Electron Tomography Tutorial Part I: Instrumentation

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Electron Tomography Tutorial Part I
Instrumentation Advances in Electron Microscope
Instrumentation and Exploration of Biological
Structures
James Bouwer, PhD. and Albert Lawrence,
PhD National Center for Microscopy and Imaging
Research
June 28, 2009
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Core Instrumentation Development Team A
Collaborative Enviroment
PRINCIPAL INVESTIGATORS Mark Ellisman, Nguyen
-Hu Xuong RESOURCE STAFF SCIENTISTS James
Bouwer Albert Lawrence Sebastien
Phan Steven Peltier Tomas
Molina Masako Terada Mason
Mackey Thomas Deerinck Tristan
Shone George Yang GRADUATE STUDENTS Rick
Giuly, Anna Milazzo, Liang Jin
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Overview of the Tomography Process

• What is tomography
• 2D images to 3D volumes
• Inherent aberrations in electron microscopes
• Physics of magnetic lenses
• Aberrations resulting from typical imaging
  conditions
• Aberrations resulting directly from tilted image
  acquisition
• Inherent 3D nature of the problem and solutions
• Back-projection
• X-ray transforms
• Non-linear transforms (TxBR)
• New Wide-field imaging systems
• High-resolution 8k x 8k lens-coupled imaging
  system for EM
• Wide field driving need for image/volume
  remapping
• New tomography modalities
• Multi-axis tomography, thick section tomography
  (MPL,micro-probe STEM)

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What is Electron Tomography
Tomograpy Tilt Series
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Intermediate Voltage Electrons Microscope
Resources
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Toadfish Swimbladder Muscle -gt lead and uranyl
acetate Contrast vs. Voltage
100 keV --gt 100nm thick 200 keV --gt 200 nm
thick 400 keV --gt 500nm thick 1.25 MeV --gt
3microns 3 MeV --gt 10microns
Thickness 80nm
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Limitations of Light Microscopy
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Electron Microscopy for Ultra-wide-field
Multi-Resolution 3D Imaging
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Photo-oxidation of cys4 transfected Hela cells to
prepare for electron tomography

• Cells labeled with ReAsH and imaged live
• Live action frozen with fixatives
• Illuminated to create singlet state oxygen
• Reactive oxygen polymerizes DAB
• Label with Osmium
• embed in epoxy

Live Cell Imaging
photo-oxidized golgi
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The Electron Microscope
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Cartoon of Electron Trajectories in a Solenoidal
Field
from J. Bozzola and L. Russel Electron Microscopy
2nd edition
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Physics of Electron Lensing
Can be solved analytically through separation of
variables in cylindrical coordinates to produce
the equations of motion for the imaging electrons

• Bz z-component of the magnetic field
• ?L Larmor frequency
• e charge of an electron
• me rest mass of an electron

Bell Shaped Field
Image from L. Reimer, TEM 1993
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Effects of Varying the Current to the Objective
Lens
Focus Series of 10nm and 20nm Gold Beads with 1µm
Focus Steps Acquired on 4k x 4k CCD
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Real Electron Trajectories in Rotating Reference
Frame
Electron trajectories for electrons incident
parallel to the optical axis for various B- field
strengths
Image from L. Reimer, TEM 1993
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A Simplified Cartoon of the Electron Tomography
Geometry with a Field Immersion Lens

• Differential magnification
• Differential rotation
• More pronounced for large
• format images
• Rotation and magnification
• are more troublesome for
• tomography

Distortion Correction is Inherently a 3D Problem
(rotation)
(magnification)
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Tilt Geometry Distortions
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2-Dimensional Warping is Insufficient
2µm thick Golgi Sample
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Projector Lens Aberrations
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Inherent Electron Microscope Spatial Aberrations
Pincushion
Barrel
Spherical aberration in the projector produces
radially dependent magnification errors
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Largest Spatial Aberrations are S-type
Distortions in the Projector Lens Optics

