findbeads3d(1) General Commands Manual findbeads3d(1)NAMEfindbeads3d - Find spherical densities in a volumeSYNOPSISfindbeads3d [options] input_image_file output_model_fileDESCRIPTIONFindbeads3d will find gold particles and other spherical densities in tomograms. Given the diameter of the beads, it will search for parti- cles of this size and store their locations in a model along with the strengths of their correlations with an average particle. It will automatically determine the threshold strength that separates the gold particles from lesser densities. The search occurs in two phases. First, density is summed over small cubes centered on each pixel, about 3/8 of the bead diameter on a side, and local peaks in the summed density are found. The peak strength is measured by an integral of the densities relative to the background density. When two of these candidate peaks are too close together, the weaker one is eliminated. The integrals are scaled so that they range up to 1, a subset of the strongest peaks are selected, and an average bead is obtained by extracting a small volume at each selected peak location. By default, the subset is selected by analyzing a histogram of the peak strengths to find a dip separating the gold from weaker features; however, the -threshold option can be used to specify a num- ber of peaks to use or a relative peak strength to use as the thresh- old. In the second phase, the average bead is cross-correlated with subvol- umes at all of the candidate peak locations from the first phase. This time the value of the cross-correlation (not a normalized correlation coefficient) is taken as the peak strength, and again they are scaled to range up to 1. A selection of the strongest peak locations are then written to the output model. Again, the default method for this selec- tion is to analyze a histogram of peak strengths for a dip between beads and weaker peaks, but the number of points stored in the model can also be controlled with the -store option. The output model consists of a scattered point object with one point per contour. The peak strength is stored as a general value, and the object properties are generally set so that only points above threshold are displayed. However, points below threshold generally do exist and need to be removed before reprojecting the points with Tilt. To see all of the points, adjust the threshold, or delete points below the threshold, load the model into 3dmod and open the Bead Fixer in Seed mode. The Bead Fixer will have a slider for adjusting the threshold, a checkbox to controlling whether points below threshold will be dis- played, and a button to delete points below threshold. Since these points are located on many Z planes, be sure to check "Delete on all sections" before deleting the points. The tomogram can be built from binned down aligned images, and the model will load correctly on an unbinned tomogram. Also, a reprojec- tion of the points by Tilt will load correctly on an unbinned or a binned aligned stack. Findbeads3d will work much faster on a binned tomogram and may even work better, as long as the bead size in the binned tomogram is at least 5 pixels. The tomogram needs to be thicker than normal because the program cannot find beads too close to the surfaces of the tomogram. With the typical elongation taken into account, the program will find only the beads that are more than ~1.1 bead diameters from the surface. If gold is on two surfaces, thickness thus needs to be at least 2.2 bead diameters more than the average distance between the surfaces, which is indicated in the Surface Analysis output of Tiltalign as "Thickness at Z intercepts". To the extent that beads do not lie exactly in planes, even more thickness will be required; 3 diameters is a good starting point and is used in the interface in Etomo. In IMOD 4.8.30, the program switched to taking the square root of cor- relation peak strength. In a histogram, this change spreads out the very large histogram peak from weaker, non-bead peaks, compacts and accentuates the much smaller histogram peak from actual beads, and thus makes the histogram dip more pronounced and easier to detect. The square root values will be reported for the histogram dip and peaks and stored in the model for visualization in 3dmod. If a relative peak strength is entered for the threshold options -fBthreshold and-store, it should be in terms of the square root values. However, the minimum relative peak strength is still entered in terms of the raw correlation values before taking the square root, in order to maintain the same behavior for this routinely applied parameter.OPTIONSFindbeads3d 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.-input(-i)OR-InputFileFilenameInput file with image volume-output(-o)OR-OutputFileFilenameOutput file for model of correlation peaks-candidate(-c)OR-CandidateModelFilename-size(-si)OR-BeadSizeFloatingpointDiameter of beads in pixels, a required entry. If the volume is binned, this size can be entered in unbinned pixels and the -binning entry used to specify the binning.-binning(-bi)OR-BinningOfVolumeIntegerThe current binning of the image volume. The entered bead size is divided by this binning to obtain the actual size of the beads in the volume.-xminmax(-x)OR-XMinAndMaxTwointegersStarting and ending X coordinates of region in which to find beads. X, Y, and Z coordinates are numbered from 1. The default is to search the whole volume.