ELECTRON TOMOGRAPHY
ALIGNMENT AND RECONSTRUCTION

Introduction

These are the procedures used at Albany for making electron tomographic reconstructions from both single- and double-tilt series of projection images.

We apply these techniques to organelles such as mitochondria, centosomes, and kinetochores (using electron tomography of thick sections). We also use these techniques for electron tomography of whole- mount, frozen hydrated specimens such as isolated mitochondria, skeletal-muscle triad junctions, and sperm axonemes.

Outline

  1. Conversion of images to SPIDER format, framing, and 90-degree rotation.
  2. Creation of control files listing tilt angles and file numbers.
  3. Interactive picking of gold bead markers.
  4. Determination of alignment parameters.
  5. Image alignment.
  6. Test or final single-tilt reconstruction.
  7. Double-tilt reconstruction.

    8. After the reconstruction is made, the following steps are commonly done:

  8. Minimize the size of the reconstruction, rotate volume.
  9. Image enhancement and visualization.
  10. Save (backup or archive) important files.

Details on the operations within the procedure files (*.tom) can be found in the comments in the procedure files. A comment starts with a semicolon (;). Full details on the operations, including links to source code, can be found in the SPIDER documentation.

We routinely make a file called info.dat which includes all the specimen and microscope information, the pixel size, and notes on details of the reconstruction. This file is kept in the directory with the data. When the reconstruction is finished, only data which can't be automatically re-created with the procedure files is archived on CDROM. At Albany, the tomography database can be used to enter all the information which would otherwise be listed in info.dat

Links to sample data and output files are provided.

  1. Preprocess projection images.

    Input filesProcedure Output files
    IVEM CCD ivem2spi.tom Windowed, rotated SPIDER

    Notes:

    • The tilt axis of the output images must be approximately vertical (parallel to the image Y-axis). At Albany, the original projection images have the tilt axis horizontal, so they are rotated by the procedures used in this step.

    • The images must be square for double-tilt reconstruction. The images should be as large as possible for accuracy in marker picking, but not larger than the pixel size of the computer screen.

    • The examples are for a tilt series from -60 to +60 degrees with a 2-degree increment, resulting in 61 images. For double tilt, the second tilt series is numbered 62-122.

    • ivem2spi.tom is for 1024x1024 Tietz-format CCD images.

  2. Create control files: makesel.tom.

    Input files Output files Output description Double-
    tilt only
    Nonesel001.dat First-set selection file
    ang001.dat First-set tilt angles
    sel002.dat Second-set selection file *
    ang002.dat Second-set tilt angles *
    sel003.dat Combined selection file *
    ang003.dat Combined tilt angles *

    Notes:

    • The second-set selection file is normally not used unless a single-tilt reconstruction is to be made from the second-series of tilt images.

  3. Pick markers, using WEB, "Markers" operation.

    Input files Input description Output files Output description
    rot_***.dat Tilt images d_rot***.dat Marker location document files

    Notes:

    • First, display the zero-degree image from the first series (image 31 in the example) using WEB's "Markers". Then pick an evenly-distributed set of gold beads. The "center of gravity" of the marker beads should be near the image center for double-tilt reconstructions. The absolute minimum is 3 marker beads, and 10-12 is preferred.

    • "Markers" will ask for the output file name and tilt angle. On any subsequent images, "Markers" will automatically increment both appropriately, once it "learns" the pattern. Be sure to check that both are correct. Refer to the angles files ang001.dat and ang003.dat to check the proper tilt angle for each image.

    • After picking the marker set on the zero tilt image you can continue using: WEB's "Markers" to manually pick markers on all tilt images in the series. Alternatively you may automatically select beads on the non-zero images using: beadtracker.tom

      Input files Input description Output files Output description Double-
      tilt only
      rot_***.dat Tilted images d_rot***.dat Marker location document files
      d_rot001.dat Zero tilt marker location document files    
      ang001.dat Tilt angle doc.    

    • To verify that the correct marker beads are being picked during manual marker picking, open another WEB window and use "Annotate" to display the previous tilt image with markers that have already been picked. Compare this to the current image in "Markers"

    • For the second series in double-tilt reconstructions, display the second-series zero-degree image (image 92 in our example) in "Markers" and the first-series zero-degree image (image 31 in our case) in "Annotate". Be sure to use the same beads and the same number for each bead in both.

  4. Align tilt images: mk.tom

    Input files Input description Output files Output description Double-
    tilt only
    rot_***.dat Tilted images viewerror_1.dat First-set average error per view  
    d_rot***.dat Marker location errors_1.dat First-set average error per marker  
    sel001.dat First-set selection cca002.dat First-set image alignment  
    ang001.dat First-set angles cra002.dat First-set 3-D coordinates  
    sel002.dat Second-set selection viewerror_2.dat Second-set average error per view *
    ang002.dat Second-set angles errors_2.dat Second-set average error per marker *
    sel003.dat Combined selection ccb002.dat Second-set image alignment *
    ang003.dat Combined angles crb002.dat Second-set 3-D coordinates *
    c3c202.dat Eulerian angles between series *
    angles_1.dat Combined first-set angles *
    angles_3.dat Combined angles *
    errors_3.dat Combined alignment errors *

    Notes:

    • Look at the file errors_1.dat. If the marker errors are all about one pixel or less, the alignment was successful. If some markers have an error above 2-3 pixels, you should look for a problem.

    • Errors can be analyzed by searching the file viewerror_1.dat for projection image numbers which have a large average marker error. If an image is found to have a large error, recheck the position of the markers picked on that image.

