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= Software Programs and Narratives = | = Software Programs and Narratives = | ||
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file is name of source image file (.i2i is optional) | file is name of source image file (.i2i is optional) | ||
| − | == EPR == | + | == 3D Deconvolution (EPR) == |
Exhaustive Photon Reassignment (EPR) is a method for Image Restoration, developed by the Biomedical Imaging Group at the University of Massachesetts Medical School. | Exhaustive Photon Reassignment (EPR) is a method for Image Restoration, developed by the Biomedical Imaging Group at the University of Massachesetts Medical School. | ||
Revision as of 19:08, 26 September 2016
Software Programs and Narratives
Click here for an itemized list of some software programs with help.
Narratives about some of the BIG software with descriptions, images, and references can be gotten to by clicking here..
More Software Components
Image-File-Type-Conversion
These programs convert mulitple image file formats into our I2I format.
alltiftoi2i
Repeatedly calls cstoi2i to convert 2D and 3D TIFF files to I2I format.
Syntax: alltiftoi2i [options] files...
Options: -h Prints this message.
-z n Number of planes (default is 1)
-u Data is 16-bit unsigned (see cstoi2i, note 2)
-2 Divide 16-bit data by two (same as -u)
-4 Divide 16-bit data by four
-8 Divide 16-bit data by eight
-v Verbose mode.
-D Debug mode.
allzeisstoi2i
Repeatedly calls zeisstoi2i to convert 2D and 3D TIFF files to I2I format.
Syntax: allzeisstoi2i [options] files...
Options: -h Prints this message.
-z n Number of planes (default is 1)
-u Data is 16-bit unsigned (see zeisstoi2i, note 2)
-2 Divide 16-bit data by two (same as -u)
-4 Divide 16-bit data by four
-8 Divide 16-bit data by eight
-v Verbose mode.
-D Debug mode.
Biorad3D
Syntax: % Biorad3d <rootname> nx ny nz channels timepoints
outputfile is 4-D image file named <root><channel>.i2i
Biorad3Db
Syntax: % Biorad3d <rootname> nx ny nz channels timepoints
outputfile is 4-D image file named <root><channel>.i2i
cstoi2i
Converts CELLscan or Metamorph(note 1) TIFF format images file to I2I format. Can be used for other TIFF formats with -z and -u options.
Syntax: % cstoi2i [options] [TIFF_file] [ < TIFF_file] [ > I2I_file]
TIFF_file TIFF format filename with extension I2I_file UMASS I2I format filename (must include .i2i) [ ... ] implies optional parameter. Input is from either filename or stdin(<). Output is only to stdout(>).
Options:
-h Prints this message. -z n Number of planes (default is 1) -u Data is 16-bit unsigned (note 2) -2 Divide 16-bit data by two (same as -u) -4 Divide 16-bit data by four -8 Divide 16-bit data by eight -v Verbose mode. -D Debug mode. -T Print ASCII text IFDs.
Notes:
1) Hacked (12/3/97) to determine the number of z-planes 2) Data is converted to 16-bit signed by dividing by two 3) Hacked to include comments 7/23/98 4) Hacked to better parse Metamorph comments 3/11/2002
Disclaimer:
This is not a completely robust TIFF implimentation and can fail if the IFDs are not ordered as expected. If the user fails to enter a comment, you will get a blank history line.
imformat
Formats a binary input image as .i2i file input pixels are 16-bit signed integers
Usage: imformat n x y z e <image >image
n is decimal number of bytes in header
x is decimal size of the X dimension
y is decimal size of the Y dimension
z is decimal size of the Z dimension
e is the data endian type {L or B}
Fixed on 5/9/2013 to handle truncated input files correctly
by filling out the missing pixels with zeros
imformatb
Formats a binary input image as .i2i file input pixels are 8-bit unsigned integers
Usage: imformat n x y z <image >image
n is decimal number of bytes in header x is decimal size of the X dimension y is decimal size of the Y dimension z is decimal size of the Z dimension
imformatR
Formats a binary input image as .i2i file input pixels are 16-bit signed integers
Usage: imformat n x y z e <image >image
n is decimal number of bytes in header
x is decimal size of the X dimension
y is decimal size of the Y dimension
z is decimal size of the Z dimension
e is the data endian type {L or B}
imformatrgb
Formats a binary input image as .i2i file input pixels are 8-bit unsigned integers
Usage: imformat n x y z <image >image
n is decimal number of bytes in header x is decimal size of the X dimension y is decimal size of the Y dimension z is decimal size of the Z dimension
putalltiftoi2i
Repeatedly calls cstoi2i to convert 2D and 3D TIFF files to I2I format.
Syntax: putalltiftoi2i dest [options] files...
Options: -h Prints this message.
-z n Number of planes (default is 1)
-u Data is 16-bit unsigned (see cstoi2i, note 2)
-2 Divide 16-bit data by two (same as -u)
-4 Divide 16-bit data by four
-8 Divide 16-bit data by eight
-v Verbose mode.
-D Debug mode.
rgbtifftoi2i
Syntax: rgbtifftoi2i newfilename[.i2i] red|green|blue file.TIF [file.TIF ...]
where: newfilename[.i2i] is the file name for the created i2i stack image red|green|blue is the color channel to use from the TIF files file[.TIF] is|are the sequence, in desired stack order, of the TIFF files
tifftoi2i
Syntax: tifftoi2i file1[.tif] [file2[.tif] ...]
Create I2I format image(s) from 16-bit 2-D TIFF. Image name(s) are the retained with .i2i extension.
xmgrtoi2i
Syntax: %xmgrtoi2i infile outfile infile xmgrace-style (& separated) data-set file name. outfile new image file name. Options: -size nx ny Size of image to create (required)
The xmgrace file is ACSII text. Data sets have one intensity value per line(record). Data sets are separated by "&". The image file has one set per row (x) starting at x=0. Each "&" starts a new row (y)
zeisstoi2i
Converts CELLscan or Metamorph(note 1) TIFF format images file to I2I format. Can be used for other TIFF formats with -z and -u options.
Syntax: % cstoi2i [options] [TIFF_file] [ < TIFF_file] [ > I2I_file]
TIFF_file TIFF format filename with extension I2I_file UMASS I2I format filename (must include .i2i) [ ... ] implies optional parameter. Input is from either filename or stdin(<). Output is only to stdout(>).
Options:
-h Prints this message. -z n Number of planes (default is 1) -u Data is 16-bit unsigned (note 2) -2 Divide 16-bit data by two (same as -u) -4 Divide 16-bit data by four -8 Divide 16-bit data by eight -X Divide 16-bit data by 256 (byte shift) -v Verbose mode. -D Debug mode. -T Print ASCII text IFDs.
Notes:
1) Hacked (12/3/97) to determine the number of z-planes 2) Data is converted to 16-bit signed by dividing by two 3) Hacked to include comments 7/23/98
Disclaimer:
This is not a completely robust TIFF implimentation and can fail if the IFDs are not ordered as expected. If the user fails to enter a comment, you will get a blank history line.
