3.2.1 Pre-data
Collection Steps
Because the target is not tracked during tilting, both methods
require accurate eucentric height measurement prior to collecting
data to minimize field-of-view loss. While this is currently being
optimized, we describe one method to measure eucentric height
below.
1. Find and center the desired target in the field of view at
“Record” magnification in low-dose mode.
2. Perform a rough eucentricity adjustment routine to adjust the
Z-height to the eucentric point by clicking the “Eucentric–
Rough” command in the Tasks menu.
3. Run the “Realign to item” command to recenter the target in
the field of view after rough eucentricity.
4. Perform the fine eucentricity routine in the Trial area, at the
same magnification as data collection. First, check the “Use
Trial in LD Refine” command in the Tasks menu, then click the
“Eucentric–Fine” command. Ensure the Trial area is
unobstructed.
3.2.2 Continuous Tilting
Data Collection in SerialEM
For a unidirectional tilt-scheme, from À60
to +60
:
1. Set the exposure time in the Record settings to be long enough
to record the entire tilt-series.
2. Turn on dose fractionation and select an adequate time per
frame.
3. Tilt to À60
.
4. Run the command “TiltDuringRecord #A #D” to record while
tilting the stage to angle #A after a delay of #D in milliseconds.
For instance, TiltDuringRecord 60 1000.
For a bidirectional tilt-scheme, starting from 0
:
5. Set the exposure time in the Record settings to be long enough
to record each direction of the tilt-series.
6. Turn on dose fractionation and select an adequate time per
frame.
7. Tilt to 0
.
8. Run the command TiltDuringRecord À60 1000.
9. Return to 0
.
10. Run the command TiltDuringRecord 60 1000.
3.2.3 Fast-Incremental
Data Collection in SerialEM
SerialEM version 3.7 includes the FrameSeriesFromVar command,
which allows the collection of fast-incremental data given an input
array variable that describes each step of the tilt-scheme.
1. Generate the array variable containing the following information for each tilt, separated by spaces: Tilt Angle, Shutter open
time (exposure time), Wait time between step (before opening
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Mohammed Kaplan et al.
Collection Steps
Because the target is not tracked during tilting, both methods
require accurate eucentric height measurement prior to collecting
data to minimize field-of-view loss. While this is currently being
optimized, we describe one method to measure eucentric height
below.
1. Find and center the desired target in the field of view at
“Record” magnification in low-dose mode.
2. Perform a rough eucentricity adjustment routine to adjust the
Z-height to the eucentric point by clicking the “Eucentric–
Rough” command in the Tasks menu.
3. Run the “Realign to item” command to recenter the target in
the field of view after rough eucentricity.
4. Perform the fine eucentricity routine in the Trial area, at the
same magnification as data collection. First, check the “Use
Trial in LD Refine” command in the Tasks menu, then click the
“Eucentric–Fine” command. Ensure the Trial area is
unobstructed.
3.2.2 Continuous Tilting
Data Collection in SerialEM
For a unidirectional tilt-scheme, from À60
to +60
:
1. Set the exposure time in the Record settings to be long enough
to record the entire tilt-series.
2. Turn on dose fractionation and select an adequate time per
frame.
3. Tilt to À60
.
4. Run the command “TiltDuringRecord #A #D” to record while
tilting the stage to angle #A after a delay of #D in milliseconds.
For instance, TiltDuringRecord 60 1000.
For a bidirectional tilt-scheme, starting from 0
:
5. Set the exposure time in the Record settings to be long enough
to record each direction of the tilt-series.
6. Turn on dose fractionation and select an adequate time per
frame.
7. Tilt to 0
.
8. Run the command TiltDuringRecord À60 1000.
9. Return to 0
.
10. Run the command TiltDuringRecord 60 1000.
3.2.3 Fast-Incremental
Data Collection in SerialEM
SerialEM version 3.7 includes the FrameSeriesFromVar command,
which allows the collection of fast-incremental data given an input
array variable that describes each step of the tilt-scheme.
1. Generate the array variable containing the following information for each tilt, separated by spaces: Tilt Angle, Shutter open
time (exposure time), Wait time between step (before opening
100
Mohammed Kaplan et al.