• EM manufacturer Specs
• lt 1.5 _at_ r 5cm
• For a 4k x 4k detector 6cm diameter
• 40 pixels
• For a 8k x 8k detector 10cm diameter
• ? 80 pixels

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S-Distortion Correction Enables Image Mosaicing
Hardware Solution to S-distortion correction for
Montaging
JEM-3200 Drift Tube for S-distortion mitigation
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EM STUDY OF THE AUTOPHAGY PATHWAY AFTER BRAIN
ISCHEMIA 2 x 2 mosaic tomogram (8k x 8k pixels)
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Beam Induced Mass Loss (Shrinkage and Sample
Warping)
The Jellyfish Analogy
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Standard X-ray Backprojection The last 40 years
Sample

• Linear electron
• trajectories
• Back project image
• density
• Filtration is necessary
• to remove artifact

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A Tilt Series Shows Aberrations Inherent in
Electron Imaging Aberrations Can Cause Any Number
of Image Errors
Dividing NRK (Rat Kidney) cell photo-oxidized Golg
i ReAsH label
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Fiducial Marker Tracking
Remap the images so that the tracks are
level A. Lawrence - The Tao of TxBR
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Image Remapping Pre-alignment
Remaped Particle Tracks
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Corrected Particle Track Parameters Are Applied
to the tilt images
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Transform Based Backprojection Assumes Curved
Electron Trajectories
Calculation of R is fully parallelizable
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Application of Transform Based Backprojection
Dividing NRK cell photo-oxidized Golgi ReAsH label
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NCMIRs New 8k x 8k Wide-field Ultra-high
Resolution Detector Highlights Tomography
Challenges
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Basic Design of Commercial Slow-Scan CCD
Detectors for TEM
e-
Scintillator
Fiber Optic Relay
TEC(Peltier) Cooling
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A 64 Mega Pixel Digital Detector for TEM
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SupraCam in Cross-Section
Electron beam
JEOL interface flange
Large diameter gate valve
Self-supporting scintillator (260mm diameter)
Scintillator chamber
Pyramidal beam splitter
Leaded glass window
Optics1 Lens
90mm Shutter
Spectral Inst. CCD
Custom 13 axis stages
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8k x 8k Alzheimer's Plaque - Human Biopsy Section
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Collaborative Project 4 ENTRY, REPLICATION, AND
ASSEMBLY OF NODAVIRUS IN DROSPHILA CELLS High
Pressure Frozen, Freeze Substituted, 40kx Mag
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Montaging with the Stage and Image Shift Coils
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4 Billion Pixel Montage on 8k x 8k Camera
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Montage Tomography
-stage montaging -image shift montaging
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2 x 2 Montaged Tomogram (8k x 8k) of a Flock
House Virus Infected Drosophila Cell at 8kx
Magnification
zero - loss energy filtered
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Problem Position

• Thin slices become warped during sample
  sectioning, handling and data acquisition (beam
  induced mass loss and lens distortions)
• Difficulty in stacking volumes in a serial
  tomography

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Serial Section Tomography
Two Possible Approaches

• Transform an already reconstructed volume
• Modify the projection maps during the bundle
  adjustment procedure

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A shear based warping transformation
Orthogonal warping transformation
work by Sebastien Phan, NCMIR-UCSD
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Neuron Specimen
Shear based transformation
Naoko3A7
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Serial Section Tomography
astrocyte soma at 8k mag
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Serial Section Montage Tomography
-stage montaging -image shift montaging
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Movie Coming Soon
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The Contrast Transfer Function (CTF)
where
I(k) Image O(k) Object k spatial frequency
and
CTF(defocus, Cs ,?E, source size)
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Wide-field Images of 60 degree tilted sample show
a strong focus gradient (CTF gradient)
?z 10µm
?z -10µm
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Large Field of View Requires CTF Reconstruction
from a Thru Focus Series
2D gt Fourier Transform 3D gt Fourier Integral
Operator
Generated with CTF generator written by Wen
Jiang and Wah Chiu
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Multiple Axis Tomography
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Titan Equipped with 3 Lens Condenser Illumination
System