Image-Corrections
These programs perform various spatial and intensity-based manipulations.
autoscalexy
Autoscales each Z plane individually to 1 - 255
Usage: autoscalexy [options] image1 outimage options:
note: To view image use play. autoscalexy test - | play -
badpixel
Usage: % badpixel [options] inputfile outputfile
inputfile image file name
outputfile corrected image file name
Options:
Replace pixel (x,y) with
-a x y average of the 8 neighbors
-m x y median of the 8 neighbors
-p x y root of the sum of the squares of the 8 neighbors
-r x y square of the sum of the roots of the 8 neighbors
Replace each pixel of column (x,y1->y2) with
-A x y1 y2 average of the 6 neighbors
-M x y1 y2 median of the 6 neighbors
-P x y1 y2 root of the sum of the squares of the 6 neighbors
-R x y1 y2 square of the sum of the roots of the 6 neighbors
bgcor3d
bgsub3d Subtract Correct 3D (Z or time) bg from data
Input file 3D data image file
Output file The actual 3D data (above bg)
Options:
-Z n Z plane for BG
-E BG subtract each Z plane separately
-C x y Center of ROI
-R n Radius of ROI
-S n Number of sigmas(std.dev.) above mean bg (default=0)
-N sf Normalize by mean bg: image=sf*(image-bg)/bg (exclusive of -S)
-V value Subtract value from image (exclusive of other options)
-M n {file|-} Subtract the mode (histogram peak) of each Z plane (exclusive of other options).
n is stdev for histogram Gaussian smoothing (default=0, no smoothing)
file is filename for mode value(s), or - for stderr
bleachima
Usage: % bleachima [options] input_file output_file
Exponential bleaching correction for 2-D times series
Options:
-sets n Specifies the number of 3-D image sets
-A A0 A1 Single exponential: A0*exp(-z/A1)
-B B0 B1 Optional 2nd exponential: A0*exp(-z/A1)+B0*exp(-z/B1)
-C A0 A1 B1 (where B0 = 1 - A0)
-Z z1 z2 Fit z-planes z1-z2 with A0*exp(-z/A1)
Can be used repeatedly to specifiy disjoint sets of planes
(overrides -A and -B)
-R x1 x2 y1 y2
Restrict -Z to region (x1,y1) to (x2,y2)
-D n Image background value(baseline, def=0)
-V Verbose operations(def=not verbose)
flip
Flips image about X and/or Y, or Z axes
Usage: % flip [options] input_image output_image
where input_image Image file to flip output_image Flipped image file options: -sets n Specifies the number of 3-D image sets. -X Flip along X axis -Y Flip along Y axis -Z Flip along Z axis -T Transpose X and Y axes
Use flip -Y to display images right-side-up. Flipping modes are mutually exclusive.
imsets
Sets the number of images sets in an image file.
Usage: % imsets imagefile.i2i #
scratchng
Usage: % tranima [options] inputfile outputfile
Input file ScratchNG image file name
Output file black-level and gain corrected image file name.
Options:
-gains B4 B3 B2 B1 A4 A3 A2 A1
where the ports are labeled:
+-------------------+
2 | A4 | A3 | A2 | A1 |
|----+----+----+----|
1 | B4 | B3 | B2 | B1 |
+-------------------+
1 2 3 4
-nogains Sets all port gains to 1
Notes:
in-place corrections (12/05/2005)
default gains as of 8/10/2010:
1.000 0.947 0.942 0.966 1.018 1.055 1.035 1.024
replaces bad columns x=[160,481] with average of adjacent columns (3/24/2011)
updown3d
Syntax: updown3d file[.i2i] zsize [plateau]
file is name of source image file (.i2i is optional)
3D Deconvolution (EPR)
Exhaustive Photon Reassignment (EPR) is a method for Image Restoration, developed by the Biomedical Imaging Group at the University of Massachesetts Medical School. All versions of the program are based on the algorithm originally developed by Walter Carrington(1-3) with Kevin Fogarty (1-3) and Fay Fay (2,3). Basically, it is an iterative, least-squares reconstruction method with tikhonov regularization and a non-negativity constraint (no negative fluorescence). The micropscope (the forward problem) is modeled as a shift-invariant linear system (convolution).
The inputs are a series of optical section images of fluorescence acquired with a suitably equipped microscope (i.e. the data), a 3-D point spread function image, either theoretical or empirical (the PSF), and a "smoothness" parameter ("alpha"). The algorithm finds the object (true 3-D fluroescence distribution) that minimizes a weighted sum of the least-squares fit to the data and the "smoothness" (total energy) of the object. As the algorithm as constructed is minimizing a strictly convex function, it is garranteed to converge to a single global minimum (a unitque object).
The data optical sections do not have to be sampled on a regular grid, in x or y (laterally) or in z (axially). The object can be reconstructed on a finer grid than the data (sparsedata, binsample, superresolution-epr). When ER is combined with Structured Light epi-fluorescence microscopy (SLeEPR) it can achieve resolutions of 50nm in x,y and 150 nm in z.
(1) Carrington WA, Fogarty KE. 3D molecular distribution in living cells by deconvolution of optical sectioning using light microscopy.
In: Foster KR, editor. Proceedings of the Thirteenth Annual Northeast Bioengineering Conference. Vol. 1. New York: IEEE; 1987. pp. 108–110
(2) Three-dimensional molecular distribution in single cells analysed using the digital imaging microscope.
Fay FS, Carrington W, Fogarty KE. J Microsc. 1989 Feb;153(Pt 2):133-49
(3) Superresolution three-dimensional images of fluorescence in cells with minimal light exposure.
Carrington WA, Lynch RM, Moore ED, Isenberg G, Fogarty KE, Fay FS. Science. 1995 Jun 9;268(5216):1483-7
binsample
BINSAMPLE bins pixels n x,y and z while maintaining the original sampling Input file Image file name. Output file Image file name. Options: -bin x y z Specifies the spatial binning factor (default is 1)
Notes: The output image size is n-1 smaller where n is the binning factor for each axis Pixels are averaged when binned
epr_i2i
epr UMMC/BIG Jul 12 2012
NAME epr - Exhaustive Photon Replacement (EPR) restores contrast by removing residual out-of-focus light and improves resolution while maintaining numerical accuracy of 3-D images of specimens obtained with serial optical sectioning from wide-field or confocal light microscopy.
SYNOPSIS epr [options] before-image[.tif] after-image[.tif]
DESCRIPTION epr performs regularized, iterative image restoration with a non- negativity constraint. The before-image is a three dimensional (3-D) TIFF image composed of rectangular, regularly spaced, optical sections. Large images are decomposed into smaller, overlapping (in x and y only) image segments for restoration, and the restored segments are recomposed. The after-image is the restored 3-D image. All options must appear before the image file names, but the order of options is not important.
epr has the following options:
-h Prints basic help on syntax and available options.
Required Parameters
-psf file[.tif] The 3-D point spread function image file. The psf must
match the optical configuration used for the before-
image. The psf depth (number of z-planes) need only be
less-than or equal-to twice that of the before-image.
Extra z-planes are symmetrically discarded.
-smoothness alpha The alpha value where alpha is typically 0 < alpha < RNL.
-sm alpha RNL is the residuals noise limit as reported by prepdata
(see also). Smaller values of a correspond to less
smoothing. (RNL)^2 is usually a good starting choice for
alpha.
-iterations n Maximum number of iterations to perform. Iterating may
-it n terminate earlier if convergence is detected. (see
-convergence)
Control Parameters
-scaling n where n is after image scaling factor. Used to prevent
-sc n integer overflow when saving after image to file.
Default=1.0
-convergence n Criteria for terminating iteration, where n is << 1.
