ctfphaseflip(1)             General Commands Manual            ctfphaseflip(1)



NAME
       ctfphaseflip - correct tilt series for microscope CTF by phase flipping

SYNOPSIS
       ctfphaseflip  options

DESCRIPTION
       This program will correct the CTF of an input tilt series by phase
       flipping, with an option to attenuate frequencies near the zeros of the
       CTF.  The user can select a subset of projection views of the input
       tilt series to correct and can specify a defocus value for each
       selected view through a text defocus file.  The output file of Ctfplot-
       ter(1) is usually used as the defocus file.

       Ctfphaseflip corrects each view strip by strip.  A strip is defined as
       an image region whose defocus difference is less than a user specified
       value, the defocus tolerance.  Normally, the strips are vertically ori-
       ented and defocus is assumed to be the same along a vertical line.
       Thus, the tilt series must be aligned so that the tilt axis is vertical
       before applying this correction.  The original thinking was that an
       image region with defocus difference less than the tolerance could be
       considered to have constant defocus and could be corrected as one
       strip.  However, the validity of the correction at the center of the
       strip probably does not depend on whether it contains material beyond
       this focus range, since only vertical lines near or at the center are
       used in the corrected image.  The program may limit the width further
       to reduce computation time, or expand it to retain enough resolution
       between successive zeros in the X direction of frequency space.

       Through most of the image, each strip is corrected based on the defocus
       at the center of the strip.  However, the strips at the left and right
       edges of the image may be corrected repeatedly, at different defocus
       values, in order to extend the correction close enough to the edges of
       the image.  In fact, it is possible with the -maxWidth option to make
       the program correct only whole images, using the defocus value appro-
       priate for successive positions across the image.

       Two entries can make the program correct along diagonals instead of
       vertical strips: a tilt around the X-axis (more specifically, the one
       orthogonal to the tilt axis), and a rotation of the tilt axis from ver-
       tical.  The latter would be entered if unaligned images are being cor-
       rected.  If only a tilt axis rotation is entered, the diagonal strips
       are at that angle.  If an X axis tilt is entered, the diagonals are at
       an angle that depends on the angle of tilt around the tilt axis.  In
       either case, full images will be corrected to obtain the diagonal
       strips at different defocus.

       Ctfphaseflip can be run in a parallel mode similiar to the Tilt pro-
       gram by setting the -TotalViews option.  The script Splitcorrection
       can be used to prepare command files for running the program in paral-
       lel.  It can also be run on the GPU of an NVIDIA card.

       A sample command file for running the program with "subm" can be found
       in the IMOD/com directory (ctfcorrection.com).

       Originally, the program limited strip widths to 256, but the power
       spectra of low tilt images often showed rings of very low values right
       around the zeros of the CTF.  As of IMOD 4.8.17, the strip width is
       limited dynamically in a way that should minimize this effect.  Making
       the strips wider eliminates or reduces this effect, provided that the
       shift in the position of the first zero across the width of the strip
       is big enough.  At very low tilt, the maximum allowed width is
       increased to a value that would make the first zero shift by 0.6 pixel
       in the Fourier transform.  At higher tilts, the criterion shift is
       reduced by (1 - tan(tiltAngle)).  Near zero tilt, the strip width is
       simply half of the image size in X.  (However, if tilt is exactly zero,
       the program does only a single correction of the whole image.)  The
       spacing between adjacent strips is also adjusted to be larger for wider
       strips, to keep the computational time down (see the -iWidth option
       below).


OPTIONS
       Ctfphaseflip uses the PIP package for input (see the manual page for
       pip).  Options can be specified either as command line arguments
       (with the -) or one per line in a command file (without the -).
       Options can be abbreviated to unique letters; the currently valid
       abbreviations for short names are shown in parentheses.

       -input (-inp) OR -InputStack   File name
              Input stack that will be corrected.  It must be aligned so that
              the tilt axis is vertical.

