Quick Tomography

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Name	Quick Tomography
Permalink	https://serialemscripts.nexperion.net/script/42
Category	Tomography
Language	SerialEM Scripting
Description	This script acquires a tomogram without additional tracking steps. The speedup compared to setting up a traditional tomo will typically be 30-50%. Currently works with FEI compustages only. A fast CMOS-based camera with drift/frame correction is recommended.
Author	Martin Schorb
Literature reference	https://doi.org/10.1038/s41592-019-0396-9

Version History

Version 2018.10 (October 10, 2018; 616 download(s); SerialEM version ≥ 3.7)

Version 2018.10

Download

Upload date	October 10, 2018
Download count	616
SerialEM version ≥	3.7

MacroName quicktomo_master
# EMBL Heidelberg 2016  Martin Schorb

########## SETTINGS ##########

#-----------------------------------------------------------

## ENTER THE TARGET DIRECTOY HERE, MAKE SURE IT EXISTS

dir @= D:\data\
# -----------------------------------------------------------

## DUAL AXIS TOMOGRAPHY ?

dual = 0            # 0 - single axis,   1 - dual tomo first axis,    2 - dual tomo second axis 
map = 0            # 1 - take additional map image for dual axis realignment

# ------------------------------------------------------------

## MINIMUM / MAXIMUM TILT AND TILT STEP (HM tomogram)

hm_step	  := 1	# stage tilt step in degrees
hm_maxtilt  := 60	# maximum/minimum tilt

# -----------------------------------------------------------

## MAGNIFICATION AND BEAM SETTING (for HM tomo)

hm_mag  := 15500     # magnification
hm_C2      :=  59.1            # Intensity C2 value in %

# ------------------------------------------------------------

## AUTOFOCUS SETTINGS

focus_step := 5 # how often to focus during tilt series (default: every 5 degrees)

# ------------------------------------------------------------

## LOW MAGNIFICATION TOMOGRAM (taken after the HM)

lowmag = 1        # 0 - no LM tomo taken, 1 - LM tomo acquired after HM, 2 - single LM image

# IF LM TOMO DESIRED:
## MINIMUM / MAXIMUM TILT AND TILT STEP (LM tomogram)

lm_step	  := 10	# stage tilt step in degrees
lm_maxtilt    := 60

## FOCUS, MAGNIFICATION AND BEAM SETTING (for LM tomo)

lm_focus  :=  -50               # target defocus for LM tomo (default -5)
lm_mag    :=  4700         # magnification
lm_C2        :=  71             # Intensity C2 value in %