-co n 0.00001 represents true convergence. 0.001 usually
achieves 90-95% convergence in about half the number
of iterations. Values of n larger than 0.001 are not
generally recommended. Default=0.001
-axial n1 n2 The z-axis object extrapolation beyond the sectioned
-ax n1 n2 image data where n1 is before first plane and n2 is
after last plane. n is in planes. By default,
extrapolation extends +-1/2 the z-axis extent of the point
spread function image and should be sufficient. Smaller
values of n1 or n2 have the effect of spatially
constraining the restored object and should be applied
carefully.
-time n1 The timepoint where you want the restoration to occur (0 indexed) -t n1
-threads n The number of threads FFTW should use. Default=1 -th n
-transverse n The x-axis and y-axis object extrapolation in pixels.
-tr n The epr process extrapolates the restoration beyond the
bounds of each image segment in order to account for
exterior out-of-focus contributions. Default = 1/4 of X or Y
dimsenion PSF width, which ever is larger.
EXAMPLES To see the command line syntax
% epr
or
% epr -h
Given specimen image mycell_.i2i and point spread function image mypsf_.i2i
let smoothness equal 0.0005, maximum number of iteration equal 250
and convergence equal 0.001. Perform single-resoltion(default) EPR:
% epr -psf mypsf_ -smoothness 0.0005 -iterations 250 mycell_ mycell_r
FAQ (Frequently Asked Questions)
see http://...
FILES
xcomp:/home/epr/epr
xcomp:/home/epr/cs_epr.lo
SEE ALSO
prepdata
preppsf
Usage:
%epr [options] before-image after-image
For help on syntax and available optionos:
%epr -h
epr_gpu
epr UMMC/BIG Aug 8 2012
NAME epr - Exhaustive Photon Replacement (EPR) restores contrast by removing residual out-of-focus light and improves resolution while maintaining numerical accuracy of 3-D images of specimens obtained with serial optical sectioning from wide-field or confocal light microscopy.
SYNOPSIS epr [options] before-image[.tif] after-image[.tif]
DESCRIPTION epr performs regularized, iterative image restoration with a non- negativity constraint. The before-image is a three dimensional (3-D) TIFF image composed of rectangular, regularly spaced, optical sections. Large images are decomposed into smaller, overlapping (in x and y only) image segments for restoration, and the restored segments are recomposed. The after-image is the restored 3-D image. All options must appear before the image file names, but the order of options is not important.
epr has the following options:
-h Prints basic help on syntax and available options.
Required Parameters
-psf file[.tif] The 3-D point spread function image file. The psf must
match the optical configuration used for the before-
image. The psf depth (number of z-planes) need only be
less-than or equal-to twice that of the before-image.
Extra z-planes are symmetrically discarded.
-smoothness alpha The alpha value where alpha is typically 0 < alpha < RNL.
-sm alpha RNL is the residuals noise limit as reported by prepdata
(see also). Smaller values of a correspond to less
smoothing. (RNL)^2 is usually a good starting choice for
alpha.
-iterations n Maximum number of iterations to perform. Iterating may
-it n terminate earlier if convergence is detected. (see
-convergence)
Control Parameters
-scaling n where n is after image scaling factor. Used to prevent
-sc n integer overflow when saving after image to file.
Default=1.0
-convergence n Criteria for terminating iteration, where n is << 1.
-co n 0.00001 represents true convergence. 0.001 usually
achieves 90-95% convergence in about half the number
of iterations. Values of n larger than 0.001 are not
generally recommended. Default=0.001
-axial n1 n2 The z-axis object extrapolation beyond the sectioned
-ax n1 n2 image data where n1 is before first plane and n2 is
after last plane. n is in planes. By default,
extrapolation extends +-1/2 the z-axis extent of the point
spread function image and should be sufficient. Smaller
values of n1 or n2 have the effect of spatially
constraining the restored object and should be applied
carefully.
-time n1 The timepoint where you want the restoration to occur (0 indexed) -t n1
-cuda n1 The cuda device that you would like to use (1 indexed). -cu n1 The highest device number would be your display card.
FFTW will be used if a cuda device isn't specified or less than 1.
Restoration will fail if there isn't enough free memory on
your cuda device.
-threads n The number of threads FFTW should use. Default=1
Only applicable if using FFTW, and not CUDA.
-th n
-transverse n The x-axis and y-axis object extrapolation in pixels.
-tr n The epr process extrapolates the restoration beyond the
bounds of each image segment in order to account for
exterior out-of-focus contributions. Default = 1/4 of X or Y
dimsenion PSF width, which ever is larger.
EXAMPLES To see the command line syntax
% epr
or
% epr -h
Given specimen image mycell_.i2i and point spread function image mypsf_.i2i
let smoothness equal 0.0005, maximum number of iteration equal 250
and convergence equal 0.001. Perform single-resoltion(default) EPR:
% epr -psf mypsf_ -smoothness 0.0005 -iterations 250 mycell_ mycell_r
FAQ (Frequently Asked Questions)
see http://...
FILES
xcomp:/home/epr/epr
xcomp:/home/epr/cs_epr.lo
SEE ALSO
prepdata
preppsf
EPR
EPR is a shell script that checks to see if you are logged into either zirconium (the workstation in the analysis room) or barium (in Roughua's office). If yes, then it runs the CUDA version of epr using either the GTX 680 (zirconium) with 2Gb of memory, or the GTX 480 (barium) with 1.5 GB of memory, with options to show you iteration by iteration results. If you're using any other computer then it runs the Intel-cpu only version of epr. It passes the remainder of your command line on to the chosen epr program.
To see the help for the epr program itself
% EPR -h
Usual syntax:
% EPR -it 999 -sm # -co 0.003 -psf your_psf_file[.i2i] input[.i2i] output[.i2i]
Put 999 for iterations to make sure it doesn't stop too early. The smoothness value is whatever you would usually choose. I recommend using 0.003 for the convergence (rather than 0.001 or smaller) when using the NVIDIA version (zirconium or barium) as the lack of double-precision calculations can make it hard to squeeze the last bit out of the algorithm, and sometimes it will stop converging and you won't get an output image. If this happens anyway, then either try 0.005 for convergence and/or make the smoothness value a little larger, or ask Kevin for help.
If your image is really large then it might not fit into the memory of the NVIDIA systems. An image of 1300 x 1000 with fewer than 20 slices will work on zirconium (with 2GB), but to do more slices than this you will need to segment the image to something like 960 x 960 first (the reason has to do with allowable FFT sizes and the dimensions of the PSF, but this is a guideline).
You CAN to run more than one NVIDIA version of EPR at a time if there is enough available memory on the graphics board. Otherwise you'll get the error:
CUDA: out of memory error in file 'epr.cu' at line 1020
It's first-come first-serve.
linux_epr_fftw3_sse_z
epr UMMC/BIG 03/30/95
NAME epr - Exhaustive Photon Replacement (EPR) restores contrast by removing residual out-of-focus light and improves resolution while maintaining numerical accuracy of 3-D images of specimens obtained with serial optical sectioning from wide-field or confocal light microscopy.
SYNOPSIS epr [options] before-image[.tif] after-image[.tif]
DESCRIPTION epr performs regularized, iterative image restoration with a non- negativity constraint. The before-image is a three dimensional (3-D) TIFF image composed of rectangular, regularly spaced, optical sections. Large images are decomposed into smaller, overlapping (in x and y only) image segments for restoration, and the restored segments are recomposed. The after-image is the restored 3-D image. All options must appear before the image file names, but the order of options is not important.
epr has the following options:
-h Prints basic help on syntax and available options.
