mtoverlap(1) mtoverlap(1)NAMEmtoverlap - to sdisplay and analyze overlap of spindle MTsSYNOPSISmtoverlapDESCRIPTIONMtoverlap allows one to display sets of "bundles" of microtubules (MT's) and to compute overlap between MT's coming from the two differ- ent directions. It has a lot of flexibility but one can select a default, standard display format fairly easily. Before running the program, you must figure out how to specify which MT's are in a bundle. If all of the MT's in a model belong to one bun- dle, then this task is easy. If you have several bundles in one model, then you have several alternatives. One is to determine the lower and upper X, Y and Z coordinates of a box, such that the bundle consists of all MT's that contain at least one point within the box. Another way is to make a model contour within the plane of one section to serve as a boundary contour. This contour, together with a lower and upper Z coordinate, specifies a "cylinder", and this program will include in the bundle any MT with at least one point inside this cylinder. The most elaborate way is to make a series of model contours for boundary contours in different sections. The program will then include in the bundle any MT that is included within any one of the contours. For each bundle that the program deals with, it will want to know a center Z coordinate; this center value is used to align different bun- dles for display and to compute the average distance past center that each class of MT extends. The program can compute the center value that makes two classes of MT's extend past the center by the same amount (in opposite directions). It can do this computation for each bundle separately, for all bundles pooled together, or for any combina- tion of bundles that you desire. Alternatively, you may enter the cen- ter Z coordinates. When you enter X, Y or Z coordinates for either of the above purposes, they must be index coordinates of the image file. That is, X and Y values must be in terms of pixel coordinates, and Z values must be in units of the original section numbers, before adjustment for tilt or scaling by section thickness. If the sections were significantly tilted during microscopy, the pro- gram can adjust for these tilts given the proper information. Prepare a file in which the first line shows the Z value and the tilt of the first tilted section (or of the first section, if that one was tilted), and each successive line shows the Z value and tilt for each section on which tilt was changed. Z values should occur in ascending order. The program refers to different kinds of MTs as "types". For an IMOD model, the type is simply the object number; for data from a WIMP model file, the type is 256 minus the object color, or the negative of this value if the WIMP object is turned off. A default display format is set up to be used with either kind of model file. To use the defaults with an IMOD model, MTs starting at low and high Z should be in objects 1 and 2 respectively; continuous MTs in object 3, and free MTs in object 4. With a WIMP model, MTs from low and high Z should have col- ors 250 and 251 (types 6 and 5 in the program), and continuous and free MTs 252 and 255 (types 4 and 1). When you start the program, you will have to make a standard series of entries until you get the first display. From there, you can select a number of options to loop back and change those entries. Initial entries in order are: Name of command file to take entries from, or Return to continue making entries from the keyboard. The program can read entries from a file instead of from the keyboard, then switch back to keyboard input if the file ends with the appropriate entry. A list of types to be mapped, or changed, into new types, or Return for no mapping of one type into another. This option is useful if you have several different types that you want to combine into one. For exam- ple, if you want to treat types 11 and 13 like type 1, and types 12 and 14 like 2, and if you also have some existing MT's of types 1 and 2 that you don't want to include with these types, then you need to remap all of these types by entering 11-14,1,2 IF you entered some types to remap, next enter the types to change them into. For the example just described, you would enter: 1,2,31,32 Number of bundles to read from model files, or 0 if the entries speci- fying all of the bundles are in yet another file. IF you enter a positive number, then enter for each bundle: Name of model file with bundle in it, or Return to use same file as previous bundle IF you enter the name of file, make the following 1-3 entries: Name of file with information on tilt angles, or Return if there is no such file (pictures taken at 0 tilt) IF the model header has no scaling information, make the next two entries as well to specify scaling: Section thickness in nm, to scale Z coordinates to microns; or / to leave Z values unscaled Magnification of negatives, and scale of digitization (the value of microns/pixel from VIDS), to scale the X/Y coordinates correctly; or / to leave X/Y coordinates unscaled. This entry makes no difference unless you choose to calculate one of the special three-dimensional overlap factors. Number of limiting regions (boundary contours or rectangles defined by X/Y coordinates) needed to specify the bundle, or 0 to take all of the objects in the model. For each limiting region, then enter: Either IMOD object number and contour number of the boundary contour, or a WIMP object number and 0 for data taken from a WIMP model file, or 0,0 to enter limiting X and Y coordinates of a box. IF you entered 0,0 next enter the lower and upper X index coordinates and the lower and upper Y coordinates of the box, or enter / to have no limit on the X and Y coordinates THEN enter the lower and upper Z coordinates of the box (in units of sections), or / to have no limits on Z coordinates IF you entered numbers for a boundary