       -output (-o) OR -OutputFileName     File name
              Name of output file for the corrected views

       -angleFn (-an) OR -AngleFile   File name
              File containing tilt angles for the input stack.  Each line of
              this file is the tilt angle for a view of the input stack.  The
              angles are listed in order starting from view 1.  If no file is
              entered, angles will be assumed to be 0.  If all views have tilt
              angles of 0, the program will do a single correction of the full
              image for each view instead of doing strips.

       -invert (-inv) OR -InvertTiltAngles
              Invert the sign of the tilt angles to compensate for a left-
              handed coordinate system in the microscope.  When the sign of
              the tilt angles and the value of the tilt axis rotation angle
              are such that reconstructions are generated with inverted hand-
              edness, then this option is needed for a proper correction.
              One way to assess this need is to examine the aligned stack
              being corrected, which has its tilt axis vertical.  At positive
              tilt angles, the right side of the images should be more under-
              focused than the left side; if not, then this option is needed.

       -axis (-ax) OR -AxisAngle      Floating point
              Angle that the tilt axis is rotated from vertical (counterclock-
              wise positive), for correcting unaligned images in their origi-
              nal orientation.

       -xtilt (-xt) OR -XAxisTilt     Floating point
              Angle (counterclockwise positive) that the specimen is tilted
              around the axis in the plane perpendicular to the tilt axis,
              which is along the X axis in aligned images.  This angle should
              correspond to the X axis tilt found in the positioning step in
              Etomo.  With an X axis tilt, the program will process full
              images instead of strips, interpolating or copying corrected
              pixels along diagonals of constant focus.  This option can be
              entered with an axis rotation angle for correcting unaligned
              images, but in that case the angle entered here must be for a
              tilt around a corresponding rotated axis, not around the X axis
              in the images.

       -defFn (-defF) OR -DefocusFile      File name
              File with list of tilt angle ranges and defocus values in
              nanometers, such as was output by Ctfplotter.  The full spec-
              ification of the defocus file is given below.  Each line should
              have a starting and ending view number (numbered from 1), a
              starting and ending tilt angle, and a defocus value.  Defocus is
              in nanometers, with positive values for underfocus.  The program
              will assign that defocus value to the midpoint of the range of
              views.  For a view at a given tilt angle, it will find the defo-
              cus either by interpolating between two surrounding midpoint
              angles, if there are such angles, or by taking the nearest defo-
              cus value, if the angle is beyond the range of the available
              midpoint angles.  To correct a tilt series with a single value
              of defocus, supply a file with a single line containing "20 20
              0. 0. defocus_value".  If you prepare a defocus file with more
              than one line, be sure to use the exact angles from the tilt
              angle file specified with the -angleFn option; do not round to
              one decimal place.  Alternatively, add the number "2" as an
              extra value at the end of the first line of the file; this will
              prevent the program from thinking that the view numbers might be
              off by one.  If you use the -invert option, you must do one of
              two things: 1) either invert all the tilt angles in this file,
              2) or start the file with the line "16 0 0.  0. 0 3" to indicate
              that it is a version 3 file in which the angles need to be
              inverted.

       -zoff (-zo) OR -OffsetInZ      Floating point
              Adjust defocus values for the given displacement in Z, in pix-
              els.  The default CTF correction is correct only for image data
              in the middle of the specimen.  With this entry, each defocus
              value will be adjusted to be correct for positions higher or
              lower in Z.  When viewing a tomogram in its original X/Z slices,
              a Y value below the center corresponds to a positive Z displace-
              ment.  The sign of this entry is thus the same as the sign of Z
              in a SHIFT entry to Tilt to bring this position to the middle
              of the tomogram.  However, the SHIFT entry is in unbinned pixels
              whereas the entry to this option is in actual pixels of a tomo-
              gram (i.e., binned pixels if the aligned stack is binned).

