newstack(1) General Commands Manual newstack(1) NAME newstack - Make a new mrc image stack. SYNOPSIS newstack [options] input_file(s) output_file DESCRIPTION Newstack is a general stack editor to move images into, out of, or between stacks. It can float the images to a common range or mean of density. It can bin images and apply a general linear transformations as well as a specified rotation or expansion. It can put the output into a smaller or larger array and independently recenter each image separately from the transformation. Images can be taken from multiple input files and placed into multiple output files. OPTIONS Newstack uses the PIP package for input (see the manual page for pip) and can take input interactively only for options available before conversion to PIP, to maintain compatibility with old command files. The options accepted by the old newst script are all accepted by newstack. Almost all options can be abbreviated to one or two let- ters. 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 paren- theses. However, if you are using short names in a script or command file, always use at least 3 letters, because new options might use the same one or two initial letters. In Windows, Newstack expands wildcards ('*' and '?') for input file- names internally, which is particularly useful without Cygwin. It can also be used in Cygwin when the command line is too long after expan- sion; enclose the names with wildcards in quotes to keep the shell from expanding then. On other systems, when there are too many input files, use the script Expandargs to produce a file that can be used with the -filein option. That script can also handle ranges enclosed in square brackets. INPUT AND OUTPUT FILE OPTIONS These options are involved in specifying input and output files. -input (-in) OR -InputFile File name Input image file. Input files may also be entered after all arguments on the command line, as long as an output file is the last name on the command line. Files entered with this option will be processed before files at the end of the command line, if any. (Successive entries accumulate) -output (-ou) OR -OutputFile File name Output image file. The last filename on the command line will also be treated as an output file, following any specified by this option. (Successive entries accumulate) -fileinlist (-filei) OR -FileOfInputs File name Name of file with list of input files and sections to read. The format of the file is: number of input files name of first file to read list of sections to read from first file name of second file to read list of sections to read from second file ... If this option is used, no input filenames may be specified with the -input option or as non-option arguments on the command line. The section lists are allowed to have 8 times as many characters as the maximum number of sections, which is 1,000,000 by default but can be changed with the -megasec option. A file list can be constructed for a set of input files with the Expandargs script. -fileoutlist (-fileo) OR -FileOfOutputs File name Name of file with list of output files and number of sections to place in each. The format of the file is the number of output files on the first line, the first file to write on the next line, number of sections to write on next line, next filename on next line, etc. If this option is used, no output filenames may be specified with the -output option or as the last file on the command line. -reverse (-rev) OR -ReverseInputFileOrder Integer Process some or all of the input files in reverse order. Enter 0 to reverse the order for all files, a number N greater than 0 to process only the first N files in reverse order, or a number N less than 0 to reverse only the last N files. This option allows you to stack sequentially numbered files in a desired order and still enter them with wild cards on a command line. No other entries, such as multiple section lists, are reversed; they all apply to the files in the order that they are pro- cessed. There must be at least the given number of input files, entered with -input or as non-option arguments. -split (-sp) OR -SplitStartingNumber Integer Starting number for a series of numbered output files, where one section will be placed in each file. There must be only one output file name entered, and it will be taken as the root name for the output files. A dot and the number will be appended to the root name for each file unless an extension is entered with the -append option. The number of digits used for the number will be the same for all files so that they will list in order. -append (-appe) OR -AppendExtension Text string Extension to append to the filenames in a series of numbered files. With this option, files will be named like "rootnnn.ext" instead of "root.nnn", where "root" is the root name entered as the output file name, and "ext" is the extension entered with this option. -format (-fo) OR -FormatOfOutputFile Text string Set output file format to MRC, TIFF, HDF (if the package sup- ports HDF), or JPEG (if the package supports JPEG) by entering MRC, TIF, TIFF, HDF, JPG, JPEG, or any lower case form of these. Only a single image can be output to a JPEG file. This entry overrides a default format set with the environment variable IMOD_OUTPUT_FORMAT. -compression (-com) OR -HDFCompressionIndex Integer Sets the level of ZIP compression when writing to an HDF file. Compression values range from 1 (fastest, least compressed) to 9 (slowest, most compressed), or 0 for no compression. This entry overrides a value for compression set by the IMOD_HDF_COMPRES- SION environment variable. -volumes (-vo) OR -VolumesToRead List of integer ranges If any HDF files containing multiple volumes are included in the input file list, this option must be entered to specify which volume to read from such a file. Enter a volume number for each input file, numbered from 1, at least up until the last multi- volume HDF file in the input file list. Enter 1 for an HDF file with only one volume or a stack of 2D images, or for other file types. -3d (-3) OR -Store3DVolumes Integer This option controls whether data are stored in a 3D volume in an HDF file. If 1 is entered, a new HDF file will be created (overriding the default output type) and data will be stored as 3D volume rather than a stack of 2D images. If 2 or 3 are entered, the output file must be an existing HDF file with data stored as volumes, and data will be stored in a new 3D volume. A value of 3 is used by Makepyramid to cause Newstack to insert the attribute "image_pyramid". By