• Triple Condenser Illumination STEM Mode
• Ability to adjust probe convergence angle
• lt 1mrad convergence angle/parallel probe long
  depth of field
• Micro-probe STEM Tomography
• Highly parallel probe, 1mrad convergence angle,
  2-4 nm resolution
• Dynamic focus
• 4k x 4k STEM will increase ability to image wider
  fields

Much thicker specimens (gt 6 µm) increase the z
dependent aberrations in micro-probe STEM
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Copper-Lead Stained Spiny Dendrite - MicroProbe
STEM Tomography
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Very High Resolution Tomography tomography in ice
TxBR Reconstruction using a Cubic
Backprojection Shows Actin Sub-units
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Summary
Electrons travel along curved paths
Linear Backprojection Methods are insufficient
Especially true for large format images and for
high resolution tomograms!
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Procedure for Solving this Set of Eqs.

• Seed computer with the knowns
• -particle positions (xj, yj) in each tilt
• image
• -The approximate (x,y,z) coordinates
• for each particle in the object are
• compute using triangulation

• Use conjugate gradient optimization to compute
• aij? bij? cij? dij? and precise (xj,
  yj, zj)
• This method of solution is called Bundle
  Adjustment

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Transform Based Back-Projection
(TxBR)
A Parallel Algorithm for Volume Computation
In GTS
Albert Lawrence James Bouwer Able Lin Masako
Terada Guy Perkins
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The Story of Transform Based Back Projection
(TxBR).
Errors inherent to tomography with magnetic
lenses result in errors which cause smearing of
the object density.These errors cannot be
corrected using linear back-projection algorithms.
- A. Rick Lawrence
Electrons travel along curved paths
resolution is the goal
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TxBr (Transform Based Backprojection)

• Very High Resolution Tomography
• High Resolution Wide Field Tomography
• Challenging Data Sets
• Non-eucentric goniometers
• Critical Data (sample warping)

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Computation of the backprojection (R) requires
the polynomial approximations to the projection
(R) along electron paths (?)
26 Unknowns!
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Sample calculation of the required number of
fiducal markers

• Quadratic model with 60 tilts and 20 fiducials
• Unknowns (26)(tilts) (particles)(3coords)
• 156060 1620
• Knowns (2coords)(20fiducails)(60tilts) 2400
• Cubic Model (cubic projector lens distortions
• number of unknowns (43)(60)(40)(3) 2700
• number of fiducials 40

• Highly Parallelizable!
• Well Suited for Grid Abstraction

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Very High Resolution Tomography
TxBR Reconstruction of ice embedded actin using
a cubic backprojection shows actin sub-units
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Imod vs. TxBR High-resolution Wide-field
Tetracystine labeled Actin Fibers and Bundles
TxBR with remap
Linear Methods (IMOD)
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Continued Progress in Electron Optical Sectioning

• Collecting data to building accurate point-spread
  function models
• Working on techniques to deconvolve the sections
• Working with Angus Kirkland to explore techniques
  for controlling the stigmator coils to reduce
  aberrations

Dev Team J. Bouwer, T. Molina, G. Yang, B.
Smith, Y. Hakozaki
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Challenges in Electron Microscopy New
Approaches to the Tomography Process

• Physics of Magnetic Lenses in EM Tomography
• Inherent aberrations in electron microscopes
• Challenges in tomography
• Contrast Transfer Function is inherently 3D
• New Data Collection Modes
• Large field montaging tomography
• Most Probably Loss Tomography
• Electron Energy Loss Imaging
• Multiple Axis Tomography
• New Instrumentation
• Ultra-wide field Cameras
• HAADF STEM microprobe microscopy
• EELS

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Serial Section Tomography
astrocyte soma at 8k mag