       -xform (-xf) OR -TransformFile      File name
              File with the linear transformations that were used to align the
              images.  Using this option has two consequences: 1) Astigmatism
              angles found by analyzing the raw stack will be properly rotated
              to the right angles for the aligned stack.  If this option is
              not entered, astigmatism must be found on the aligned stack.  2)
              The program will use the shifts in X to adjust the middle posi-
              tion that is considered to be at the nominal defocus value for
              each image.

       -defTol (-defT) OR -DefocusTol      Integer
              Defocus tolerance in nanometers, which is one factor that gov-
              erns the width of the strips. The actual strip width is based on
              the width of this region and several other factors: a fixed min-
              imum width of 128, a minimum width required to achieve suffi-
              cient resolution in the Fourier transform, governed by the the
              0zero option, and the dynamic adjustment of maximum width
              described above.

       -maxWidth (-m) OR -MaximumStripWidth     Integer
              Maximum width of strips in pixels.  This entry disables the
              dynamic adjustment of maximum width described above and applies
              a fixed maximum at all tilt angles.  As a special case, a width
              at least as wide as the full image size in X will make the pro-
              gram do each correction on full images instead of strips.

       -iWidth (-iW) OR -InterpolationWidth     Integer
              The distance in pixels between the center lines of two consecu-
              tive strips.  A pixel inside the region between those two center
              lines resides in both strips. As the two strips are corrected
              separately, that pixel will have 2 corrected values. The final
              value for that pixel is a linear interpolation of the 2 cor-
              rected values.  If a value of 1 is entered, there is no such
              interpolation.  For a value greater than one, the entered value
              will be used whenever the strip width computed from the defocus
              tolerance is less than 256 (i.e., at high tilt), and the value
              will be scaled proportional to the strip width for widths above
              256.  This scaling keeps the computational time down and is rea-
              sonable because the defocus difference between adjacent wide
              strips at wider intervals is still less than that between the
              narrower strips at high tilt.  However, strips at constant spac-
              ing can still be obtained by entering the negative of the
              desired spacing, which disables the scaling of the spacing.

       -zero (-ze) OR -MinimumZeroSpacing       Integer
              The minimum spacing between successive zeros at 0.4/pixel, in
              pixels of frequency space.  If necessary, the strip width will
              be increased above the value required by the defocus tolerance
              to maintain this much resolution in the Fourier transform at
              high frequency.  If a maximum width is entered with -fBmaxWidth,
              however, strips will not be wider than that maximum.  A higher
              number decreases the fraction and the width of frequency pixels
              that straddle a zero.  The default is 8.

       -pixelSize (-pi) OR -PixelSize      Floating point
              Image pixel size in nanometers

       -volt (-vo) OR -Voltage   Integer
              Microscope voltage in kV

       -cs OR -SphericalAberration    Floating point
              Microscope spherical aberration in millimeters.  A value of 0
              can be entered; it will be made slightly larger to prevent divi-
              sion by 0 in the CTF equations.

       -ampContrast (-am) OR -AmplitudeContrast      Floating point
              The fraction of amplitude contrast. For Cryo-EM, it should be
              between 0.07 and 0.14.  The value should be the same as was used
              when detecting the defocus.

       -phase (-ph) OR -PhasePlateShift    Floating point
              A fixed value for the phase shift imposed by a phase plate, in
              radians.  It can be entered with either positive or negative
              sign; the absolute value of the entered value will be subtracted
              when computing phase within the program.  This entered value
              will be overridden by the presence of any view-specific phase
              shift values in the defocus file.

       -degPhase (-deg) OR -PhaseShiftInDegrees      Floating point
              The expected value for the phase shift imposed by a phase plate,
              in degrees. See the -phase option, which should have been in
              degrees.  The two may not both be entered.