contour, next enter lower and upper Z coordinates of the "cylinder", or / to set those limiting coordinates to the Z coordinate of the boundary contour. The latter is typical if one uses several contours in different sections to specify the bundle. IF you entered 0 for the number of bundles, next enter instead the name of a file. The first line of this file should have the number of bun- dles specified there. The rest of the file should be all of the entries just described for each bundle. Enter 0 if you want to specify EVERYTHING or 1 to use the default for- mat for types, display colors, etc. with an IMOD model, or 2 to use defaults for a WIMP model. With an entry of 1, you will get centers of bundles calculated from MT types 1 and 2, overlap calculated from types 1 and 2, and a display occupying the whole screen with, from top down, type 4 in order by increasing length, types 1 and 2 interleaved with 1 in order by increasing Z of endpoint and 2 in order by decreasing Z or startpoint, then type 3 in order by increasing length. Enter a list of numbers of the bundles to work with. Ranges may be entered, e.g. 1-3,7-9. Enter 1 to have each bundle's center computed separately, 2 to have a single center Z value computed with all bundles pooled together, 3 to specify a single center Z coordinate for all bundles, or 4 to control center specification more intimately. IF you entered 3, next enter the Z value to use as center for all bun- dles, in units of original section numbers. IF you entered 4, next enter a set of numbers, one for each bundle: either a specific Z center section value for that bundle, or the nega- tive of a specific Z center value in microns, or 0 to have its center computed separately from other bundles, or a negative number less than -100; all bundles with the same negative number will be pooled and given the same computed center value. IF you did not select default display, next enter two lists of types to calculate the center from, where ranges may be entered: List of types coming from low Z List of types extending to high Z IF you did not select default display, next enter two lists of types to compute the overlap from, or 2 Returns to omit computing overlap: List of types coming from low Z List of types extending to high Z Enter 0 for simple overlap factor (without considering proximity in the X/Y plane), or 1, 2 or 3 for a 3-D overlap factor, where the amount of overlap between two MT's per section decays with increasing distance between them in the X/Y plane, either as a step function (1 within a certain distance and 0 beyond it), an inverse power, or exponentially. IF you entered 1-3, next enter 0 to compute an average overlap factor for each MT, then average those values over the MT's, or 1 to compute the sum of overlap factors for each MT, then average those sums over the MT's. In the latter case, the resulting values may depend heavily on bundle size. IF you entered 1-3, next enter the distance in the X/Y plane at and below which overlap will equal 1. The distance should be in microns if you have scaled X/Y values, or in pixels if you have not. For the step function option, enter the maximum preferred distance between MT's. IF you entered 2, next enter the power for the decay (e.g., with a power of 2, overlap will decay as the inverse square of distance) IF you entered 3, enter instead the space constant for exponential decay. Overlap will be 1/e less for MT's separated by 2 space constants than for MT's separated by 1 space constant. Distance should be in microns if you have scaled X/Y values, or in pixels if you have not. IF you did not select default display, make the following entries to control the display: List of types to display, or Return for no display. Ranges OK. Colors to display them as, or / to take standard colors. Colors are specified as numbers from 0 to 255. 0-240 correspond to gray scales from black to white, then 237-255 give olive, dim yellow, orange, red, green, blue, yellow, magenta, and cyan. For data from an IMOD model, / will assign colors as 256 minus the type. For data from a WIMP model, / will give the same colors as in the model, unless types have been remapped. Enter a number for each type to control the ordering of the MT's from the top down: 1 or -1 to have in order by increasing or decreasing Z of the starting point; 2 or -2 for order by increasing or decreasing ending Z; 3 or -3 for order by increasing or decreasing length Enter a positional value for each type, where positions are numbered from the top down; two types with the same position number will be displayed with their MT's interleaved. Enter 1 to plot all bundles in the same graph, 2 to plot each bundle in a separate graph, or 3 to specify more complicated combinations IF you entered 3, enter a graph number for each bundle included in the display, where graphs are numbered from the top down. Bundles with the same graph number will be pooled for display. IF you did not select default display, make three more entries Either the negative of the total horizontal size of display, in pixels, or the number of pixels per unit of Z, or / to use the default indicated (initally 1280 pixels). Total vertical size of display, in pixels, or / to use the default Line spacing in regions where MT's are interleaved relative to spacing in non-interleaved regions, line thickness, axis thickness, label thickness, and lengths of major and minor ticks. (It will tell you what the defaults are.) A thickness of 2 IS available, but higher even thicknesses are rounded up by 1 (so only odd thicknesses are available above 3). To get lines drawn in order from the bottom up instead of from the top down, enter the negative of the desired value for interleaved line spacing (typically, the negative of the indicated default value.) Colors for the axes, the labels, and the fitted lines; size of labels; # of pixels of additional shift leftward and downward for labels; intervals (in # of ticks) at which to have major ticks and labels. It will tell you the defaults; enter / to use them. At this point you will get the display and some output: the number of each type of tube in each graph and the mean and standard deviation of their lengths, and computed overlap values for each bundle separately and for all bundles together (the last line of output). Four overlap values are computed (mean, S.D., and # of MT's contributing to each value are printed). The first is the distance past the center that each MT extends. The other three are overlap values for MT's coming from low Z (from the left), for MT's coming from high Z (from the right), and for both of those sets of MT's combined. With the simplest overlap computation, the overlap value for a single MT is the average amount of Z overlapping with other MT's, where the average is only over those MT's from the other direction that actually do overlap with the given MT. The values printed out are the mean and S.D. of these aver- ages for all the MT's from the given direction. With the inverse power or exponential decay options, instead of count- ing 1 unit of overlap per section of overlap between two MT's, the amount of "overlap" in each section is computed from the distance between the two MT's in that section, giving a number that is 1 for nearest neighbor MT's and less for more separated MT's. This overlap factor is then summed over all sections in which both MT's appear. For a given MT, the program will then form either the mean or the sum of the summed overlap factor between that MT and all other overlapping MT's. The sum is probably a more meaningful measure. Finally, these means or sums are averaged over all MT's from a given direction, including MT's with 0 overlap. Now you can loop back to various parts of the program. Enter: 1 to combine the overlap calculation for a group of bundles 2 to change the display size or interleave/non-interleave spacing 3 to specify which bundles should go in which graphs 4 to specify the types to display, and their colors, positions and ordering parameters 5 to specify the types to compute overlap from, or the way of computing the overlap factor 6 to change which bundles are included in the display or computations 7 to control output of numbers of MT's and overlap values to a file 8 to read in new bundles and add them to existing ones 9 to read in new bundles and replace previously read ones 10 to take commands from a file (next enter filename, or Return to take input from the keyboard) 11 to exit 12 to fit lines to the starting and ending points of certain types 13 to change the mapping of one type into another 14 to plot the graph to a postscript file 15 to display such a postscript file on the screen 16 to print the postscript file IF you enter 1, next enter the list of bundles to combine for computing overlap (ranges are ok). If there are, say, 4 bundles included in the computation and/or display, they are referred to as numbers 1 to 4, regardless of their numbers among the entire set of bundles that have been read in. IF you enter 7, on the first such occasion, enter the name of a file to store output into. Then enter: 0 to turn off output to the file 1 to output only the overlap calculations to the file 2 to output only the numbers of MT's to the file 3 to output both overlap and numbers. IF you enter 6, 8, or 9, you will loop back and have to make all of entries that follow the point to which you looped back; other options involve re-entering only a subset of the parameters. IF you enter 12, the program will fit a line to the starting points of one type of MT, and another line to the ending points of another type of MT. It will display the fitted lines and report two factors: the slope, in units of percent of that type of MT starting (or ending) per unit of Z; and the distance past the center at which the line crosses the level of 50% of the MT's. It reports these factors separately for the two lines, and also shows the average of the values for the two lines. If there are too few MTs to derive a value, the value is reported as 0. It also reports the number of MT's used to derive the factors. Each line displayed on the screen occupies the vertical extent of the MT's included in the fit. When you enter 12, you next make two entries: The type to whose starting points a line will be fit, and the type to whose ending points a line will be fit, or / to accept the defaults, which are initially the types used to calculate overlap. The lower and upper percentile limits for the MT's to be included in the fits, or / to accept the defaults shown in parentheses. MT's are counted from the top of the display downward. For example, if you enter 5 and 85, then the top 5% and the bottom 15% of MT's in each type will NOT be included in the fits. IF you enter 13 to change type mapping, you should then select option 6 in order to make sure that the new types are being used correctly for display or computation. IF you enter 14 to plot the graphs, the program will ask for the X and Y size and lower left X and Y coordinates, in inches, of the location on paper corresponding to the full screen display. You can use these entries to change the size or aspect ratio of the display. Next the program will ask for a label for the X axis; enter Return for no label. The size and spacing of the axis numeric and text labels can be con- trolled by the entries that one sets when displaying the graphs on the screen.HISTORYWritten by David Mastronarde, 10/3/90 2/21/92: changes to scale data into microns, add line fits 5/1/92: implemented simple distance-dependent overlap 6/9/92: implemented mapping of types 11/5/94: fixed interset and intergraph spacing, aligned interleaves at the bottom of each set to obviate need to invert drawing 6/14/96: added plotting output 4/28/97: changes for IMOD modelsBUGSEmail bug reports to mast at colorado dot edu. BL3DEMC 4.7.3 mtoverlap(1)