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 Low Dose Record mode.

SerialEM:

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

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

Setting up maps for all areas of interest

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. Keep in mind this is on the record area so do not use too much dose or too many images!!!!

When 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.

That's it, Focus/Tracking for eachpoint will be done later, so find next area of interest.

When done with all positions, close file alignimages.mrc:
File menu , Close.

With all areas of interest are setup/mapped

In the Navigator window, select the first position, click Shift + T.

Now select the very last View map, click Shift + T again.

Choose Single frame images:

Don't change anything:
Save as TS_001.mrc (as an example), click save.

Setup the tilt series controler, click here for some ideas first, when done click Ok.

Now all Preview maps are selected as TS (Tilt Series) with numbered file name TS_xxx.mrc:

Setting up the Focus/Trial positions

Double click the View map.
Select the Preview map.
Select Edit Focus in the Navigator window (Low Dose needs to be on!).
Click in the View map to set the Focus/Trial position for this tilt series.
The Navigator item should have TSP set now, continue for all points.

Focus target needs to be set per tilt series, no magic command yet, waiting for David.....

Save the Navigator file: Navigator menu, Save.