default, if an HDF output file is being created, it will be stored as a volume if the first input file is an HDF file with volume storage. A value of -1 will cause an output HDF file to be organized as a stack even if the input file has volumes. -chunk (-ch) OR -ChunkSizesInXYZ Three integers Set target chunk sizes in X, Y, and Z for 3D volumes stored in HDF files. Based on the target, an actual chunk size will be chosen in each dimension that minimizes the amount of extra image area created by making a set of full tiles. When the tar- get value already evenly divides the image size in a dimension, that target will be used. If this option is entered, the output file will be an HDF file with data stored as a 3D volume, making it unnecessary to enter -3d or -format. The default is for 3D volumes to be stored with no tiling in X and Y and a chunk size of 1 in Z, or with the tiling and chunk size of the first input file if it is an HDF file stored as a 3D volume. -mdoc (-md) OR -UseMdocFiles This option allows data about each image section in metadata autodoc (.mdoc) files to be transferred and managed much as data in the extended header of an MRC file are. With it selected, the program will search for a matching .mdoc file for each input file that is not HDF, and create a matching .mdoc file for each output file that is not HDF. (A matching file is one with .mdoc appended to the image filename, as SerialEM creates). Metadata about each image slice in ZValue sections will be transferred between autodocs and the ZValue will be renumbered appropri- ately. Thus, data can be transferred from one .mdoc to another, if input and output files are MRC; from an .mdoc into the attributes of an HDF file if input is MRC and output is HDF; or from HDF attributes into an .mdoc if input is HDF and output is MRC. When input and output are both HDF, attributes are trans- ferred automatically. As of IMOD 4.11, metadata in autodoc sec- tions other than ZValue ones will be transferred in their entirety.^ If there is a single image in the input file, an .mdoc file produced by SerialEM along with a stack of frames will also be accepted as a valid source of metadata for the image. These .mdoc files have a single section named "FrameSet" and no global information about the image size or mode at the top. Their contents will be transferred to ZValue sections in the new .mdoc file -remove (-rem) OR -RemoveForMdocName Text string This entry and the next one allow use of .mdoc files whose name endings do not match those of the corresponding image files. The two entries specify how to get from the current name of the input image file to the image file name upon which the .mdoc is based. For this option, enter the text to be removed from the end of the image file name. If necessary, use the -addback option to restore the full original image file name. For exam- ple, if a stack of frames is saved into "originalName.tif" and the metadata into "originalName.tif.mdoc", then they are aligned and summed into a single image file "originalName_ali.mrc", the entry to this option would be "_ali.mrc" to get to "original- Name". The entry to -addback would be ".tif" to restore the original extension and get back to "originalName.tif". The pro- gram would then look for and find the .mdoc "original- Name.tif.mdoc". -addback (-ad) OR -AddBackForMdocName Text string String to add back to image file name after removing the string specified by the -remove option (which must also be entered), to obtain a file name for which there is a matching .mdoc. -pixel (-pi) OR -PixelSizeFromMdoc Set the pixel size in the header of each output file from the value of "PixelSpacing" in an .mdoc file associated with the input file, or in the metadata of an HDF file. Specifically, the pixel size for an output file is set from the value for the first section going into that file. When outputting one image per file, such as with the -split option, each file will have the correct pixel size. Output .mdoc files are not created unless -mdoc is also entered. -tilt (-ti) OR -TiltAngleFile File name Name of file with tilt angles, one per line, to substitute or insert into the metadata of the output file(s). The successive angles in the file will be placed into the output in order, so the angles in the file should be in the same order as the intended order of the output. Angles can subsequently be retrieved with Extracttilts. If the input file(s) have extended headers with either the SerialEM or Agard/FEI format, the new values will be substituted for existing angles and other extended header items will be preserved. (There must already be angles in the SerialEM header, and all input files must have the same type of extended header.) If the input file(s) have no extended header, angles will be placed into an extended header in the Agard/FEI format (just 1 floating point number per sec- tion). (In this case, all input files must have no extended header.) Tilt angles can also be replaced or inserted if output file(s) are HDF, with or without an incoming .mdoc file; and tilt angles in .mdoc files associated with MRC files will be maintained as well. If just an extension is entered (for exam- ple, ".tlt"), the program will look for a file with the root name of the input image file and this extension. -reorder (-reo) OR -ReorderByTiltAngle Integer Reorder sections by tilt angle, which are taken either from an extended header or from a file of current angles specified with the -angle option. If any input file does not have angles in its extended header, the angles must be supplied with the -angle option. Enter 1 or -1 to have sections put in increasing or decreasing order by angle. If angles are provided with the -angle option, you can also enter 2 or -2 to have the reordered angles inserted into the extended header. Reordering is compat- ible with the section selection options below except -twodir; the subset of sections specified by them will be output in order by their angles. The -tilt option cannot be combined with this one. There must be only one output file. If there is more than one input file, the sections will be ordered separately for each one. -angle (-ang) OR -AngleFileToReorder File name File with tilt angles to use for reordering sections by angle with the -reorder option. The file must contain one angle per output section, each on its own line (not one per section in the input file, if a subset of sections is being used.) The angles should be in the same order as the input sections. The angles in this file override any angles