to open it (can take a few seconds).
Search for targets, the display behaves like IMOD, + and - to zoom, left-click-hold to drag the map, single click to add point (when Add points is selected).
De-select Add points.

Setup positions for batch acquisition

Titan UI:

Dial in -4 micron defocus in Record mode.

SerialEM:

For lamellae, make sure the stage is at the pre-tilt!
In Imaging State control panel, click "forget prior state".

In File menu open new (first time) or existing file called alignimages.mrc

In Navigator window, select a point, Go to XYZ.

Center feature with View image: right click and drag image.
Center feature with Preview image: right click ad drag image.

we centered: Navigator window, New Map (= Preview image), first time will throw a dialog about saving the image, ok.

First time Anchor Map: take a View image, In navigator window click New Map, select For anchor state.
Next time Anchor map: click Anchor Map, SerialEM will automatically acquire a view image and save it as anchor map.

NOTE: the View image may look slightly shifted compared to the Preview image, this is not a problem! Final alignment when revisiting this position for acquisition will be done on the Preview image map.

Take another View image, set the Low Dose Trial/Focus position (Low Dose Control, Define position of area).

In the Navigator window:

Select the Preview image map as acquisition point "Acquire (A)".
Select "New file at item", Single Frame Images.
- First time you have to give it a name, “TS_01.mrc”).
- It will auto number on later selections.
Click "Imaging State" to save the previously set Trial/Focus position.

Save the Navigator file: Navigator menu, Save.
Close file alignimages.mrc: File menu , Close.