# -----------------------------------------------------------------

# DELAY AFTER TILTING
# this is microscope/camera-specific. For our Tecnai-OneView, we use 150

tilt_delay := 150

########## END SETTINGS ##########

# start Script

ReportFocus foc1

ReportTargetDefocus def1

ReportSpotSize spot

NoMessageBoxOnError 1

ReportNavItem

# generate file names

If  $dual == 0 
   
   filename @= $dir\$navLabel_hm.st
   
 ElseIf $dual == 1
   
   filename @= $dir\$navLabel_hma.st
   
 ElseIf $dual == 2
   
   filename @= $dir\$navLabel_hmb.st
   lowmag = 0
   
Endif

# Echo $filename

OpenNewFile $filename

# Set HM microscope state

SetMag $hm_mag
SetPercentC2 $hm_C2
SetSpotSize $spot

# run high-mag quick tomo

CallFunction quickHMTomo-nav_AF

If $lowmag == 0
   
   TiltTo 0
   
 ElseIf $lowmag == 1
   
   #run additional low-mag tomt right afterwards
   
   filename @= $dir\$navLabel_lm.st
   #Echo $filename
   
   SetTargetDefocus $lm_focus
   
   OpenNewFile $filename
   
   SetMag $lm_mag     
   SetPercentC2 $lm_C2
   
   CallFunction quickLMafterHM-nav
   
 Else
   TiltTo 0
   
   If $lowmag == 2
      
      # acquire additional single low-mag image
      
      
      filename1 @= $dir\$navLabel_lm.mrc
      
      SetTargetDefocus $lm_focus
      
      OpenNewFile $filename1
      
      SetMag $lm_mag     
      SetPercentC2 $lm_C2
      
      G
      R
      S
      
      CloseFile 
   EndIf
   
   SetTargetDefocus $def1
   
   
   SetDefocus foc1
   ResetImageShift 
   ResetAccumShift 
   
   
Endif

# ================================================
# ================================================
# ================================================

Function quickLMafterHM-nav
   # EMBL Heidelberg 2016  Martin Schorb
   
   ########## SETTINGS ########## 
   
   step	= $lm_step	# stage tilt step in degrees
   maxtilt	= $lm_maxtilt	
   
   ########## END SETTINGS ########## 
   
   
   
   tilttimes = 2 * $maxtilt / $step
   step = $step
   tiltangle = -1 * $maxtilt
   G
   TiltTo $tiltangle
   
   T
   
   Loop $tilttimes
      
      TiltTo $tiltangle
      tiltangle = $tiltangle + $step
      
      # acquire tilt image
      TiltDuringRecord $tiltangle 0 5
      AlignTo B
      S
   EndLoop
   TiltTo 0
   ResetAccumShift
   NormalizeAllLenses 0
   CloseFile
   
EndFunction

# ================================================
# ================================================
# ================================================

Function quickHMTomo-nav_AF
   # EMBL Heidelberg 2016  Martin Schorb
   
   ########## SETTINGS ########## 
   
   step	= $hm_step	# stage tilt step in degrees
   maxtilt	= $hm_maxtilt	
   AF_step     = $focus_step
   
   
   ########## END SETTINGS ########## 
   
   
   ResetAccumShift 
   ResetImageShift 
   
   tilttimes = 2 * $maxtilt / $step + 1
   tiltangle = 1 * $maxtilt
   
   MoveToNavItem 
   
   Eucentricity 1
   UpdateItemZ
   
   ResetAccumShift
   NormalizeAllLenses 0
   ResetImageShift 
   
   # Realign after Eucentricity for better precision
   RealignToNavItem 1
   
   If $map == 1
      
      OpenNewFile $dir\$navLabel_map.st
      G
      R
      S
      NewMap 
      CloseFile 
   EndIf
   
   # calculate where to take AF
   
   c = 0
   
   div = $maxtilt / $AF_step * 2
   
   Loop $div
      
      a = $maxtilt - $AF_step * $c
      c = $c + 1
      AFv$c = $a
   EndLoop 
   
   WalkUpTo $tiltangle
   
   Loop $tilttimes
      
      TiltTo $tiltangle                 
      
      # check if at AF tilt angle
      c = 0
      Loop $div
         c = $c + 1
         testvar = $AFv$c
         If $tiltangle == $testvar
            Copy A O
            G
            Copy O A
         Endif 
      Endloop
      
      tiltangle = $tiltangle - $step
      
      # acquire tilt image
      TiltDuringRecord $tiltangle 50
      AlignTo B
      S
      Delay $tilt_delay
   EndLoop
   
   ResetAccumShift
   NormalizeAllLenses 0
   
   CloseFile
   
EndFunction

Documentation

Quick Tomography

Acquires a tomogram without additional tracking steps. We acquire tilt series from + to -60 deg with 1 deg increment in about 7 minutes using a TF30 and a OneView camera.

* The script will name the files according to the Navigator Label.

* It allows to automatically append -a or-b depending on the axis for dual tomo.

* A second tomo with lower mag and different tilt increment or defocus can be acquired right after the first in opposite tilt direction.

License

This code can be shared and adapted under the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (https://creativecommons.org/licenses/by-nc-sa/4.0/)