present in the extended header, but they will replace the angles copied over in the extended header only if -reorder is entered with 2 or -2. -newangle (-new) OR -NewAngleOutputFile Integer File for output of tilt angles after reordering with the -reorder option. This option can be used regardless of whether original angles are supplied with the -angle option. SECTION SELECTION OPTIONS These options are used to specify which sections are read and written -secs (-se) OR -SectionsToRead List of integer ranges List of sections to read from an input file, numbered from 0 unless -fromone is entered. Ranges may be entered (e.g., 1-3,5,8), and / may be used to specify all sections in the file. If multiple lists are entered with multiple input files, each one will be applied to the respective input file. If no list is entered for a file, all sections will be read. When there is a single input file, multiple entries will simply be concatenated to make one list. Such entries may be necessary for entering very long lists, because each list entry is limited to 100,000 characters. (Successive entries accumulate) -samesec (-sa) OR -SameSectionsToRead Read the same set of sections from all input files, without hav- ing to enter a section list for each file. The -secs options must be entered only once. -fromone (-fr) OR -NumberedFromOne With this option section numbers entered with the -secs, -replace, and -exclude options, line numbers entered with -use- lines, and field numbers entered with -fields are numbered from 1 instead of 0. -exclude (-exc) OR -ExcludeSections List of integer ranges List of sections to exclude from the input file(s). Exclusion is applied to the entire section list after it has been composed from other section-specifying options. Any number in the sec- tion list contained in this exclusion list is removed from the section list, no matter how many times that number appears. Thus, one exclude entry can be used to exclude the same set of section numbers from multiple input files. The list can consist of ranges; sections are numbered from 0 unless -fromone is entered. -twodir (-tw) OR -TwoDirectionTiltSeries Combine two files from the two halves of a bidirectional tilt series, where each half is taken in opposite directions of tilt from a common starting angle. The sections will be stacked in inverted order for the first file. There must be two input files, and section lists cannot be entered. -skip (-sk) OR -SkipSectionIncrement Integer Increment for reading a regularly spaced subset of the sections in the list from the input file. Enter 2 to take every other section, 3 to take every third section, etc. Note that the increment is used to select from the input list, not from all the sections in the file. -numout (-nu) OR -NumberToOutput Multiple integers Number of sections to write to each output file, when there are multiple output files. These numbers may be listed in sequence in one entry, or in several entries. (Successive entries accu- mulate) -replace (-rep) OR -ReplaceSections List of integer ranges List of section numbers at which to write images into an exist- ing output file, numbered from 0 unless -fromone is entered. There must be only a single output file, its size in X and Y must be appropriate, and the given sections must already exist in the file. The minimum and maximum density in the file header will be modified by the min and max of the sections being writ- ten, so they can become more extreme but not less extreme. The mean density will not be changed. -blank (-bl) OR -BlankOutput Output blank sections for nonexistent section numbers in input file, namely for section numbers less than 0 or more than the NZ - 1. The sections are filled with the mean value of the input file or with a fill value if one is entered with the -fill option; this value is then scaled in the same way that actual data are. OPTIONS FOR OPERATING ON IMAGES These options specify geometrical operations performed on the images. -offset (-of) OR -OffsetsInXandY Multiple floats X and Y center offsets for each section. This is an offset to the center of the area from which image will be taken, so a pos- itive offset in X will move images to the left. If images are being reduced with -bin, -shrink, or -ftreduce, the entry should be in unbinned pixels. Enter one X,Y pair to apply a single offset to all sections. If other transformations are being applied to the sections, these offsets will generally be com- bined with those operations, and non-integer offsets are mean- ingful. Otherwise, the nearest integer offset will generally be applied, except when the -phase, -ftreduce, or -ftexpand option is used and the offsets are applied in Fourier space. (Succes- sive entries accumulate) -applyfirst (-appl) OR -ApplyOffsetsFirst Apply offsets in X and Y before transforming image, which will make a rotated image come from a predictable location. The default is to apply offsets after, which shifts the transformed image instead. This option is relevant only when offsets are combined with either general linear transformations or rotation, expansion, or shrinkage; when shifts are applied in Fourier space with the -ftreduce option, they are always considered as unbinned shifts and thus occur before the reduction. -xform (-x) OR -TransformFile File name File with one or more linear transformations to apply to images; if there is only one transform it will be applied to all images. -uselines (-use) OR -UseTransformLines List of integer ranges A list of the line numbers of lines of transforms to use in the transform file (numbered from 0 unless -fromone is entered). Ranges are allowed. The default is for the line numbers to match the input section numbers, unless there is just one trans- form in the file. To have a single transform applied to all of the sections, just enter a single number. (Successive entries accumulate) -onexform (-on) OR -OneTransformPerFile Apply one transform line for each input file. The default is for the first transform to be applied to the first file, the second transform to the second file, etc., in which case the transform file must have at least one line per input file. Oth- erwise, -uselines can be used to specify which transform to apply for each file, in which case at least one line must be specified per input file. -phase (-ph) OR -PhaseShiftFFT Shift images by taking a Fourier transform, changing phases appropriately, and taking an inverse Fourier transform, rather than by interpolating in real space. In principle, this method preserves high frequencies