howflared(1)                                                      howflared(1)

       howflared - measure microtubule end flaring and curvature

       howflared  [options]  output_file

         Howflared analyzes tracings of the walls of microtubules (MTs) to
       compute various measures of the amount of curvature or "flaring" at the
       MT end.  The IMOD model with MT tracings must meet a number of require-
       ments.  The first step is to extract the MTs into subvolumes so that
       they are oriented vertically, with the end facing downward.  This can
       be done with Mtrotlong.  The orientations do not need to be perfect.
       Once subvolumes have been extracted as a set, they can be loaded into
       3dmod together as a 4-dimensional data set.  The 4th dimension is
       referred to as "time".  Alternatively, a central slice can be extracted
       from each and a stack made with these central slices.

       The program will also work with lines drawn in an arbitrary plane in
       the Slicer window, although it cannot analyze pairs of left and right
       walls with such data.  Each contour is fit to a plane and rotated so
       that it lies in an X/Y plane, then X/Y coordinates are analyzed as

         The tracings must all be drawn starting near the top of the image in
       Y and ending at the MT end.  Each tracing defines two regions of an MT
       wall: a linear segment before the end, and a potentially curved seg-
       ment.  A line is fit to a defined part of the linear segment and
       extrapolated into the region of the end.  The quantitative measures are
       based on the deviation of the curved segment from this extrapolated
       line.  Thus, a tracing must include a linear segment, and there must be
       some way to indicate where the linear segment ends and the measurement
       of the deviation should begin. There are essentially three different
       ways to indicate this boundary.

         One way is to specify how far down from the top of the tracing the
       beginning and the end of the line fit should be.  This method is suit-
       able if all the tracings have similar starting points near the top of
       the subvolumes and their curvature starts at similar locations as well.
       The starting and ending points can be specified as either absolute
       units (in pixels) or as a fraction of the distance from the maximum to
       the minimum Y value in the tracing.  The second method is to use a hor-
       izontal line, consisting of only two points, entered as a separate con-
       tour, to specify the boundary point.  With this method, one might still
       enter an ending limit, in which case the maximum of the ending limit
       and the Y position of the marker line are used for the end of the lin-
       ear fit, but the measurement of deviation starts at the marker posi-
       tion.  The third method is to model the linear segment with just two
       points.  In this case, one would enter 0,0 for starting and ending lim-
       its.  The second and third methods can both used in the same model; the
       second point will be used as the boundary only if there is no horizon-
       tal marker line.

         The program can analyze either two paired walls of a MT, or a single
       wall.  When two walls are paired, the linear fit finds a single slope
       for the two walls, and a width between them.  This width is used for
       normalizing some of the flaring measurements.  If there are also sin-
       gle, unpaired walls, a width is assumed when normalizing these measure-
       ments (see -width option).  When a horizontal marker is used for a pair
       of walls, it should be drawn from near the boundary point on the left
       wall to near the boundary point on the right wall.  A single wall is
       treated like the wall on the left of a pair; so in this case a marker
       line should have its left end near the boundary point on the wall.

         The program needs to be able to tell when two MTs are paired, and
       when a marker line belongs with one or two MTs.  Nothing special needs
       to be done when modeling in only a single plane per MT.  If you are
       modeling in multiple sub-volumes, the program will segregate the trac-
       ing based on their times; if you are modeling in a stack of single
       slices from different MTs, the segregation will be based upon Z value.
       However, if you want to trace multiple, unpaired walls in the Slicer,
       either you need to segregate them by placing them in either separate
       objects or separate surfaces, or you need to use the -nopairs option.
       Thus, you could have one object for all of the first tracings in each
       MT, a second object for the second tracing, etc.  Similarly, you could
       use the surface number 0 for the first tracing in each MT, 1 for the
       second, etc.; or you could simply put each tracing (or tracing pair)
       with associated marker into a separate surface.  If you are willing to
       have no marker lines, then you can use the -nopairs option to indicate
       that there are neither pairs nor markers.

