A Detailed Introduction to the Basic Operation of SerialEM

1
A Detailed Introduction to the Basic Operation of
SerialEM
2
Image Buffers in SerialEM

• Images are saved in buffers, referred to as A
  N
• Camera acquisitions and processed images go into
  buffer A, and the top few images are rolled into
  higher buffers
• Typical situations (enforced during tilt series)
  are

Normal operation
Low dose mode
(Home) (Insert) (Delete) (End)
(Home) (Insert) (Delete) (End)
A B C D E G H
New image -gt
New image -gt
A B C D E F G H
Align reference
Record reference
Read-in image -gt
Trial reference
Read-in image -gt
3
Image Display Panel

• The available data range in the displayed image
  is controlled by the Black and White levels
  everything outside this range is truncated
• To determine Black and White, it analyzes pixels
  in a central area and truncates a certain
  fraction
• Contrast and brightness are of limited usefulness
  because of this truncation
• Most useful option is to set Area Fraction to
  smaller value, like spot metering on smaller
  central area
• To see contrast at extreme end of range, enter
  image minimum as Black value or image maximum as
  White value
• Zoom pretty much takes care of itself with
  default settings

4
Microscope Status Panel

• Screen current is more useful than exposure time
  Float button floats a screen meter, good when
  tuning scope
• Dose button floats a cumulative dose meter
• The Defocus is based on a microscope readout, not
  the actual focus on the specimen
• On Tecnai, it matches TUI defocus reading, can be
  reset in TUI or SerialEM
• On JEOL, it is arbitrary and set to zero on
  program startup, can be reset
• IS is image shift in microns, important to know
  on 300KV Tecnai and JEOL 2100 without high power
  image shift

5
Tilt Control Panel

• The delay time is the delay after tilting by
  basic increment delay will be longer or shorter
  for bigger or smaller tilts

Camera and Macro Tools

• Buttons for camera parameter setup and 5 camera
  acquisition modes (good for low dose)
• STOP is the one general stop button for all kinds
  of operations
• Preview morphs into Montage when montaging,
  unless low dose is on
• Macro buttons also morph into useful buttons
  during tilt series

6
Camera Parameter Setup

• Continuous acquisition is only mildly useful (no
  access to fast camera modes)
• Processing options useful for testing and
  troubleshooting otherwise always use gain
  normalization
• Binning is used for speed. Tracking correlations
  are always binned down to 512 so there is no
  point taking Trials bigger than 512.
• A full-width image gives fastest readout per
  pixel hence the wide options for fast subarea
  pictures
• A separate dark reference is kept for every
  binning and exposure time.
• Force new dark reference next time is the way
  to remove a dark reference
• Averaging dark references is helpful for a
  high-noise camera (not typical)

7
Pre-exposure

• Pre-exposure (drift settling) requires two
  shutters
• The Gatan shuttering mode that allows
  pre-exposure is inflexible and exposes specimen
  during readout so SerialEM uses beam blanking
  to provide selected pre-exposure while blanking
  during readout
• Dual shuttering minimum exposure mode
  provides for this and just does beam blanking
  when drift settling is 0 so it can always be
  used
• But the camera timing must be calibrated so the
  beam blanking does not occur during exposure time
• Tietz cameras require Shutterbox II for
  pre-exposure to work

8
Image Alignment Focus

• Autoalign always changes microscope image shift
  Clear alignment undoes that
• Reset Image Shift moves the stage to keep the
  same specimen point centered
• Moving stage for mouse shifts avoids having to
  reset image shift
• Happens automatically if option selected and
  movement is above either absolute (micron) or
  relative (fraction of field) threshold
• You can do it intentionally with Shift key
• Trimming dark borders keeps autoalign from being
  thrown off by beam edge or grid bar
• Used automatically in low dose and during tasks
  done at lower than working mag
• Will not detect less extreme contrast differences
  that can throw off autoalign alternative is to
  use smaller area (higher trim fraction)
• Center image shift on tilt axis will give fewer
  movements during tilt series but requires an
  offset calibration

9
Autofocusing with Beam Tilt

• Autofocus is done by taking two images with the
  beam tilted in opposite directions and measuring
  the shift between images
• It relies on the microscope being aligned such
  that the image of a specimen in the focal plane
  of the objective lens does not move when the beam
  is tilted.