better than interpolation does. The method can be used only if images are just being shifted or scaled with the -ftreduce or -ftexpand option, not when they are rotated, expanded, shrunk, stretched, warped, or undistorted. The shifts can be specified in two ways: with the -offset option, or as linear transforms that contain only shifts (i.e., transform lines containing "1 0 0 1" followed by the shifts in X and Y). This option can be used together with either -bin or -shrink, but not both, because the data are scaled down when read in. The option need not be entered when doing image reduc- tion in Fourier space with the -ftreduce option, as any shifts will then be applied as addition phase shifts in the cropping operation. -rotate (-ro) OR -RotateByAngle Floating point Rotate all images by the given angle in degrees, positive for a counterclockwise rotation. Rotation is applied after binning and transformation, if any. It can be combined with linear transforms but not with warping transforms. If the rotation is by +90 or -90 degrees and no output size is specified, then the X and Y sizes of the image will be transposed so the the output file will show the whole rotated image. For any other rotation angles, you will get the same output size as the input image unless you enter a new size explicitly. -expand (-exp) OR -ExpandByFactor Floating point Scale all images up in size by the given factor, greater than 1 to expand, or less than 1 to shrink with ordinary interpolation. Scaling is applied after binning and transformation, if any. It can be combined with linear transforms but not with warping transforms. If no output size is specified, the size of the input image will be scaled appropriately so that the output will contain the entire image. -shrink (-sh) OR -ShrinkByFactor Floating point Scale all images down in size by the given factor, a value greater than 1, using antialias filtering. This option should be used for large image reductions, because using ordinary interpolation will give aliasing artifacts that appear primarily as noise. Even binning will give some aliasing, especially for images rich in information above the highest frequency that will be retained in the reduced image. Shrinking can be applied either after binning or in place of binning. With no binning, it can be followed by rotation, general transformation, or dis- tortion correction, although only an integer shrinkage factor can be used with warping transformations. With binning, it can- not be used with these other operations. If no output size is specified, the size of the input image will be scaled appropri- ately so that the output will contain the entire image. -antialias (-ant) OR -AntialiasFilter Integer Type of antialiasing filter to use when reducing images with the -shrink option. In addition, if this option is entered along with -bin and without -shrink, it will cause image reduction to be done with antialias filtering instead of by simple binning. The available types of filters are: 1: Box - equivalent to binning 2: Blackman - fast but not as good at antialiasing as slower filters 3: Triangle - fast but smooths more than Blackman 4: Mitchell - good at antialiasing, smooths a bit 5: Lanczos 2 lobes - good at antialiasing, less smoothing than Mitchell 6: Lanczos 3 lobes - slower, even less smoothing but more risk of ringing The default is 6 for Lanczos 3 as of IMOD 4.7. Although many people consider Lanczos 2 the best compromise among the various factors, that sentiment may be based on images of natural scenes where there are sharp edges. Enter a negative number to select the default, whatever it is set to. -bin (-bi) OR -BinByFactor Integer Use ordinary binning to reduce images in size by the given fac- tor, or use antialias filtering to reduce images by this factor if the -antialias option is entered and -shrink is not. The value of a binned pixel is the average of pixel values in each block of pixels being binned. Binning is applied before all other image transformations. If no output size is specified, the size of the input image will be scaled appropriately so that the output will contain the entire image. If the -oddeven option is not entered, the binned size in a dimension will be made even or odd depending on whether the input file's size is even or odd, respectively, which can result in an output size one pixel bigger even when the input size divided by the binning is an integer. -oddeven (-od) OR -AllowOddEvenChange Allow the output size from binning in a dimension to be odd when the input size is even, or even when the input size is odd, but only when this will add just 1 pixel to the size that results from integer division instead of 2. -ftreduce (-ftr) OR -FourierReduceByFactor Floating point Scale all images down in size by the given factor by cropping the Fourier transform. This reduction method provides complete removal of frequencies above the highest frequency being retained (i.e., antialiasing) with no attenuation of the retained frequencies. However, this option is slower than image reduction in real space and cannot be used with any other image transformation, reduction, or warping except simple shifting. Shifts to keep the image centered will be applied in Fourier space during the cropping operation, so this option implies the -phase option and any other shifts that you choose to enter will be applied along with this. Shifts can be entered either with the -offset option or as linear transformations containing only shifts. Currently, the reduction factor must be either an inte- ger, or an integer divided by 2, 3, 4, 5, 6, 8, or 10. Thus, 2.7, 1.375, 3.75, 1.333, and 2.167 are all acceptable factors. Factors that are integers divided by 3 or 6 must be entered with three decimal places, as in the last two examples. The actual factor applied in those cases will be a ratio of integers, not the entered number, and be within 0.001 of the entered factor. If the factor is not acceptable, the program exits with an error. -ftexpand (-fte) OR -FourierExpandByFactor Floating point Scale all images up in size by the given factor by padding the Fourier transform. This expansion method provides complete preservation of the existing spatial frequencies without intro- ducing any higher frequencies in the result, as linear or cubic interpolation would. However, this option is slower than real- space interpolation and has all the features and limitations just described for the -ftreduce option. -noise (-no) OR -NoisePadForFFT Use tapered noise based on nearby pixels along the edge of