       -cuton (-cu) OR -CutOnFrequency     Floating point
              A fixed value for cut-on frequency that attenuates phase at low
              frequency, in reciprocal nanometers.  Whenever the cut-on fre-
              quency is non-zero, the phase shift value used for a view, from
              either the defocus file or the -phase entry, will be treated as
              the phase at a fixed frequency of 0.3/nm rather than the shift
              at infinite frequency.

       -scale (-sc) OR -ScaleByCtfPower    Floating point
              Scale frequency components by calculated CTF to the given power,
              in order to attenuate frequencies near the zeros of the CTF.
              The scaling does not occur below 0.9 times the frequency of the
              first zero, and it is introduced gradually between that point
              and the first zero to avoid a sharp transition in scaling.
              Lower powers give a narrower range of frequencies over which the
              strongest attenuation occurs around each zero.  This is not a
              Wiener filter; it will only attenuate signals and not boost any.

       -gpu (-g) OR -UseGPU      Integer
              Use the GPU (graphical processing unit) for computations if pos-
              sible; enter 0 to use the best GPU on the system, or the number
              of a specific GPU (numbered from 1).  If the GPU is not avail-
              able, the program will use the CPU or take the action specified
              by the -action option if that is entered.  The program will
              respond in the same way if a failure occurs in the initial GPU
              access for each slice, where memory allocation is done, but for
              any GPU errors after that, it will exit with an error message.

       -action (-ac) OR -ActionIfGPUFails       Two integers
              The action to take when the GPU cannot be used after being
              requested: 0 to take no action, 1 to issue a warning prefixed
              with MESSAGE:, and 2 to exit with an error.  Enter 2 numbers:
              the first for the action when the GPU is requested by the UseGPU
              option; the second for the action when the GPU is requested only
              by the environment variable IMOD_USE_GPU, or by the variable
              IMOD_USE_GPU2.  The default is 0,0.

       -views (-vi) OR -StartingEndingViews     Two integers
              The starting view and the ending view to correct in this run of
              the program, numbered from 1.  This is an optional field.  The
              default is to correct all views in the input stack.

       -totalViews (-t) OR -TotalViews     Two integers
              The starting view and the ending view that need to be corrected
              in all parallel runs of the program.  This field is only needed
              when doing parallel runs with direct writing to a single output
              file.  The program is run initially with a StartingEndingViews
              entry of -1,-1 to set up the output file.  Then it is run with
              actual starting and ending views for each parallel run, and each
              run writes to the existing output file.

       -boundary (-b) OR -BoundaryInfoFile      File name
              Name of file with information about boundaries between chunks
              and files in which to write lines at the boundaries, when multi-
              ple runs are writing in parallel to an output file.

       -skipFlag (-sk) OR -SkipCorrectedFlag    Integer
              1 to not set flag that CTF was corrected, 2 to ignore flag if
              set, 3 for both

       -debug (-deb) OR -DebugOutput       Integer
              Value to control debug output: 1 for timing, 2 for column
              detail, 0 for neither; add 10 to suppress initial list of angles
              and defocus values.

       -param (-pa) OR -Parameter     Parameter file
              Read parameter entries from this file.