Required Parameters
-psf file[.tif] The 3-D point spread function image file. The psf must
match the optical configuration used for the before-
image. The psf depth (number of z-planes) need only be
less-than or equal-to twice that of the before-image.
Extra z-planes are symmetrically discarded.
-smoothness alpha The alpha value where alpha is typically 0 < alpha < RNL.
-sm alpha RNL is the residuals noise limit as reported by prepdata
(see also). Smaller values of a correspond to less
smoothing. (RNL)^2 is usually a good starting choice for
alpha.
-iterations n Maximum number of iterations to perform. Iterating may
-it n terminate earlier if convergence is detected. (see
-convergence)
Control Parameters
-scaling n where n is after image scaling factor. Used to prevent
-sc n integer overflow when saving after image to file.
Default=1.0
-convergence n Criteria for terminating iteration, where n is << 1.
-co n 0.00001 represents true convergence. 0.001 usually
achieves 90-95% convergence in about half the number
of iterations. Values of n larger than 0.001 are not
generally recommended. Default=0.001
-axial n1 n2 The z-axis object extrapolation beyond the sectioned
-ax n1 n2 image data where n1 is before first plane and n2 is
after last plane. n is in planes. By default,
extrapolation extends +-1/2 the z-axis extent of the point
spread function image and should be sufficient. Smaller
values of n1 or n2 have the effect of spatially
constraining the restored object and should be applied
carefully.
-time n1 The timepoint where you want the restoration to occur (0 indexed) -t n1
-threads n The number of threads FFTW should use. Default=1 -th n
-transverse n The x-axis and y-axis object extrapolation in pixels.
-tr n The epr process extrapolates the restoration beyond the
bounds of each image segment in order to account for
exterior out-of-focus contributions. Default=16 pixels
-OP wavelength NA n dx dz ideal
The optical configuration of the data and psf where
wavelength, dx and dz are in microns, n is one of
1.0 air
1.33 water
1.47 glycerin
1.515 oil
and ideal is < 1 for spherically aberrated systems.
-znear n The z-axis distance to first resolution reduction stage. -zn n Default=1.25 microns.
EXAMPLES To see the command line syntax
% epr
or
% epr -h
Given specimen image mycell_.i2i and point spread function image mypsf_.i2i
let smoothness equal 0.0005, maximum number of iteration equal 250
and convergence equal 0.001. Perform single-resoltion(default) EPR:
% epr -psf mypsf_ -smoothness 0.0005 -iterations 250 mycell_ mycell_r
FAQ (Frequently Asked Questions)
see http://...
FILES
xcomp:/home/epr/epr
xcomp:/home/epr/cs_epr.lo
SEE ALSO
prepdata
preppsf
linux_epr_pentium4
epr UMMC/BIG 03/30/95
NAME epr - Exhaustive Photon Replacement (EPR) restores contrast by removing residual out-of-focus light and improves resolution while maintaining numerical accuracy of 3-D images of specimens obtained with serial optical sectioning from wide-field or confocal light microscopy.
SYNOPSIS epr [options] before-image[.tif] after-image[.tif]
DESCRIPTION epr performs regularized, iterative image restoration with a non- negativity constraint. The before-image is a three dimensional (3-D) TIFF image composed of rectangular, regularly spaced, optical sections. Large images are decomposed into smaller, overlapping (in x and y only) image segments for restoration, and the restored segments are recomposed. The after-image is the restored 3-D image. All options must appear before the image file names, but the order of options is not important.
epr has the following options:
-h Prints basic help on syntax and available options.
Required Parameters
-psf file[.tif] The 3-D point spread function image file. The psf must
match the optical configuration used for the before-
image. The psf depth (number of z-planes) need only be
less-than or equal-to twice that of the before-image.
Extra z-planes are symmetrically discarded.
-smoothness alpha The alpha value where alpha is typically 0 < alpha < RNL.
-sm alpha RNL is the residuals noise limit as reported by prepdata
(see also). Smaller values of a correspond to less
smoothing. (RNL)^2 is usually a good starting choice for
alpha.
-iterations n Maximum number of iterations to perform. Iterating may
-it n terminate earlier if convergence is detected. (see
-convergence)
Control Parameters
-scaling n where n is after image scaling factor. Used to prevent
-sc n integer overflow when saving after image to file.
Default=1.0
-convergence n Criteria for terminating iteration, where n is << 1.
-co n 0.00001 represents true convergence. 0.001 usually
achieves 90-95% convergence in about half the number
of iterations. Values of n larger than 0.001 are not
generally recommended. Default=0.001
-axial n1 n2 The z-axis object extrapolation beyond the sectioned
-ax n1 n2 image data where n1 is before first plane and n2 is
after last plane. n is in planes. By default,
extrapolation extends +-1/2 the z-axis extent of the point
spread function image and should be sufficient. Smaller
values of n1 or n2 have the effect of spatially
constraining the restored object and should be applied
carefully.
-time n1 The timepoint where you want the restoration to occur (0 indexed) -t n1
-threads n The number of threads FFTW should use. Default=1 -th n
-transverse n The x-axis and y-axis object extrapolation in pixels.
-tr n The epr process extrapolates the restoration beyond the
bounds of each image segment in order to account for
exterior out-of-focus contributions. Default=16 pixels
-OP wavelength NA n dx dz ideal
The optical configuration of the data and psf where
wavelength, dx and dz are in microns, n is one of
1.0 air
1.33 water
1.47 glycerin
1.515 oil
and ideal is < 1 for spherically aberrated systems.
-znear n The z-axis distance to first resolution reduction stage. -zn n Default=1.25 microns.
EXAMPLES To see the command line syntax
% epr
or
% epr -h
Given specimen image mycell_.i2i and point spread function image mypsf_.i2i
let smoothness equal 0.0005, maximum number of iteration equal 250
and convergence equal 0.001. Perform single-resoltion(default) EPR:
% epr -psf mypsf_ -smoothness 0.0005 -iterations 250 mycell_ mycell_r
FAQ (Frequently Asked Questions)
see http://...
FILES
xcomp:/home/epr/epr
xcomp:/home/epr/cs_epr.lo
SEE ALSO
prepdata
preppsf
prepdata
prepdata UMMC/BIG 06/19/02
NAME prepdata - prepares a 2-D, 3-D or 4-D image data sets, acquired using a Digital Imaging (light) Microscope, for further processing (e.g. image restoration/3-D reconstruction)
SYNOPSIS prepdata [options] before-image after-image
DESCRIPTION prepdata performs all necessary corrections on a image data set based on the fluorescence D.I.M image formation/acquisition model, and can be used to perform basic imaging corrections to many forms of digitally acquired image data. The before-image argument is the name of a 3-D image set as acquired (DIM-1, DIM-2, CELLscan, UFM, or other). The after-image argument is the file name for the corrected image set, ready for further processing. The normal order of application of corrections (see -before) is basic[->background[->temporal]]. All calculations are performed using single-precision floating-point arithmetic. All options must precede input and output image file names.
Usage:
%prepdata [options] before-image after-image
For help on syntax and available options:
%prepdata -h
preppsf
preppsf UMMC/BIG 12/20/02
NAME preppsf - prepares a 3-D point spread function (psf) image data set, acquired using a Digital Imaging (light) Microscope, for use with image restoration/3-D reconstruction. Normally psf images are first processed using prepdata (see below) to apply basic corrections and any background corrections, but not temporal corrections.