an image to pad an image for reduction and/or shifting in Fourier space. The default is to pad with a simple taper that makes streaks outside the edge of the image, which may appear as structure if shifted into the image area. For low-dose images, these streaks may look odd or otherwise cause trouble, which can be avoided by using this option. This is the padding method used in Alignframes. -distort (-d) OR -DistortionField File name Image distortion field file to use for undistorting images. The undistortion is applied before any transformations. -imagebinned (-im) OR -ImagesAreBinned Floating point The current binning of the images, so that the distortion field can be applied correctly. This entry is required unless the program can determine the binning unambiguously from the image size. -fields (-fie) OR -UseFields List of integer ranges A list of the distortion fields to apply for each section (num- bered from 0 unless -fromone is entered). Ranges are allowed. The default is for the field numbers to match the input section numbers, unless there is only one field in the file. To have a single field applied to all of the sections, just enter a single number. This option was added when the only way to warp images was with distortion field files but it is not needed with cur- rent warping files. (Successive entries accumulate) -subarea (-su) OR -SubareaOffsetsXandY Multiple floats X and Y offsets between the center of a distortion field and the center of each section. A subarea from the upper right of the full image on which the field was based will have a positive offset in X and Y. If images are being binned, the entry should be in unbinned pixels. Enter one X,Y pair to use a single off- set for all sections. (Successive entries accumulate) -gradient (-g) OR -GradientFile File name File with magnification gradients to be applied for each image. This should be a file listing the tilt angle, the percent magni- fication change per micron of Z height, and the degrees of rota- tion per micron of Z height for each image, such as is produced by Extractmaggrad. The mag gradient correction is applied before a distortion field correction and before any transforma- tions. -origin (-or) OR -AdjustOrigin Adjust the origin values in the image file header for changes in image size or the centering of a subarea. With this adjustment, a model built on the input stack should be correctly located when loaded onto the output stack in 3dmod. Model points will be correctly located in Z provided that a contiguous set of sections is output. They will be correct in X and Y provided that the only operations on the data are a change in size of the output, binning with -bin, scaling with -expand, or -shrink, and shifting with -offset. -linear (-l) OR -LinearInterpolation Use linear instead of cubic interpolation to transform images. Linear interpolation is more suitable when images are very noisy, but cubic interpolation will preserve fine detail better when noise is not an issue. Images are transformed when the -xform, -expand, -rotate, -distort, or -gradient option is entered. -nearest (-nea) OR -NearestNeighbor Use nearest neighbor interpolation instead of cubic interpola- tion to transform images. This method simply picks the nearest existing pixel value instead of interpolating between surround- ing values, so it can be used when pixels have discrete or mean- ingful values that need to be preserved. This option and -lin- ear are mutually exclusive. Images are transformed when the -xform, -expand, -rotate, -distort, or -gradient option is entered. OUTPUT CONTROL OPTIONS These options control the size, form, or density scaling of the out- put. -size (-si) OR -SizeToOutputInXandY Two integers The dimensions of the output image in X and Y. The default size is the same as that of the first input file, except in two situ- ations where the input sizes are transposed for the output: when -rotate is entered with an angle within 40 degrees of plus or minus 90, or when non-warping transforms are entered that all contain a rotation closer to plus or minus 90 degrees than to 0. -mode (-mo) OR -ModeToOutput Integer The storage mode of the output file; 0 for byte, 1 for 16-bit signed integer, 6 for 16-bit unsigned integer, 2 for 32-bit floating point, 12 for 16-bit floating point, or 101 for 4-bit data. The default is the mode of the first input file, with two exceptions. 1) For a 4-bit input file, the default is to output as bytes. If you create a file in mode 101, the standard header listing will show the "Map mode" as 0, but there will be a line above that indicating that the mode is truly 101. 2) The default mode of floating point output for MRC files is governed by the value of environment variable IMOD_WRITE_FLOATS_16BIT. Without this set non-zero, mode 2 is output by default for any floating point input. Mode 12 is allowed only if the output for- mat is MRC. The range for mode 12 is -65504 to 65504; values are stored with 11-bit precision down to 6.1e-5 and 10-bit pre- cision down to 6.0e-8. -bytes (-by) OR -BytesSignedInOutput Integer This entry controls how bytes are stored in the output file and overrides both the default for this version of IMOD and the set- ting of the environment variable WRITE_MODE0_SIGNED. Enter 0 for unsigned numbers or 1 for signed numbers. Unsigned output is needed to work with versions of IMOD before IMOD 4.2.23; signed output may be needed for compatibility with external software that has followed recent documentation of the MRC for- mat. Regardless of the representation in the file, bytes are read into IMOD programs as unsigned with a range of 0 to 255. -strip (-st) OR -StripExtraHeader Do not transfer extra header information in input file(s) to output file(s). The default is to transfer this information whenever possible. -float (-fl) OR -FloatDensities Integer Adjust densities of sections individually. Enter 1 for each section to fill the data range, 2 to scale sections to common mean and standard deviation, 3 to shift sections to a common mean without scaling, or 4 to shift sections to a common mean then rescale the resulting minimum and maximum densities to the Min and Max values specified with the -scale option (which must be entered in this case). When floating to mean and SD, the program will express the minimum and maximum densities for each image as the number of SDs from the mean and analyze these min- ima and maxima for extreme outliers. Images with extreme ranges will be truncated to preserve the dynamic range for the rest of the images. If no truncation