A
Specimen in focal plane
Specimen out of focus
B
C
Image plane
Image plane
A appears here with both directions of tilt
B or C appears here depending on direction of
tilt
10
Focus Parameters and Operations

• The target is the defocus it sets to after
  measuring
• The beam tilt needs to be big enough to get good
  image displacements, but is limited by objective
  aperture
• Autofocus Offset lets you measure defocus at
  focus different from target
• Can protect against correlation failures by
  focusing at zero
• Can be used to focus at a defocus with more
  contrast
• Taking three pictures lets it subtract off drift
  Drift protection
• Report shift and drift takes the three pictures
  and just tells you the beam-induced shift and the
  drift
• Report on existing reports the shift and drift
  from pictures just acquired
• Autofocus measures and changes to target
  Measure defocus just measures without changing
• Autofocus sometimes works poorly at high tilt -
  Check autofocus assesses what fraction of a
  known focus change can be detected
• Move Focus Center lets you click on spot at
  desired focus and change focus so center of field
  is at that focus

11
File Operations

• You can open new image files or existing files
  (up to 8 now)
• Opening a new file brings up File Properties
  dialog, where you set the data type and items to
  save in header
• Anything selected for saving can be extracted
  with extracttilts (options -st, -ma, or -in for
  stage position, mag, or intensity)
• After opening an old file, the Set options can
  be used to change the properties that would be
  set in the File Properties dialog
• Save to Other and Read from Other can be used
  to save one image or read from a file without
  leaving it open
• You can save an image to a specific Z value with
  Overwrite
• Saving a log is useful. Read Append lets you
  accumulate a log across several runs of the
  program

12
Camera Commands

• The most commonly used item in the camera menu is
  Prepare Gain Reference
• Gain reference can be done at typical exposure
  for non-low dose work
• Having lots of counts averaged (e.g., 10 x 1/3
  of dynamic range) is more important than matching
  the counts for actual images
• Binned gain reference works for normalizing
  images with even binning useful for slow 4K
  camera that is being used as 2K camera
• If camera counts per electron is calibrated, this
  can do automatic dose calibration
• It can be useful for troubleshooting to view gain
  reference or dark reference of image just taken
• Post-actions controls whether image shift, mag
  change, or stage move are taken during camera
  readout
• Requires correct camera timing parameters and
  with that, it is surprisingly robust
• But it can be turned off to troubleshoot problems

13
Montaging The Camera Is No Limit

• To start montaging, define the number of frames
  in X and Y in Montage Setup dialog
• Parameters start at current state, based on
  current Record parameters
• The default overlap (10) is fine for image-shift
  based montage
• Once data has been taken, or if you open an
  existing montage, most parameters in the dialog
  are locked
• After taking a montage, you will see overview
  in buffer B, a subsampled version of the whole
  area
• A (composed) image of the center is left in A for
  alignment
• Either image can be mouse-shifted
• Prescan takes a fast highly binned montage of
  the same area without storing it
• Options are mostly important when using montages
  as navigation maps, except
• Change focus with height will take each picture
  at same focus, but this may make it harder to
  correct for distortions due to magnification
  gradients

14
Image Processing

• In general, processing works on the active image,
  the processed image is put in A, and the top
  images are rolled
• Binned FFT bins the image to 512 pixels
• It is faster, less noisy but only shows central
  part of frequency space
• Probably most useful for assessing drift in image
• Live FFT will do FFT immediately in Continuous
  acquisition mode (version 2.6.4?)
• X-ray items are to help set up removal of X-rays
  in dark references and images
• X-ray removal from images should only be done for
  low-dose images from low gain cameras where
  X-rays screw up tracking
• Show cross-corr will do an autoalignment with
  current A and align buffer images, then show
  correlation