    • Errors of individual markers on each image can be analyzed by selecting full output when running MK interactively, which displays the individual marker coordinates and errors on each projection image. A marker with an unusually high error is likely picked in the wrong position. This data can be saved as a series of files if the corresponding prompt is answered.

    • An easy way to identify incorrectly-picked markers is to use b62.spi. This procedure file makes a series of images of the marker positions, with a line drawn from marker to marker on each image. When viewed with WEB, using montage with movie turned on, the shape of the lines will change more than usual when the image with the bad marker position is reached.
    • Using b63.spi, you can align the images made with b62.spi, and view them with WEB, using montage with movie turned on. This will show what the final alignment of the tilt series will look like. The pattern of lines should appear to rotate smoothly.

    • If the errors shown with 'MK' start to increase rather continuing to decrease, use b64.spi, to change the initial tilt angle. Then use b65.spi instead of b20.spi (i.e., use the file cct002.dat, made by b64.spi as the "previous corrections" file for MK. Each time, be sure to delete cca002.dat and cra002.dat before using b20.spi or b65.spi again.

    • If all of these fail to show where the problem is, run MK again and save cca002.dat. Then run MK once more interactively using cca002.dat as input when prompted for "previous corrections". If the errors are improved, rename the final output files to the standard names (cca002.dat and cra002.dat)

    • If you decide that one or more markers shouldn't have been chosen, use editmar.tom to create a new set of marker files with the undesired markers removed. Note that you will then have to enter the new names chosen for the marker files when using: mk.tom to align the tilt images.

    • The 3-D errors for alignment of the two sets are found in errors_both.dat. The average error is shown on the last line.

    • If the errors for the individual sets were low, these errors should also be low. If the errors are very high, the same markers may not have been picked in both series, or there might have been a difference in image dimensions.

    • The angles_both.dat file will include the angles for all the projections, even if they won't all be used in the reconstruction.

  5. Apply corrections to align the tilt images : align.tom

    Input files Input description Output files Output description Double-
    tilt only
    rot_***.dat Tilted images rdb_***.dat Aligned images  
    sel001.dat First-set selection doc      
    cca002.dat First-set alignment doc      
    sel002.dat Second-set selection doc     *
    ccb002.dat Second-set alignment doc     *
    sel003.dat Combined selection doc     *
    c3c202.dat Combined alignment doc     *

    Notes:

    • Look at the aligned images, rdb***.dat, with WEB montage operation, with movie turned on. There should appear to be a smooth rotation, and the artifacts on the edges of the images (from wrapping during alignment) shouldn't extend too far into the center of the image.

  6. Single-tilt or test reconstruction : singlerec.tom

    Input files Input description Output files Output description
    rdb_***.dat Aligned images vol001.dat Reconstructed single-tilt volume
    sel001.dat Selection    
    ang001.dat Tilt angles    

    Notes:

    • First use singlerec.tom to make a test reconstruction. Select about five rows, usually in the middle of the volume by specifying the Beginning row & Ending row. Look at a central Y slice of the output volume to see if you guessed correctly with the Reconstruction depth setting. If you do not see the top and bottom of the specimen, increase the Reconstruction depth and try again. If you think the specimen may have been tilted when the microscope stage was at zero tilt (e.g. due to a bent grid), it would be wise to check test reconstructions nearer the edges of the input images, as well. Such a test reconstruction only takes a few seconds.

    • Use singlerec.tom gain to make a full single tilt reconstruction. For the 507x507x200 volume shown in the example, the reconstruction takes about 45min on a single R5000 processor. Remember that SPIDER images are 32-bit, so the size of the volume in bytes is the pixel length x width x depth x 4.

    • The Frequency cut-off for the Parzen filter value used in singlerec.tom is 0.3. This seems to be optimal.

  7. Double-tilt reconstruction: doublerec.tom

    Input files Input description Output files Output description
    rdb_***.dat Aligned images] vol002.dat Reconstructed double-tilt volume
    sel003.dat Combined selection    
    ang003.dat Combined tilt angles    

    Notes:

    • If you want to reconstruct a partial volume, use the full Output volume dimensions, and indicate the desired Beginning slice and Ending slice
      • .
    • The SNR filter value used in the reconstruction is: 1.0E+4. This seems to be optimal. If you want to experiment, see SPIDER documentation for BP 3D.

    • A double-tilt reconstruction takes about 10 times longer than a single-tilt reconstruction. If you have a slow machine you may wish to do a small (e.g. 128x128x128) test volume first.

  8. Minimize the size of the reconstruction, rotate volume

    • Window (WI) the reconstruction to eliminate blank or unwanted volume.

    • If the specimen is slightly tilted, rotate the volume (RT 3D), and re-window.

  9. Image enhancement and visualization

    • You will usually need to threshold the volume to optimize the contrast range for display. First use the "contrast" slider in WEB to get an idea how to adjust the top and bottom of the density range. Then note the density range from FS. Use TH and experiment to find the best min and max values to use.

    • If desired, reverse the contrast using the SPIDER operation: NEG.

    • As a minimum, an animation of the slices can be made by picking slices with PS X, PX Y, or PS Z, then playing the slices as a movie).

  10. Save (backup or archive) the following files:

    1. Original files from the CCD camera or scanner.
    2. All procedure files.
    3. All document files (marker document files, all list files, correction and coordinate files, information file).
    4. The final windowed, thresholded, reconstructed volume.

Source: docs/techs/lgstr/tomo.html     Last update: 29 Mar. 2025