is warranted from this analysis, the output when transforming images while floating to bytes may have a variable SD to keep the data after transformation within the byte range. If this is problematic, float to an integer mode output and then restack into bytes with the default or a specified constant scaling. -meansd (-mea) OR -MeanAndStandardDeviation Two floats Scale all images to the given mean and standard deviation. This option implies -float 2 and is incompatible with all other scal- ing options. There is no check that the scaling is sensible for the data mode, so be sure to change data modes to prevent exces- sive truncation or loss of resolution. For example, change from unsigned bytes to integers if setting the mean to 0 or the SD to a large number (> 50); change from bytes or integers to floating point if setting the SD to a small number (< 10). -contrast (-con) OR -ContrastBlackWhite Two integers Rescale densities to match the contrast seen in 3dmod with the given black and white values. This works properly only when the output file will be bytes. If the data were loaded into 3dmod with intensity scaling, add the -map option with the intensity range given to 3dmod. If you already have a byte file and want to reverse the contrast, you can do so with "contrast 0,255 -map 255,0". -scale (-sc) OR -ScaleMinAndMax Two floats Without the -float option, this option will rescale the densi- ties of all sections by the same factors so that the original minimum and maximum density will be mapped to the Min and Max values that are entered. The Max can be less than or greater than the Min. With "-float 4", the minimum and maximum result- ing from the initial shifting of densities will be mapped to the entered values. If you want to reverse the contrast of the file, use Header to determine the existing minimum and maxi- mum of the file and then use "-scale Max,Min". -map (-ma) OR -MapFromRange Two floats Scale from the given range of densities rather than the current min and max when using -contrast or -scale without "-float 4". The second number can be either less than or greater than the first. Combined with -scale, this option allows an arbitrary mapping of two density values to two other values. Combined with -contrast and "-mode 0", the option has the same effect as using mrcbyte with its -s and -c options. -multadd (-mu) OR -MultiplyAndAdd Two floats Rescale the densities of all sections by multiplying by the first entered value then adding the second value. This option must be entered either once only, or once per input file. (Suc- cessive entries accumulate) -fixrange (-fix) OR -FixRangeIfNeeded Two floats This option controls the conditional scaling and shifting of data described in detail in the section below on Density Scal- ing. Such operations will occur only for integer image data that are being transformed in some way, and only if the data distribution fits some criteria. The first value entered is the number of standard deviations from the mean within which data should be protected from truncation after image transformations; it must be at least 2. The second value is a scaling factor that will be applied when the SD is below a criterion value (10 by default); the default scaling is 1. This option cannot be used with any other scaling specifications. -rfparam (-rf) OR -RangeFixingParams Two floats This option sets two parameters relevant to the -fixrange option: the criterion SD value below which data should be scaled (default 10), and the factor by which the range of the data might expand on each end upon interpolation or shifting by phase shifts in Fourier space (i.e, the default of 1.2 allows for an increase in total range of 1.4). -fill OR -FillValue Floating point Value to fill areas of the output image that have no image data. The filling is done before intensity scaling, so the value in the filled areas will be modified by any scaling that is done. As of IMOD 4.12.7, the default is to fill with the mean at the edge of the input image if it can be loaded entirely into memory at once, otherwise to use the image mean. -taper (-ta) OR -TaperAtFill Two integers To taper an output image at a border between real image and a filled area, just as is done with Mrctaper, enter this option with two values: 1) the distance over which to taper or 1 for a default distance, and 2) a 0 for tapering outside or 1 for tapering inside the border. The default distance is 0.5% of the mean of the X and Y sizes of the output image, but at least 16 pixels and no more than 127. The program will exit with an error if there is not enough memory to hold the entire output image. OTHER OPTIONS These include options for testing and memory control, and generic PIP options -memory (-mem) OR -MemoryLimit Integer Use this entry to specify the amount of memory used by the pro- gram for its main array, in megabytes. The use of memory and the default limits for memory allocation are described above in the section "Memory Usage and Size Limitations." Limiting the memory can keep it from using more memory than available but may result in it operating on the images in chunks. Entering a value larger than its default limit will allow it to use more memory and avoid writing to a scratch file. The main array is used to hold both the input image (in whole or in part) and a transformed image (in whole or in part). -test (-te) OR -TestLimits Two integers To test the code for reading in and binning images in chunks, enter limits for the total size of the working array, and for the size of the array used for binning. The sizes are the num- ber of 4-byte elements. -megasec (-meg) OR -MaxMegaSections Integer Maximum number of sections that will be processed, divided by 1,000,000. The default is 1, which means that the program will set up arrays for a million section numbers. If you get the error message "ERROR: PARSELIST - TOO MANY LIST VALUES FOR ARRAY" and are trying to process more than a million sections, enter this option with a higher value. -quiet (-q) OR -QuietOutput Suppress image file opening and min/max/mean output -print (-pr) OR -PrintXYSizeAndExit Determine output file size in X and Y, print it, and exit -verbose (-ve) OR -VerboseOutput Integer 1 for diagnostic output -param (-pa) OR -ParameterFile Parameter file Read parameter entries as keyword-value pairs from a parameter file. -help (-h) OR -usage Print help output -StandardInput Read parameter entries from standard input. INTERACTIVE INPUT If no command line arguments are entered, inputs are prompted interac- tively. The