4.1.2 Coarse Alignment
The aim of this step is to align the images of the tilt-series using
cross-correlation before producing a finer-aligned stack using the
fiducials. Click first “Calculate Cross-Correlation” and then “Generate Coarse Aligned Stack.” Check the aligned stack by clicking
“View Aligned Stack in 3dmod.” Remove any bad images from the
stack and create and use a new stack lacking the bad images.
4.1.3 Fiducial Model
Generation
If the sample has gold fiducials, these can be tracked, usually using
the “Make seed and track” or “Raptor” option. Generally, we select
between 10 and 30 beads. We usually begin with “Make seed and
track”, activate the “Refine center with Sobel filter” provide a value
of 8, and then click “Generate Seed Model” in the “Track beads”
subwindow. After that, click “Fix Fiducial Model” to fix any
untracked or badly tracked beads manually. If the tracking using
“Make seed and track” is bad, one can try tracking the beads using
“Raptor”. Tracking with “Patch Tracking” is usually used for
eukaryotic samples which lack gold beads in many cases.
4.1.4 Fine Alignment
Click “Compute Alignment” to correct the inappropriately placed
fiducials by clicking “View/Edit Fiducial Model.”
4.1.5 Tomogram
Positioning
A tomogram thickness value is provided (usually a thickness of
600 is used for many single-small-cell samples). Click “Create
Whole Tomogram” with a binning of three and after that “Create
Boundary Model”. Rotate the tomogram 90
, save the model, and
click on “Create Final Alignment”.
4.1.6 Final Aligned Stack
Here, we only perform “Create Full Aligned Stack”. However, one
can also apply “Contrast Transfer Function (CTF) correction”,
“Erase Gold”, and/or “2D Filter”.
4.1.7 Tomogram
Generation
The aligned image stack is used to build the tomogram by using
either: (a) Weighted Back Projection or (b) Simultaneous Iterative
Reconstruction Technique (SIRT-like). The generated tomogram
can be viewed by clicking “View Tomogram in 3dmod.”
4.2 Fiducial-Less
Patch Tracking
Using IMOD
Sometimes it is difficult to add gold fiducial markers to a specimen,
particularly FIB-milled lamellae or cryosections. In these cases, the
alignment is usually done by image correlation within. Here, we
briefly describe how to reconstruct data without fiducials by using
the patch tracking method in IMOD. More detailed information
can be found on the IMOD website: https://bio3d.colorado.edu/
imod/doc/patchTrackExample.html
1. Raw tilt-series are usually dose-weighted to boost the contrast
of higher angle tilt images.
2. Data are imported and pre-processed in IMOD as described
before.
Methods in Cryo-Electron Tomography
103
The aim of this step is to align the images of the tilt-series using
cross-correlation before producing a finer-aligned stack using the
fiducials. Click first “Calculate Cross-Correlation” and then “Generate Coarse Aligned Stack.” Check the aligned stack by clicking
“View Aligned Stack in 3dmod.” Remove any bad images from the
stack and create and use a new stack lacking the bad images.
4.1.3 Fiducial Model
Generation
If the sample has gold fiducials, these can be tracked, usually using
the “Make seed and track” or “Raptor” option. Generally, we select
between 10 and 30 beads. We usually begin with “Make seed and
track”, activate the “Refine center with Sobel filter” provide a value
of 8, and then click “Generate Seed Model” in the “Track beads”
subwindow. After that, click “Fix Fiducial Model” to fix any
untracked or badly tracked beads manually. If the tracking using
“Make seed and track” is bad, one can try tracking the beads using
“Raptor”. Tracking with “Patch Tracking” is usually used for
eukaryotic samples which lack gold beads in many cases.
4.1.4 Fine Alignment
Click “Compute Alignment” to correct the inappropriately placed
fiducials by clicking “View/Edit Fiducial Model.”
4.1.5 Tomogram
Positioning
A tomogram thickness value is provided (usually a thickness of
600 is used for many single-small-cell samples). Click “Create
Whole Tomogram” with a binning of three and after that “Create
Boundary Model”. Rotate the tomogram 90
, save the model, and
click on “Create Final Alignment”.
4.1.6 Final Aligned Stack
Here, we only perform “Create Full Aligned Stack”. However, one
can also apply “Contrast Transfer Function (CTF) correction”,
“Erase Gold”, and/or “2D Filter”.
4.1.7 Tomogram
Generation
The aligned image stack is used to build the tomogram by using
either: (a) Weighted Back Projection or (b) Simultaneous Iterative
Reconstruction Technique (SIRT-like). The generated tomogram
can be viewed by clicking “View Tomogram in 3dmod.”
4.2 Fiducial-Less
Patch Tracking
Using IMOD
Sometimes it is difficult to add gold fiducial markers to a specimen,
particularly FIB-milled lamellae or cryosections. In these cases, the
alignment is usually done by image correlation within. Here, we
briefly describe how to reconstruct data without fiducials by using
the patch tracking method in IMOD. More detailed information
can be found on the IMOD website: https://bio3d.colorado.edu/
imod/doc/patchTrackExample.html
1. Raw tilt-series are usually dose-weighted to boost the contrast
of higher angle tilt images.
2. Data are imported and pre-processed in IMOD as described
before.
Methods in Cryo-Electron Tomography
103