SYNOPSIS preppsf [options] before-image after-image
DESCRIPTION preppsf performs further corrections on a psf image data set based on the fluorescence D.I.M image formation/acquisition model. The before- image argument is the name of a 3-D psf image set, acquired (DIM-1, DIM-2, CELLscan, UFM, or other) and processed using prepdata (see below) without the -norm option. The psf is extracted as a symmetric sub-region (square in XY) centered at the psf origin (-center), optionally normalized for constant total intensity (-norm), and optionally masked (-mask) to exclude any extramural data. The after-image argument is the file name for the processed psf image set. All options must precede input/output image files
Usage:
%preppsf [options] before-image after-image
For help on syntax and available optionos:
%preppsf -h
SNR
Usage: SNR [options] imagefile
imagefile data for SNR calculation
Options:
-gain n CCD gain (e/ADU)
-read n CCD readout noise (e RMS)
-bias n CCD bias value (ADUs)
-dark imagefile dark current image
-flat imagefile flatfield image
-offset dx dy offset of lower-left corner of data image
from lower-left corner of flatfield image
sparsedata
sparsedata UMMC/BIG 01/29/97
NAME
sparsedata - re-formats sparsely sampled images for EPR interpolation
and/or extrapolation
SYNOPSIS
sparsedata [-{h,sets,magnify|interpolate,extrapolate,z]
source-image[.i2i] destination-image[.i2i]
DESCRIPTION
The source image is re-formatted for EPR(see also) interpolation
and/or extrapolation by creating a "sparse" destination image sampled
on a finer(integer multiple), regularly-spaced spatial grid. Existing
data are placed in their corresponding sample positions. The
remaining grid positions are set to a unique value (-32768) that
indicates missing data to EPR. sparsedata is normally applied
following prepdata(see also) and before EPR. EPR must be provided
with a corresponding PSF (see note 1 below). For 4-D images,
re-formatting is repeated for each 3-D image set.
sparsedata has the following options:
-h Prints basic help on syntax and available
options.
-sets n Specify the number of 3-D image sets in
source-image. Overrides the sets number (if)
present in source-image and assigns the sets
number in new-image.
-magnify x y z Create intervening voxels between existing
-mag x y z voxels for each spatial axis. A value of 1
indicates no magnification for that axis, a
value of 2 doubles the sampling (equivalent to
-interpolate 1), etc.
-interpolate x y z Create intervening voxels between existing
-int x y z voxels for each spatial axis. A value of 0
indicates no interpolation for that axis, a
value of 1 create one intervening voxel
(equivalent to -magnify 2), etc.
-extrapolate x y z Create surrounding voxels outside the bounds of
-ext x y z the existing voxels for each spatial axis. The
new voxels are created symmetrically (+/-axis).
A value of 0 indicates no extrapolation for that
axis.
-zed file[.zed] Re-format the z-axis based on sampling protocol
described in the named file (see note 2 below).
Any z-axis interpolation (-interpolate or
-magnify) is applied AFTER -zed re-formatting
and cumulatively.
The -magnify and -interpolate options describe the same process and
are thus mutually exclusive. Use of both results in an error.
EXAMPLES
1) Interpolate one optical section between existing sections:
% sparsedata -int 0 0 1 in_image.i2i out_image.i2i
which is equivalent to doubling the magnification in z:
% sparsedata -mag 1 1 2 in_image.i2i out_image.i2i
2) Now also extrapolate four sections, two before and two after
the data:
% sparsedata -int 0 0 1 -ext 0 0 2 in_image.i2i out_image.i2i
3) Re-create a z-axis sampling protocol (7 optical sections at
-5,-3,-1,0,1,3,5) positioned so that -5 becomes the first
plane:
protocol.zed (see note 2 below)
+------------
| 6
| -5 1
| -3 1
| -1 1
| 0 1
| 1 1
| 3 1
| 5 1
+------------
% sparsedata -zed protocol.zed in_image.i2i out_image.i2i
The out_image has 11 optical sections (-5,-4,-3,...,5) .
4) Now also double the original z-axis spacing:
% sparsedata -zed protocol.zed -mag 1 1 2 in_image.i2i out_image.i2i
The out_image has 21 optical sections (-10,-9,-8,...,10).
FILES
SEE ALSO
prepdata
preppsf
epr
NOTES
1) The PSF must have the same sampling grid as new-image. However,
CCD detectors (generally) integrate over the entire area of a
pixel, as compared to optical sectioning, which measures at
discrete positions in z. The PSF in x and y must be sampled at the
new grid spacing, but the new pixels should be area integrations
corresponding to the original (source-image) pixel area.
2) The .zed files are simple text files describing the optical
sectioning protocol used for acquisition. They have the following
format:
The first line is the "origin" in z. This is added to all
following z positions to create an index(plane number)
greater than or equal to one.
Subsequent lines have two value separated by a (white)space:
the relative z position and the relative exposure time for
the corresponding image plane.
For 4-D images, the protocol is repeated for each 3-D image set.
3. A - in place of an image name means stdin or stdout.
superresolution-epr
Usage: superresolution-epr [-h|H] image[.i2i] psf[.i2i] smoothness XY-fold Z-fold
Description:
Superresolution EPR is a technique for restoring (deconvolving) an image using a finer pixel sampling
than the original image. This gets around the limitation on acheivable resolution imposed by the imaged pixel size.
Example: Given the command
$ superresolution-epr foo.i2i psf.i2i 1e-3 3 2
The program creates these files in your directory:
psf_3x3x2.i2i (interpolated psf.i2i) psf_3x3x2_sr.i2i (interpolated psf convolved with a 2-D square of the original pixel size) foo_3x3x2.i2i (foo.i2i resampled with NODATA (-32768) at new pixel positions) foo_3x3x2_sr1e-3.i2i (the restored image with the new pixel count and size)
The arguments XY-fold and Z-fold are the pixel sampling increase; e.g. a value of 2 means double the number of pixels in the restored image and half the pixel spatial size (double the sampling resolution). It is limited to 1, 2 or 3 in XY and Z for reasons of calculation time (proportional to fold increase) and storage space (it uses lots of random access memory).
Notes:
The program takes care of everything. You give it properly prepared image and psf files ready for regular EPR,
and it handles all the things need to make it work. I only recommend running this on large RAM machines and on images where the resulting superres (original size times the requested XY and Z fold increases) image will be under 1000x1000x100. And run it on the fastest CPU you can (that would be the new mizar and alcor).
Authors:
Karl D. Bellve <karl.bellve@umassmed.edu> Kevin E. Fogarty <kevin.fogarty@umassmed.edu> Walter A Carrington
References:
Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. WA Carrington, RM Lynch, ED Moore, G Isenberg, KE Fogarty, and FS Fay Science 9 June 1995: 268 (5216), 1483-1487
Copyright:
© 2014 Biomedical Imaging Group, Univ. of MA Medical School, Worcester MA, USA
See also:
epr_i2i sparsedata preppsf preppdata
SLeEPR
Usage: superresolution-epr [-h|H] image[.i2i] psf[.i2i] smoothness XY-fold Z-fold
Description:
Superresolution EPR is a technique for restoring (deconvolving) an image using a finer pixel sampling
than the original image. This gets around the limitation on acheivable resolution imposed by the imaged pixel size.