input is as follows; lines starting with IF are entered only if a particular option is selected. Number of input files, or -1 to enter list of files from a file IF you enter a number >0, then for each input file, enter two lines: File name list of section numbers to take from that file. The first section is numbered 0 and ranges may be entered (e.g. 0-4,5,7-9) OTHERWISE, IF you enter -1, then enter the name of the file with list of files and sections. The format of this file should be Number of input files File name list of section numbers (numbered from 0), ranges allowed next file and list of sections, etc. Number of output files, or -1 to enter list of files from a file IF you enter 1, next enter the name of the single output file. OR IF you enter a number >1, then for each output file, enter two lines: File name Number of sections to put in that file OR IF you enter -1, then enter the name of the file with the list of files and numbers of sections. The format of this file is: Number of output files File name number of sections to place in that file next file and number of sections, etc. The X and Y dimensions of the output file, or / to take the same dimensions as the first input file The data mode of the output file, or / to take the same mode as the first input file. Mode can be 0 for bytes, 1 for 16-bit integers, 2 for 32-bit real numbers, or 9 to 15 for 9 to 15 bit values. 0 for no offsets, or 1 to apply X and Y offsets separately to each section, or -1 to apply the same X and Y offset to all sections IF offsets are selected, next specify the X and Y center offsets of each of the sections (they need not be on separate lines), or the single offset to apply to all sections. Hint: offsets of 50,50 will take the center of the output image from the upper right quadrant of the input image. 0 for no transforms, or 1 to transform images using cubic interpolation, or 2 to transform using bilinear interpolation IF transforming is selected, next specify the file for transforms, and on a separate line, a list of lines to use in the file (numbered from 0). Ranges are allowed here, but all must be on one line. To have a single transform applied to all of the sections, just enter a single number. 0 for no floating of densities, 1 to float densities so that each section occupies the same RANGE of densities (the optimum range for the particular data mode), 2 to float so that all sections have the same MEAN and STANDARD DEVIATION of density (which requires the input sections to be read twice), -1 to specify a single scaling that will be applied equally to all sections, -2 to scale all sections by the same amount, determined by black and white contrast settings, in order to convert to bytes with stretched contrast, 3 to SHIFT sections to the same mean value without rescaling, or 4 to SHIFT sections to the same mean then rescale all sections by the same specified scaling. IF single rescaling is selected with "-1", next specify the density values to which the minimum and maximum in the data file should be mapped, or just enter "/" to have them mapped to the full-scale range for the data mode, or enter "1,1" to override any scaling of data that would otherwise occur because of a change of mode. This scaling method can be used to invert densities by entering, e.g., 255,0. IF scaling by black and white contrast settings is selected with -2, next enter the black and white settings (see mrcbyte). OR, IF shifting and rescaling was selected with "4", next specify the density values to which the new, shifted minimum and maximum values should be mapped, or just enter "/" to have them mapped to the full- scale range for the data mode. Additional Notes Pixel size. The program will adjust the pixel spacing in the header of the output file if binning and/or the expansion or shrinkage options are used. Models built on the input file in 3dmod should display cor- rectly on a file scaled by these three options. However, the image origin is not adjusted for images that are a subset of the input image unless the -origin option is entered as well. Without this option, it would be necessary to shift a model manually in 3dmod to display it on a subset. Density Scaling When there is a change of modes, data are rescaled optimally in most cases. If floating or other scaling is not specified, the scaling is by an amount that would map the range for the input data mode into the range for the output data mode (e.g., going from mode 0 to mode 1, it would scale data up by a factor of 128). Generally, all sections will be rescaled by the same amount, thus preserving relative differences between sections. However, if either the input or the out- put mode is 2 (real), this kind of rescaling will not occur. Data will also be scaled down by default if being stored in 4-bit mode (101), but not scaled up when going from 4 bits to bytes. In general, if your input mode is real and you are outputting to bytes or integers, you need to specify some kind of floating, shifting, or scaling in order to get usable values. These same kinds of automatic scalings upon change of mode will occur with shifting to a common mean (option 3), but here the program is more careful to apply the same scaling to all sections. To disable the scaling upon change of mode, use the option "-mult 1,0," or the "-1" floating option with interactive input. At the end, the program will report how many pixels have been truncated at the low or high limits of the output range. If this happens after shifting with a specified output range, try again with a slightly smaller range. When the image is being transformed, automatic scaling or shifting of integer values can be selected with the -fixrange option to avoid trun- cation of values or loss of intensity resolution. Truncation can occur when densities are at the bottom of the range for the data mode, because transformations (especially with phase shifting in Fourier space) can produce values outside the original range. Loss of resolu- tion can occur if input values with few gray levels (such as from elec- tron counting cameras) are interpolated but then rounded to the same few gray levels. When given the -fixrange option, the program will decide whether the data need to have their range shifted, their values scaled, or both to avoid these effects. The decision is controlled by the two values entered with that option. The first is the number of standard deviations from the mean within which data should be protected from truncation. (If the actual minimum or maximum of the data are within this range, they