-yminmax(-ym)OR-YMinAndMaxTwointegersStarting and ending Y coordinates of region to find beads in-zminmax(-z)OR-ZMinAndMaxTwointegersStarting and ending Z coordinates of region to find beads in-light(-l)OR-LightBeadsBeads are light on dark background-angle(-a)OR-AngleRangeTwofloatsStarting and ending tilt angles, for computing the elongation factor due to the missing wedge in single-axis tomograpy. The elongation is computed using a formula in Radermacher (1988), with the maximum tilt angle for that formula taken as the mean of the absolute values of the minimum and maximum angles of the series.-tilt(-ti)OR-TiltFileFilenameFile with tilt angles, for computing elongation factor from the minimum and maximum tilt angle, as just described. Only one of -angle and -tilt may be entered.-ylong(-yl)OR-YAxisElongatedFeatures are elongated by the missing wedge along the Y axis instead of the Z axis. This entry is needed when analyzing a tomogram as produced by Tilt with Z slices along the tilt axis and Y perpendicular to the plane of the specimen.-peakmin(-pe)OR-MinRelativeStrengthFloatingpointMinimum relative peak strength for keeping a peak in the analy- sis. The square root of the specified value is used for compar- ing with the square root of peak strength, for compatibility with existing command files. The default is 0.05, which corre- sponds to a relative square root peak strength of 0.22. Too many weak peaks can prevent a dip from showing up in the smoothed histogram of strengths. If the program fails to find a histogram dip, one strategy is to try raising this value.-threshold(-th)OR-ThresholdForAveragingFloatingpointThreshold relative peak strength or number of beads for averag- ing. With a non-zero entry, selected beads from the first round of peak finding and integral measurement are averaged to produce a reference for a second round. If a negative value is entered, the program will analyze the histogram of peak strengths and find the dip indicating the best boundary between actual and false beads. The value has 4 different meanings depending on the range: Greater than 1: an absolute number of beads with the strongest peaks Between 0 and 1: minimum relative square root of peak strength Between 0 and -1: negative of strongest fraction of peaks above histogram dip (e.g., -0.33 for the strongest 1/3 of the peaks above the dip) -2: 1/4 of way from histogram dip to histogram peak (the default)-store(-st)OR-StorageThresholdFloatingpointThreshold relative peak strength for storing peaks in model. With a value of 0 (the default), the program will find the dip in the histogram of peak strengths, find the mean and SD of the strengths above the dip, and store all of the beads above the dip plus additional ones below the dip. The latter will be up to the 5 SD's below the mean or up to the number of ones above the dip. Enter a number between 0 and 1 to specify a relative square root strength above which peaks will be stored. Enter a negative number to specify the number to store as a fraction of the number above the histogram dip (e.g., -1 for all points above the dip, -0.33 for the strongest 1/3 above the dip, -1.33 for all above plus 1/3 that many below the dip.) If you use the default, or a number below -1, then you must examine the points in 3dmod, adjust the threshold if necessary, and delete points below the threshold.-fallback(-f)OR-FallbackThresholdsTwofloatsThresholds to use as fallbacks when no histogram dip is found. The first number is a threshold to use for averaging when no dip is detected in the histogram of peak strengths in the first round. It can be between 0 and 1 to specify a relative peak strength, or a number greater than 1 to specify a number of beads. The second number is a threshold relative strength for peaks to store, between 0 and 1, when no dip is found in the histogram of second-round peak strengths. If a fallback is desired for only one of these two rounds, enter a zero for the other value.-spacing(-sp)OR-MinSpacingFloatingpointMinimum spacing between peaks as a fraction of the bead size. When two peaks are closer than this distance apart, the weaker one is eliminated unless the -both option is entered. The default is 0.9. A value less than 1 is helpful for picking both beads in a pair.-both(-bo)OR-EliminateBothEliminate both peaks when their spacing is below the minimum spacing-guess(-g)OR-GuessNumBeadsIntegerA guess for the number of beads. This entry may sometimes be required to help the program find a dip in the histogram, espe- cially if there are very few beads. The number can be an under- estimate and a very rough estimate. Use this entry if a his- togram dip is found at a rather high peak strength with few peaks above it.-max(-m)OR-MaxNumBeadsIntegerThe maximum possible number of beads to look for. Only this number of peaks will be retained and used for finding the his- togram dip. If the program fails to find a histogram dip, try entering a value that is at least twice the estimated number of beads. The default is 50000.-verbose(-v)OR-VerboseOutputIntegerEnter 1 for diagnostic output, 2 for listing of peaks, and 3 or 4 for verbose output from the histogram routines-param(-pa)OR-ParameterFileParameterfileRead parameter entries from file-help(-h)OR-usagePrint help output-StandardInputRead parameter entries from standard input.HISTORYWritten by David Mastronarde 12/10/08BUGSEmail bug reports to mast at colorado dot edu. IMOD 4.9.7 findbeads3d(1)