Multishot Tomography using SerialEM script commands (old)

Following workflow describes Multishot data acquisition as depicted in Fig. 3 of Multishot Tomography preprint

../_images/manu_fig_3.png

Fig 1. Multishot tilt series acquisition provides high throughput way to sub-nanometer in situ subtomogram average from FIB-milled lamellae.

We want to achieve following goals:

  • Setup custom pattern for Multishot acquisition.

  • Modify SerialEM dose-symmetric acquisition script for Multishot acquisition.

Setting up custom Multishot pattern

We will use Multiple Record setup dialog. Please familiarize yourself with available options.

Open the dialog using Navigator > Montaging & Grids > Set Multi-shot parameters.

../_images/multishot_fig1.png

Fig 2. Multiple Record setup dilog in SerialEM

If you don’t have any saved custom pattern, Use custom pattern will be grey and show (NONE DEFINED).

First, we will setup a custom pattern to acquire three-shot (3X multishot) tilt series as depicted in Fig 1. For this purpose, one has to calculate Image shift for each shot as shown in Fig 3.

SerialEM/images/imageshifts_fig1.png

Fig 3. Image shifts for 3x Multishot acquisition.

To start, make sure your is Low dose mode Record [Rec]. Open a blank script using Script and type following script command.

ImageShiftByMicrons 0 0

Click on For Custom Pattern under section Save image shift values at holes

../_images/multishot_fig2.png

Fig 4. Setting up custom pattern

It will promt to Shift to first acquire position in the pattern. In this case the first acquire position is without any Image shift. Hence, we will continue by clicking Save Iamge Shift.

For the second shot we will have to Image shift by 1.2 µ. Edit the script to Image shift by 1.2 microns and execute using Run.

ImageShiftByMicrons 1.2 0

Continue by clicking Save Iamge Shift to save image shift for second shot.

For the third shot we will have to Image shift by -2.4 µ with respect to second shot. Edit the script to Image shift by -2.4 microns and execute using Run.

ImageShiftByMicrons -2.4 0

Continue by clicking Save Iamge Shift to save image shift for second shot. Click on End Pattern.

You can always Abort, and start again.

Once the custom pattern is saved you should be able to select checkbox for Use custom pattern.

Finally, Check following boxes:

  • Save Record image

  • Adjust beam tilt & astig to compensate for image shift

  • Use Record illuminated area for circles in Low Dose

In the Navigator menu, select Montaging & Grids > Show Shots when Show Acquire. you should be able to see the the custom pattern when you move the Navigator marker.

../_images/multishot_fig3.png

Fig 5. Multiple records shown for marker position.

Multishot acquisition script