       -help (-h) OR -usage
              Print help output

       -StandardInput
              Read parameter entries from standard input

   Defocus File Format
       The defocus file consists of a series of lines, each containing at
       least 5 numbers: starting and ending view numbers for the range of
       views fit to (numbered from 1), starting and ending tilt angles for the
       range of angles fit to, and the defocus value in nanometers (underfocus
       positive). The first line of the file can have a version number at the
       end.  For versions 1 and 2, the first line must have fitting data as
       well.  The typical file currently written by Ctfplotter thus starts
       with lines like
       61      61      -58.94  -58.94    4376  2
       60      60      -56.94  -56.94    4599
       A version 3 file can have columns for astigmatism values, or the phase
       shift from a phase plate, including an optional cut-on frequency, or
       both.  Past version 2, the first line is a header line without data.
       Thus a version 3 file should start with
         flags  0 0. 0. 0  3
           where "flags" is the sum of:
              1 if the file has astigmatism values
              2 if the astigmatism axis angle is in radians, not degrees
              4 if the file has phase shifts
              8 if the phase shifts are in radians, not degrees
             16 if tilt angles need to be inverted to match what the
                 program expects (what Ctfplotter would produce)
                 with the -invert option
             32 if the file has cut-on frequencies attenuating the phase
                 at low frequencies
       The other values on the header line could be used in the future but
       should be 0.  If there are astigmatism values, the convention is for
       the higher defocus value to be in the standard column for defocus, fol-
       lowed by the lower defocus value, then by the angle (counterclockwise
       positive) of the axis with the higher defocus (the short axis of the
       ellipse in an FFT).  However, either defocus value can be in the first
       column as long as the axis angle is that of the first defocus value.
       If the lower defocus is first, both Ctfphaseflip and Ctfplotter will
       swap the two values and subtract 90 from the axis angle, so that astig-
       matism will always appear as a positive value.

       Either the astigmatism angles must be correct for the aligned stack, or
       the angles must be correct for the raw stack and the -xform option must
       be entered with the file of transforms used to produce the aligned
       stack from the raw one, so that the program can rotate these angles
       appropriately.  A file with just astigmatism values could start with:
       1  0 0. 0. 0  3
       61      61      -58.94  -58.94    4652    4376  -43.2

       If there are phase shifts, they go in a column at the end of the line.
       They can be positive or negative; only the absolute value will be used
       and equations using these shifts subtract the absolute value of the
       number.  A file with just phase shifts in radians could start with:
       12  0 0. 0. 0  3
       61      61      -58.94  -57.94    4376  1.47
       while a file with both astigmatism and phase shifts could start with:
       13  0 0. 0. 0  3
       61      61      -58.94  -57.94    4652    4376  -43.2  1.47
       If an expected phase shift is entered to Ctfplotter (either through
       the user interface or with the -phase option) and phase is not searched
       for, it will put out a defocus file with a phase shift that is the same
       on every line.

       A cut-on frequency in reciprocal nanometers may be included in the file
       only if it includes phase shifts.  This value goes on the end of each
       line after the phase shift.  Whenever a file contains cut-on frequen-
       cies, the phase values read in from the file are interpreted differ-
       ently: they are treated as being the phase at a particular frequency,
       0.3/nm.  The model applied is
          phase(freq) = phaseAtInfinity * (1. - exp(-freq / cuton))
       where
          phaseAtInfinity = phaseInFile  / (1. - exp(-0.3 / cuton))

       The reason for this convention is that cut-on frequency is difficult to
       find accurately, and the implied phase at infinity will vary consider-
       ably because of the random variations in cut-on frequency.  The phase
       at this higher frequency will be much more stable and allows a better
       assessment of how accurately phase has been found.

       As for phase, if a cut-on on frequency is entered to Ctfplotter
       through either the user interface of the -cuton option, the it will put
       out a defocus file with the same cut-on frequency on each line.  A file
       with astigmatism, phase in degrees, and cut-on frequency could start
       with:
       37   0    0.0     0.0     0.0     3
       1    1   -50.00  -50.00   3665    3472   -62.10    118.54  0.1686

AUTHORS
       Quanren Xiong and David Mastronarde

SEE ALSO
       ctfplotter

BUGS
       Prior to IMOD 4.0.29, Ctfplotter had a bug in which the view numbers
       written to the defocus file were numbered from 0, not 1.  When Ctf-
       phaseflip reads in the defocus file, it will do its best to detect this
       situation and adjust all the view numbers up by 1.  If it does detect
       an inconsistency between view numbers and angular ranges, it will issue
       a warning.

       Email bug reports to mast at colorado dot edu.



IMOD                                4.10.22                    ctfphaseflip(1)