15
Tasks

• Tasks are (mostly) operations required for doing
  tilt series, involving a sequence of stage
  movements and tracking images
• Eucentricity has two different routines
• Rough eucentricity will work from 100 µm away or
  more in Z
• Refine eucentricity does a 24 tilt series to
  measure Z height with lateral offset of tilt axis
  taken into account and estimated also
• Works up to 10 µm away or more
• Refine Realign runs it in a way that restores
  the image position at the end
• Can use result to Set Tilt Axis Offset

Optic axis
Tilt axis
24
?Z
Axis offset
Specimen feature
0
-24
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Tasks 2

• Walk Up goes from one tilt to another while
  keeping an image feature centered
• Anchor is an image at intermediate tilt that
  can be a reference when coming back down in a
  tilt series
• Reset IS Realign and Reverse Tilt keep an
  image feature centered when resetting image shift
  or reversing direction of tilt
• Set Intensity lets you change intensity based
  on the counts in the current image
• Enter a factor to change intensity by (e.g.,
  0.75, 1.5)
• Or enter desired counts in a Record image (e.g.,
  4000)
• Move Beam - if you click at the center of the
  beam, this will move the beam to the center of
  the field

17
The Problem of Tilt Series Acquisition

• The goal in acquiring a tilt series is to have
  every picture centered on the same specimen
  feature, and at the same focus
• The basic sequence of operations required is
  thus
• Tilt to a new tilt angle
• Correct change in specimen position
• Correct change in specimen focus
• Acquire final (Record) image
• For full automation, the desired features must
  stay centered during initiation and after
  interruptions of the series the tasks are
  components for ensuring this

18
Approaches to Automated Tomography

• Traditional approach track and focus at every
  tilt
• Used in original Agard/Koster software and in
  Tietz software
• May have to track at low mag time-consuming
• Precalibration take a coarse tilt series to
  determine X, Y, and focus, then take full tilt
  series without tracking or focusing
• Used in new Koster software for Tecnai and in
  FEI software
• Can be fast but is susceptible to drift and
  calibrations being off hard to accommodate user
  intervention
• Z-prediction calibrate tilt axis location and
  assume specimen moves in a circle around it (new
  Agard method)
• - Very fast, but many scopes do not meet the
  assumptions
• Robust prediction predict X/Y/Z position on
  next tilt from changes in position on previous
  tilts
• Must adapt to non-ideal conditions and user
  interventions
• Goal is to achieve the reliability of
  traditional approach and speed of precalibration

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The Robust Prediction Method in SerialEM
Tilt 2
Y
Actual position
Predicted position
Make and use prediction but track
Tilt 3
Y
Make and use prediction but track
Tilt 4
Y
Rely on prediction do not track
Tilt 5
Y
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Tilt Series Preliminaries

• Adjusting beam and exposure parameters
• Tradeoff between specimen damage and
  signal-to-noise ratio in images and
  reconstruction
• For plastic section work, additional constraints
  of dynamic range of camera and difficulty of
  getting drift-free high-exposure image
• Whether to let program start at zero or go to
  high tilt manually
• Manually means you refine eucentricity and run
  walk-up
• You would do this if
• You are not sure you can get to the highest tilt
  angle
• You may need to adjust drift settling at high
  tilt
• You are unsure of what counts you can reach at
  high tilt
• Pre-expose plastic sections to minimize shrinkage
  and warping during series - 2000 electrons/Å2

21
Tilt Series Setup the Mother of All Dialogs

• Tilt and focus parameters are the same as the
  ones exposed elsewhere there is just one
  underlying parameter
• Some choices will be set/enabled depending on
  whether you are at zero or already at high tilt
• Low mag tracking needed only if field of view lt
  1 µm the low mag only needs to be low enough to
  give gt 2 µm field (all assuming well-behaved
  stage)

