ALIGNMENT AND RECONSTRUCTION
Method used at Albany for making reconstructions
from both single- and double-tilt series of projection images.
Conversion of images to SPIDER format, framing, and 90-degree rotation.
Creation of control files listing tilt angles and file numbers.
Interactive picking of gold bead markers.
Determination of alignment parameters.
Test or final single-tilt reconstruction.
After the reconstruction is made, the following steps are commonly done:
Minimize the size of the reconstruction, rotate volume.
Image enhancement and visualization.
Save (backup or archive) important files.
Use of tomography database (only available at Albany).
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
Links to sample data and output files are provided.
Preprocess projection images.
|Input files||Procedure ||Output files|
| IVEM CCD
||Windowed, rotated SPIDER
- 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
- 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
Create control files: makesel.tom.
- The second-set selection file is normally not used unless a
single-tilt reconstruction is to be made from the second-series of
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 |
- First, display the zero-degree image from the first series
(image 31 in the example) using
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
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:
|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.
Align tilt images: mk.tom
- Look at the file
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
- 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
again and save cca002.dat. Then run
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
- 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.
Apply corrections to align the tilt images : align.tom
- 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.
Single-tilt or test reconstruction :
- 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.
Double-tilt reconstruction: doublerec.tom
- 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
- 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.
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.
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
get an idea how to adjust the top and bottom of the density range.
Then note the density range from
and experiment to find the best min and max values
- If desired, reverse the contrast using the SPIDER operation:
- As a minimum, an animation of the slices can be made by picking
slices with PS X,
PX Y, or
then playing the slices as a movie).
- The volume can be segmented by tracing the slices using
Usually, every two Z-slices from
the volume are added, reducing the number of slices to be traced by
two. This in fact better reflects the actual Z resolution. Batch file
b80.dat makes the added-together slices and enlarges them
for more accurate tracing. It also makes a series of SGI (*.rgb) images
of the added slices, at original size, for creation of an SGI movie to
use when following structures during tracing. Note that sets of Z-slices
can also be made into stereopairs for tracing. Please contact M. Marko at
if you are interested in
Save (backup or archive) the following files:
- Original files from the CCD camera or scanner.
- All procedure files.
- All document files (marker document files, all list
files, correction and coordinate files, information file).
- The final windowed, thresholded, reconstructed volume.
Last update: 18 Dec. 2008