Example: Given the command
$ superresolution-epr foo.i2i psf.i2i 1e-3 3 2
The program creates these files in your directory:
psf_3x3x2.i2i (interpolated psf.i2i) psf_3x3x2_sr.i2i (interpolated psf convolved with a 2-D square of the original pixel size) foo_3x3x2.i2i (foo.i2i resampled with NODATA (-32768) at new pixel positions) foo_3x3x2_sr1e-3.i2i (the restored image with the new pixel count and size)
The arguments XY-fold and Z-fold are the pixel sampling increase; e.g. a value of 2 means double the number of pixels in the restored image and half the pixel spatial size (double the sampling resolution). It is limited to 1, 2 or 3 in XY and Z for reasons of calculation time (proportional to fold increase) and storage space (it uses lots of random access memory).
Notes:
The program takes care of everything. You give it properly prepared image and psf files ready for regular EPR,
and it handles all the things need to make it work. I only recommend running this on large RAM machines and on images where the resulting superres (original size times the requested XY and Z fold increases) image will be under 1000x1000x100. And run it on the fastest CPU you can (that would be the new mizar and alcor).
Authors:
Karl D. Bellve <karl.bellve@umassmed.edu> Kevin E. Fogarty <kevin.fogarty@umassmed.edu> Walter A Carrington
References:
Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. WA Carrington, RM Lynch, ED Moore, G Isenberg, KE Fogarty, and FS Fay Science 9 June 1995: 268 (5216), 1483-1487
Copyright:
© 2014 Biomedical Imaging Group, Univ. of MA Medical School, Worcester MA, USA
See also:
epr_i2i sparsedata preppsf preppdata
Convolution
The prgrams are useful for fast linear filtering, and for creating simulated data.
blur2d
Usage: blur2d [options] image psf result
Image files (do not use any file extension): image .i2i file image to blur psf .i2i file image to serve as psf result .i2i file blurred image
Option(s):
-S n scale factor for result (default=1)
n<1 prevents integer overflows
n>1 minimizes integer truncation
-N x y dimensions of convolution space
(default dimensions are image + 1/2 psf)
-M x y dimensions of output image
(defaults are dimensions of input image)
-Z pad image convolution space with zeros
(default is pad image with edge values)
-X cross-correlation instead of convolution
-xnorm xcorr with normalization: I=(I-)/
implies -X -Z -D but not -0
-D Do not normalize the psf
-0 No DC component in psf
-V verbose mode (default is not verbose)
blur2d will choose a convolution space size large enough to prevent wrap-around. This size must also 1) be even in the X axis, and 2) have largest prime factors < 19. The image is padded with the edge pixel values to avoid sudden transitions to zero, except with the -Z option.
blur3d
lurs an image by a psf. FFTs are done with 4 byte floats, so total space needed will be 4 times the padded image size (in voxels) plus the size of the two original images (image and psf) The input image can be real or short int format. The psf must be short int format. note: performs a convolution (not a correlation). note: fixed a small bug. 10/17/11
Usage: blur3d [options] image psf newimage
options:
-S #: scale (multiply) output values by # (to prevent underflow or overflow)
voxels which after scaling are > 32767 are set to 32767. voxels < -32766 are set to -32766.
-p # # #: pad the image in x, y, and z by the specified amounts
by default image is padded (with 0) by 1/4 psf width all around,
so its size for blurring becomes bigger by psf/2.
psf is always padded to the same size the image becomes.
Padding is stripped off before newimage is written, so it will be the same size as
the original image.
-N # # #: pad so that the image has the specified dimensions after padding (just prior to FFT).
-n #: pad with # instead of boundary pixel values.
-Z: pad with zero instead of the boundary pixel values.
-c # # #: the center of the psf is at (#,#,#) zero indexed.
the default is to take the brightest pixel as its center.
-C: don't center the psf at all (default is to center on the max, unless -c is specified).
-P: normalize the psf so each slice (after padding) averages to 1. -P acts like an optical PSF.
the default is to normalize so the entire psf (after padding) has an average pixel value of 1
(the default keeps total light constant).
-d: don't normalize at all.
-o: do a cross correlation instead of a blur (convolution)
-R: output image as 4 byte floating point instead of short integer.
-v: verbose. print more info to stderr.
note: entire image name (with extension) is NOT required. note: can read from stdin or write to stdout (use a dash instead of the file name) note: a compressed image will be automatically be uncompressed.
smooth2d
smooth2d 2D image smoothing
Input file The image to be filtered.
Output file The filtered image.
Options:
-3 3x3 approx to gaussian filter: [[1,2,1][2,4,2][1,2,1]]
-B n NxN box filter
-T n NxN triangle filter
-G n Gaussian kernel where n = std.dev.
-IO Nin Nout
Difference of average of inner Nin x Nin and outer Nout x Nout regions
-RMS n calculate local RMS value over n x n pixels
-S n image scale factor.
-X 1D X-axis(horizontal) smoothing.
-Y 1D Y-axis(vertical) smoothing.
-pos Positive-quad-only box filter
-v|-V Verbose
smooth3d
smooth3D 3D image smoothing
Input file The image to be filtered.
Output file The filtered image.
Options:
-3 3x3x3 gaussian approximation.
-G sd1 sd2 p sd3 Gaussian kernel where sd = std.dev. in pixels
sd1 is x,y
sd2 is z rising phase
p is z duration of peak (usually zero)
sd3 is z falling phase
-Z Z-axis only filtering (xx,y extent=0)
-S n image scale factor.
Note: The half-amplitude point of a gaussian occurs at 1.177*sd
Modeling/Simulation
These prgrams are used when modeling images of cells.
checknoise
Usage: checknoise [options] imagefile [textfile] Imagefile An image file name (extension optional) Options: -Z n -N n -M
COSINEex
Usage: % scaleima [options] input_file output_file
Options: -size x y (pixels) -period n (pixels) -phase n (degrees) default=0
Notes:
makebead
MAKEBEAD Make a 3D fluorescent bead model Output file The converted image file. Options: -dim x y z Image dimensions x,y, and z (pixels) -D n n bead inner and outer diameters (microns) -R n inner rise std.dev. (microns) -F n outer fall std.dev. (microns) -S n n n X,Y,Z pixel dimensions (microns) -X n number subpixels per pixel
makeDOG
Usage: % makeGaussian [options] output_file
Options: -DOG s1 s2 Difference-of-Gaussians sigmas (pixels) s1<s2 -scale n Gaussian unit area scale factor (default is +1.0)
makeImpulse
Usage: % makeImpulse [options] output_file
Options: -dim nx ny nz image size (pixels) -orig x0 y0 z0 impulse location(pixels) -scale n impulse scale factor (default=1)
noise
Abstract:
This program takes an image (ADUs), adds a background (ADUs),
multiplies by a scale factor (-s), and adds photon (Poisson) and read (Gaussian) noise appropriate to a given detector (-c|-e,-g)
Usage: noise [options] inp_image out_image
Options:
-c RCA DIM-1 type of camera -c TI DIM-2 type of camers -c LL128 SCRATCH type of camera -c PM512 RATCH type of camera -c LLraw SCRATCH type of camera (input in electrons) -c photons input in photons(just adds shot noise) -e e_ADU electrons per A to D -g gain gain -n sigma read noise as electrons RMS -b number value of background (added to input, not output) -s number value of scale_factor (applied to input after -b)
Image files (do not use any file extension):
inp_image .i2i input file of image data out_image .i2i output file of image data
NOTE:
'-' in place of an image name means stdin or stdout.
psf3di
Computes a 3-D microscope point spread function image.