22
Tilt Series Setup 2 - Intensity Control

• Intensity control is important because otherwise
  higher tilt images will have lower SNR
• Intensity control is complicated because it
  covers several situations
• You might want constant intensity if doing cosine
  tilting
• 1/cosine or 1/cosine to a power is preferred for
  cryo
• Use a target of counts for stained material
  1/cosine can give big variations in counts
• The problem with constant counts is that it may
  require too bright a beam at high tilt. Two
  solutions
• Taper counts down above an angle
• OK once you are familiar with usable values
• Use with Do not increase intensity above value
  for first saved image to protect against
  overconstricted beam at far end of series
• Set up intensity before starting and use Keep
  intensity below current value
• Preferred method if you are starting at high tilt
  and setting up beam there
• Can be used even if starting at zero tilt as long
  as images arent too bright there

23
Tilt Series Setup 3 Autofocus

• The autofocus interval sets how often it will
  focus anyway when predictions are reliable 5-6
  is good value
• Can focus every time above an angle not really
  needed at end of series (should have an option to
  do this only at start)
• Checking autofocus is designed to prevent runaway
  focus changes if autofocus doesnt work well
  enough at high tilt
• You can follow the advice, i.e. adjust focus
  manually, and set defocus target to measured
  defocus value so it will stop trying to change
  focus
• Using an autofocus offset that is the negative of
  the target defocus might also work
• The (rare) problem usually goes away by 50

24
Tilt Series Setup 4 Initial Actions

• It is good practice to have a centered image
  before you go into the dialog, then select the
  option to align to it
• You should refine eucentricity before a series,
  especially after pre-irradiating the specimen
• Rationale for anchor is that tracking is more
  likely to drift off at high tilt and is likely to
  be accurate below 45-50
• Aligning to an image taken during walkup at such
  an angle will result in desired area being
  centered at low tilt
• There is no data loss from the misalignments at
  higher tilt, since the drift is lateral to the
  tilt axis and lost image area is not needed for
  backprojection
• You can have it take the anchor when it walks up,
  or you can to Walkup Anchor before starting
  series and then select the anchor there

25
Tilt Series Setup 5 Tracking Control

• The defaults are good for normal work (defaults?
  What defaults? It needs a Reset Defaults
  button.)
• Repeat Record if percentage of field lost can
  be 5 for typical work
• Make it smaller, down to 2.5, if area of
  interest goes very close to borders at 0
• Make it bigger or turn it off for very high mag
  or low dose
• Maybe make it bigger for very large montages
• Get tracking image when error in X/Y prediction
  gt is typically 1.5, could be bigger for high
  mag or low dose work to reduce tracking
• Stop if Autoalign shifts more than is a
  protection against wild alignments
• Get new track reference if Record alignment
  differs by gt is relevant if low mag tracking or
  low dose is in effect could be relaxed (made
  bigger) for very high mag

26
Resuming and Backing Up a Tilt Series

• A series can be stopped with End, at the end of
  a cycle, or with STOP, right away.
• If you resume from the Setup dialog it goes on to
  the next step
• The Resume Dialog gives you options for resuming
• The next action is listed
• You can repeat steps on the current cycle or
  sometimes skip steps that you have taken care of
  while stopped
• You can shift the image in A into a new alignment
  and check Use image in Buffer A as reference for
  alignments
• If you take a new Record it automatically
  overwrites the previous one
• The Backup dialog lets you back up the tilt angle
  and overwrite multiple images
• When you back up, it tilts the stage back, and
  loads the Record from that tilt into A and the
  image from the previous tilt into D, recreating
  the situation at that tilt
• You can open the dialog repeatedly and go forward
  or backward to the desired point

27
Verbose Log at Start of Series
28
Verbose Log with Good Predictions