will be used for the decision instead of the mean minus or plus the given number of SDs.) When the program decides that a change is needed and can be done without saturating the high end of the data, it will either shift signed data (mode 1) that starts at -32768 up by 32768, or change the mode of unsigned data (mode 6) to signed (mode 1). A value around 5 should be adequate. The second value is a scaling factor, which can be 1 to have no scaling. Data will be scaled by this factor if its SD is below a criterion value (10 by default, copntrolled by the -rfparam option), and if that does not produce truncation on the high end. Scaling in the range of 10-20 should be adequate to preserve resolution in unscaled electron counting data. Warping Transformations. The transformation file entered with -xform can contain warping transformations as well as simple linear trans- forms. The transforms can consist of either displacements at a set of control points, as output directly by Midas, or displacements on a regular grid, as produced by Xftoxg. Such warping files contain information about the size of the images that were aligned and their pixel size. The transforms will be adjusted for a difference between that pixel size and the pixel size of the image file being transformed. They will also be extrapolated as necessary to cover an image area larger than that specified in the warping file; however, the program assumes that the input images are centered on the images with which the warping file was prepared. The warping will be incorrect if this is not the case. Warping cannot be used with distortion corrections. If this is neces- sary, consider using Blendmont, which can combine such operations. The -expand and -rotate options also cannot be used with warping trans- formations because such transformation would occur after the warping while the linear transform included in a warping file occurs before the warping. If these operations are necessary, you can avoid transforming the images twice by preparing a one-line text file with factor 0. 0. factor 0. 0. for expansion by the factor, or with cos(angle) -sin(angle) sin(angle) cos(angle) 0. 0. for rotation by the angle. Then use Xfproduct to multiply the warp- ing transforms by the single linear transform, and transform the images with the product. Complex and Color Data. Complex data (Fourier transforms) can be stacked but not otherwise manipulated. Don't try to change the size or center of the FFT's. Newstack can read in color data (MRC data mode 16, TIFF RGB or colormap files) as floats, but it cannot preserve the color channels and output color data again. If the input data are mode 16 and you want to com- bine the channels, you must specify a different output mode. If you want to preserve the color, there is a script, Colornewst, that will split the color channels into three byte-mode files with "clip splitrgb", use Newstack to perform identical operations on each of the three files, and recombine the data into a color file with "clip join- rgb". Memory Usage and Size Limitations. By default, the amount of memory that the program will try to use depends on the operations being done and on the amount of memory in the system. When data are being trans- formed in real space, it will seek to hold the entire input image and the transformed image in memory, at 4 bytes per pixel. For other oper- ations, including shifting and reduction in Fourier space, the same memory may be used for input and output, although there are some cases where it would need separate input and output memory. The memory to be allocated is limited to the minimum of 3/4 of system memory, system memory minus 1 GB, and 15 GB when there is up to 30 GB of system mem- ory, or to half of system memory when there is more than 30 GB. For large images, it can work with less memory than the ideal amount by reading, operating on, and writing strips of the image if necessary. The ability to work on the image in strips is limited when images are rotated by large angles (30-90 degrees); in such cases the program will need at least enough memory to hold the whole input images. If there is image scaling that requires seeing the entire image to be output before knowing how to scale it (i.e., with the -float option), the pro- gram may even write these strips to a scratch file then reread them for final scaling into the output file. On a 32-bit system, if the memory allocation fails, it will try to work with just under 2 GB of memory. Memory allocation will probably not fail on a 64-bit system. In either case, system performance will suffer if the program allocates more than the amount of memory actually available, and it may be necessary for you to limit the memory as appropriate with the -memory option. Con- versely, when you have a lot of memory for handling large images, you may be able to use that option to allow the program to use more memory and avoid writing to a scratch file. The sign that a scratch file is being used is a line of output starting with "SCRATCH image file on unit 3 :". The program can manipulate and transform images bigger than 4 gigapix- els, but distortion correction is currently limited to 2 gigapixel images. Format of Linear Transformations. Each linear transformation in a transform file is specified by a line with six numbers: A11 A12 A21 A22 DX DY where the coordinate (X, Y) is transformed to (X', Y') by: X' = A11 * (X - Xci) + A12 * (Y - Yci) + DX + Xco Y' = A21 * (X - Xci) + A22 * (Y - Yci) + DY + Yco where (Xci, Yci) and (Xco, Yco) are the center coordinates of the input and output images, respectively. HISTORY DNM (mast) for VAX 8/88 5/4/89 changed to float to same mean and sd rather than mean and min. 12/19/89 added full control of density scaling; fixed bug in interpolation that was generating half-pixel error half the time. 4/19/90 added line-by-line prompts for file and section entry and added time/date stamp 1/23/91: fixed so that it can correctly pad images and extract subsets of images with either center-shifting or transforms 6/11/91: fixed bug if trying to output scaled real values les than 0 Feb 94 : Ported to SGI by Paul Furcinitti. Jul 94 : PSF (Furcinitti) Fixed bug when writing byte files. 11/16/94: DNM added shifting to mean, rationalized scaling somewhat 9/24/01: changed to read and write in chunks if images are big 12/24/03: converted to PIP input, added undistorting and binning June 2011: Added support for warping SEE ALSO mrcbyte, tif2mrc, clip BUGS Email bug reports to mast at colorado dot edu. IMOD 5.0.2 newstack(1)