Syntax: % psf3di [options] output_image_file
Options: -help Extended abstract explaining method -verbose Show wavefront data -size x y z Pixel sizes (microns) -dim x y z Psf extents (pixels) -lambda n Wavelength in vacuum (microns) -NA n Numerical Aperture. of objective lens -sqrt Save square-root of intensities (more dynamic range)
-nominal n0 z n1 thickness n2 -actual n0 z n1 thickness n2
-cglass z z-plane poistion of the cover glass (default is (zdim+1)/2)
Nominal refers to the nominal objective lens corrections.
Actual refers to the actual imaging parameters.
Where:
n0 n(D) of object medium
(nominally same as n2)
z Distance(microns) from cover glass
(nominally 0)
n1 n(D) of cover glass
(1.522 +/- 0.004 is standard)
thickness Thickness(microns) of cover glass
(170 +/- 10 is standard no. 1)
n2 n(D) of lens immersion medium
(air=1.0, water=1.33, oil=1.515)
Example: objective is a Zeiss 63x 1.4NA Planapo % psf3di -NA 1.4 -nominal 1.515 0 1.522 170 1.515 ...
Image-Reformatting
These programs are used when changing the pixel sampling of images.
chres
chres changes image resolution(trilinear interpolation) Input file image file Output file image file Options: -S n n n X Y and Z voxel scale factors -E Operate on each Z plane separately
concateima
Usage: % concateima [options] image image... output_image
Concatenates image files together
Options: none
hugesegz
Syntax: hugesegz inputfle[.i2i] outputfile[.i2i] zstart zend
matrixima
Usage: % matrixima [-lead string] [-tail string] -row ... -col ... output_image
Assembles multiple time-course well images into a matrix that can be viewd with play
where the canonical well image name is [{lead}]{row}{col}_{position}_{objective}_{filter}[{tail}].i2i
required: -row n str [str [str...]] typically B, C, D, etc. -col n str [str [str...]] typically 02, 03, 04, etc.
important(check defaults): -pos[ition] n position in well: 0 <= n <= 99 (default is 0) -obj[jective] n object lense: 0=5x, 1=10x, 2=20x (default is 1) -fil[ter] n filter position: 0=phase, 1=GFP, 2=RFP (default is 0)
optional: -lead string filename leading string (default is none) -tail string filename trailing string (default is none)
-bin n bins pixels nxn (default is larger of rows and cols) -h[elp] additional information and examples.
Notes: The first well (row 1, col 1) image MUST exist. Skipped wells (missing image file) will be included in matrix but left blank.
Colocalization
These prgrams calculate measures of colocalization between/among images..
coloc3way
coloc3way image1[.i2i] image2[.i2i] image3[.i2i] [coloc-image[.i2i]] Two-way and three-way binary image colocation Options: -T n1 n2 n3 image1|2|3 Thresholds(>) default: -T 0 0 0 -E operate on Each image plane separately (2-D mode)
Meaning of output column headers: 1=>2 percentage of image-1 pixels found with image-2 1=>23 percentage of image-1 pixels found with both image-2 and image-3 1=>!23 percentage of image-1 pixels found with image-3 but not with image-2 1=>2!3 percentage of image-1 pixels found with image-2 but not with image-3
Object-Segmentation
The programs perform various types of segmentations/classifications of pixels into objects, etc.
addlines
Adds polyline|pixel objects to image files
Usage: % addlines image textfile [options] image image input image file textfile polyline|pixel object description file image output image file Options: -O label n select labeled object with w=n(superseeds -P) -P n default w for all objects -C n mark centroids with w=n(superseeds -P) -T truncate floating point coordinates to integer (default is to round) -0 use zero-indexed coordinates (default is one-indexed) -silent redirect stderr to /dev/null Defaults: all objects selected w = -1 centroids not marked
Object description file format: 1)file is ASCII byte stream 2)blank lines are ignored 3)anything from a # to end-of-line is a comment 4)objects begin with the object= key and continue until the next object or the end-of-file 5)x,y,z coordinates are the position of the pixel 6)w coordinate is the value assigned to the pixel(s)
Legend:
| indicates a choice of parameters
[ ] indicates an optional parameter
{ } indicates a set of number separated by spaces
do NOT include these symbols in the data
Object description format:
object=name # name may include spaces
[closed] # ensures closure of a polyline object
# applies only to ordered boundaries
[z=n] # default z when none specified in coordinate
[everyplane] # apply as 2-D(x,y) coordinate list to every z-plane in image
[w=n] # set pixel intensities to w(superseeds command line options)
[centroid=x y] # 2-D centroid position of closed boundary
boundary=ordered|unordered|chain-code [xy|xyz|xyw|xyzw]
[{x y}|{x y z}|{x y w}|{x y z w}] # one pixel/line
[{x y}|{x y z}|{x y w}|{x y z w}] # repeated...
[break] # next pixel begins disjoint curve
# not compatable with closed option
[{x y}|{x y z}|{x y w}|{x y z w}] # repeated...
countobjs
Usage:
countobjs [options] outputfile image1.i2i [image2.i2i]
Description:
Count objects in co-localized images. If only one image given the objects will be counted in that one image. Copious amounts of information will be written to the output file. A - for outputfile will write to stdout. outpufile can be analyzed with objs2bb.pl and objs2dist.pl
Options:
-C # # : set opacity and brightness level to these values (0-255 only) -k : use original images for data analysis (so scale and black level not important and
-t option can be in same units as original, unscaled, image values)
-S # # # : image # scale and black level (default autoscale image)
-r # # # # # # : region of image to count
(-r x0 y0 z0 x1 y1 z1)
( ^lower left ^upper right)
(zero indexed coordinates)
(default whole image)
-s # # : size of smallest and largest objects to count
(default 1-100000)
-m # # : min and max iod of allowable objects (applied to image1 only).
-t thresh1 thresh2 : thresholds (applied after -S or default scaling, unless -k specified).
Like left brightness slider in DAVE.
if only one image, second number still required, but ignored.
-o <image.i2i> : output image with all objects represented by the voxel
with the maximum intensity in the object
the intensity of the voxel will be the IOD of the object.
-O <image.i2i> : output image with all pixels in each object having
the intensity of the original objects IOD.
NOTE: this image will be in floating point format.
use float2int to convert it back to "normal" format.
-Q <image.i2i> : output image with all pixels in each object having
a unique id # (1-# of objects). Must be < 32766 objects.
NOTE: this image will be in standard signed short int format.
-M # : When countobjs is given 2 images, this flag will
turn of some subset of the voxels for colcalization.
calculations.
0: turn off colcalized voxels (default on)
1: turn off image 1 voxels (default on)
2: turn off image 2 voxels (default on)
-q : quiet. don't print out all objects, but just summary statistics
-Z : simple analysis, no sliders, opacity,etc. can use with -t option.
-h : show this message.
-H : show even more help.
Notes:
A - in place of an image name means stdin or stdout. Source code in /storage/big1/lml/jac/was_invitro/Projects/countobjs
DoTheTrackingAndRatios
Syntax: DoTheAnalysis.csh [--random] rootfilename wavelength threshold(edges) [threshold(newprog1)|.]