         The program computes various quantities and stores them in columns of
       a data matrix; you specify which columns you want output with -columns.
       The items and their units are:

         The area between the tracing and the extrapolation of the wall, down
       to the minimum Y value of the tracing (square nm).
         The square root of this area (nm)
         The sum, over all line segments, of the product of the line segment
       length and the angle between the segment and the extrapolation of the
         The area divided by the square of the average MT width (square MT
         The square of the area divided by the average MT width (MT widths)
         The angular sum divided by the average MT width (MT widths * radians)
         The total length of the tracing after the boundary (nm)
         The final angular deviation of the tracing from the extrapolation of
       the wall (degrees)
         The angular change in degrees per nm of length
         The average radius of curvature in nm.

         The first 6 items are placed into columns 1-6 for the left wall of a
       pair or for an unpaired wall, and into columns 7-12 for the right wall
       of a pair.  The sum of the two values for left and right in placed in
       columns 13-18.  The last 4 items are placed into columns 19-22 for the
       left wall of a pair or for an unpaired wall, and into columns 23-26 for
       the right wall of a pair.

         In the output file, these columns of output are preceded by 3 or 4
       numbers.  The first number is a model identifier; the second is the
       object number.  If you used surface numbers to segregate tracings, the
       third of 4 numbers is the surface number; if you used the -nopairs
       option, the third of 4 numbers is the contour number.  The last number
       is the time value, or the Z value.

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

       -output (-o) OR -OutputFile    File name
              Output file for results. If this option is not entered, the
              first non-option argument will be used for the filename.

       -columns (-c) OR -ColumnsToOutput   List of integer ranges
              List of columns to output (values ranging from 1 to 26).  For
              paired MT walls, columns 1-6 and 19-22 are for the left walls,
              7-12 and 23-26 are for the right walls, and 13-18 are the sum of
              values from 1-6 and 7-12.  Unpaired walls are treated as left
              walls, and their values will appear in both columns 1-6 and
              13-18.  See description of computed values above.

       -point (-po) OR -PointOutputFile    File name
              Output file for points in each wall, starting at the point where
              the flare measurement begins.  The points are rotated so that
              the wall above that point would be vertical, and they are
              expressed in nanometers relative to the starting point.  For
              each MT wall, there is a line starting with the number of
              points, then containing the 3 or 4 numbers described above as
              preceding the columns of output in the standard output file, and
              ending with 1 for a left or unpaired wall, or 2 for a right

       -pixel (-pi) OR -PixelSizeDefault   Floating point
              Default pixel size in nm, which will be used for any models that
              do not have a pixel size defined.  The default is 1, namely
              results will be in pixels.

       -width (-w) OR -WidthDefault   Floating point
              Default width between walls in nm.  This value is used to com-
              pute normalized areas for unpaired walls.  The default is 20.

       -surface (-s) OR -UseSurfaceNumbers
              Use surface numbers to determine whether two wall tracings are
              paired and whether a horizontal marker contour matches a wall
              tracing.  This is not the default because surface numbers might
              have been introduced by accident.

       -nopairs (-n) OR -NoPairsOrMarkers
              There are no paired tracings and no horizontal marker lines.
              With this option you do not need to segregate tracings in sepa-
              rate objects or surfaces.

       -model (-m) OR -ModelFile      File name
              Input model file with tracings of microtubule walls (Successive
              entries accumulate)

       -fit (-f) OR -FitTopAndBottom       Two floats
              Upper and lower limits for the line fit.  The limits are entered
              as either an absolute distance from the maximum Y for a MT or a
              fraction of the distance from the maximum to the minimum Y.  If
              the upper limit is 0, the maximum Y is used.  If the lower limit
              is 0, the limit used depends on whether there is a horizontal
              marker line or not.  If there is a line, it is used as the
              limit; if not, the second point of the contour is used as the
              lower limit.  Enter this option once to use the same limit for
              all models, or once per model.  If it is not entered, the
              default limits are 0,0.  (Successive entries accumulate)

       -id (-i) OR -Identifier   Integer
              ID number for output.  Enter this option either once to specify
              a starting ID number, or once per model.  If it is not entered,
              the ID numbers start at 1.  (Successive entries accumulate)

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

              Read parameter entries from standard input.

       Written by David Mastronarde in 1996
       Modified for PIP input and incorporation into IMOD, 10/14/06

       Email bug reports to mast at colorado dot edu.

IMOD                                 4.9.3                        howflared(1)