Finds all local 2-D maxima above 1st threshold(edges) in 491|561
Computes the vesicle ratio of TFIs of 660/{491|561} in a 5x5 box centered at each maximum
Options:
--random randomize the 660 location realive to the 491|561 Set the 2nd threshold (newprog) to a period(.) for automatic denominator TFI thresholding
newprog1
foo image1[.i2i] image2[.i2i] image3[.i2i] textfile
Two-way intensity-based vesicle colocalization
image1 primary image (denominator)
image2 secondary image (numerator)
image3 image of vesicle IDs (2-D maxima)
Options:
-IO inner outer +-size (pixels) of region for local average intensiy calculation
default: 2 3 (i.e. 5x5 and 7x7)
-random n randomly shift vesicle paths by adding uniform random variable [0..n] pixels
default: 0
-T threshold primary (denom.) image local average intensity must be > threshold
(default 0)
Notes: 8/2/2010 output to textfile includes (ix,iy) of those positions passing threshold test
trackves
trackves 2D vescile tracking Inputfile_1 Original vesicle time series Inputfile_2 Filtered vesicle time series Outputfile Tracked vesicles time series. Options: -T vesicle intensity threshold (> or =) for filtered image (default 1) -id n vesicle ID modulo n (default 256) -xy n search n pixels in x and y to link vesicle path (default 1) -z n search n pixels in time (z) to link vesicle path (default 1) -dur mark vesicels with duration rather than ID -crop n crop the filtered image n pixels from edges (default 0) Notes: Only single paths allowed (no branching, first come first servered) setting -z > 1 no longer guaranties FCFS matching and branching may occur
planimeter3
PLANIMETER3: Measure length and area of closed contours Usage:
planimeter3 [options]
Options:
-I input_file Image file name(s). A maximum of three
images can be entered. The first one is displayed
as green, the second as red and the third as blue.
Voxels colocalised between any two images are shown
as white
-R rpts file. If it exists on input, it is read in and
contours contained in it are displayed and can be deleted
or edited. If it does not exist, it will be saved on
exit unless Quit is pressed first
-S n n n Image number, display scale and black-level -X n n range of X planes (low,high) -Y n n range of Y planes (low,high) -Z n n range of Z planes (low,high) -M n Magnification (zoom)
This program allows you to draw boundaries around a cell or object The boundaries can be drawn in any order and can be erased if required.
Use: Left mouse button: move between planes (as in play). Middle mouse button: draw boundary.
Once a object has been defined pressing the middle mouse button will start drawing a contour for that object, or, if the contour has already been started, continue it. Each subsequent perimeter is associated with the previously defined object. To change the object or start a new object use the pop-up menu.
Backspace - undraw previous point (may be repeated).
Keypad + : Zoom in. Keypad - : Zoom out.
l - (toggle) show n planes in addition to the current plane.
Where n is 3 (default) but can be set from 1 - 9 by pressing the appropriate numerical key. Pressing the l key again will show just the current plane
v - (toggle) while drawing will result in a vertical line being
drawn from the current position when the mouse is moved.
h - (toggle) while drawing will result in a horizontal line being
drawn from the current position when the mouse is moved.
Control (toggle) - Toggle on and off scaling for the images.
Notes: A maximum of 20 separate objects can be defined for
contouring. Each object is represented by a different
colour.
The only limit on the number of contours that may
added to an object is the number of Z planes.
There is a limit of 4096 points per drawn contour,
however there is no limit on the number of points
of a contour in a file that is read-in
pMapping
Maps (postive only) image intensities into probability P that pixel is from G1 rather than G0.
Usage:
% pMapping [-{G|B|S|P|Q}] input_file[.i2i] output_file[.i2i]
Options: -G1 A0 x0 A1 x1 s0 Double-Gaussian parameters (floats) -G2 A0 x0 A1 x1 s0 s1 Double-Gaussian parameters (floats) -G ... Same as -G1 (for backwards compatability) -B n Camera bias level (integer, default=0) -S n Image scale factor (float, default=100) -P Make P image (default) -Q Make (1-P) image instead
Notes:
Image pixel intensities (x) are modeled as belonging to one of two Gaussian probabilty distributions (G):
-G1: G0=A0*exp(-(x-x0)^2/(2*s0^2)) and G1=A1*exp(-(x-x1)^2/(2*(x1/x0)*s0^2))
-G2: or G1=A1*exp(-(x-x1)^2/(2*s1^2))
where x1 > ix
If x>0 then the probability that pixel belongs to G1 is: P=G1/(G0+G1)
and the probability it belongs to G0 is: Q=(1-P)
Else x<=0 and P=Q=-32768 (no data)
repeat_rpts
Syntax: % repeat_rpts filename count
Filename is the .rpts file to reproduce Count is the number of Z planes to reproduce
Output is to stdout
Example:
% repeat_rpts one.rpts 10 >many.rpts
trackspots
trackspots: Command not found.
Utilities
The programs perform various useful functions.
cull
Choose and order columns of text
Usage: cull [-{h,c,a,s,n,1,2,...}
Options:
-h print this message -c ignore comments (from # to end of line) -a ignore ampersands (from & to end of line) -s suppress blank output lines -n number rows -1,-2,... next column to print
hist
This program prints image history records.
Usage: hist imagefile
hist2list
hist2list filenames
Prints history information for .i2i files
NOTE: works works with wildcards
NOTE: works with .Z files
immax
INFOCUS Find position of brightest pixel in a 3D data set Input file The 3D image file. Options:
infocus
INFOCUS Find most infocus Z plane of 3D data set Input file The 3D image file. Options: -X output infocus X position -Y output infocus Y position -Z output infocus Z position (default) -N normalize z-planes (MGL=1) Output is a number (Z coordinate) sent to stdout
printpixels
Syntax: printpixels [options] imagefile [ > textfile ]
Options:
-X x1 x2 keep pixel coordinates between x1 and x2 inclusive
-Y y1 y2 keep pixel coordinates between y1 and y2 inclusive
-Z z1 z2 keep pixel coordinates between z1 and z2 inclusive
-low n keep pixels valued > low
-high n keep pixels valued < or = high
-silent suppress output of non-warnings/errors to stderr
Output is to "stdout"
stats
Usage % stats [-h] <infile >outfile Options -h types this message Computes minimum, maximum, mean and standard deviation of a string of numbers read from stdin.
imsets
Sets the number of images sets in an image file.
Usage: % imsets imagefile.i2i #
Image-Visualization
These programs perform basic to sophisticated 2, 3, 4 and 5-D (x,y,z,t and color) image visualization.
montage
Version: ImageMagick 6.8.6-3 2014-04-08 Q16 http://www.imagemagick.org Copyright: Copyright (C) 1999-2013 ImageMagick Studio LLC Features: DPC OpenMP Modules Delegates: bzlib djvu fftw fontconfig freetype gslib jng jp2 jpeg lcms lzma openexr pango png ps rsvg tiff wmf x xml zlib
Usage: montage [options ...] file [ [options ...] file ...] file
Image Settings:
-adjoin join images into a single multi-image file
-affine matrix affine transform matrix
-alpha option on, activate, off, deactivate, set, opaque, copy
transparent, extract, background, or shape
-authenticate password
decipher image with this password
-blue-primary point chromaticity blue primary point
-bordercolor color border color
-caption string assign a caption to an image
-channel type apply option to select image channels
-colors value preferred number of colors in the image
-colorspace type alternate image colorsapce
-comment string annotate image with comment
-compose operator composite operator
-compress type type of pixel compression when writing the image
-define format:option
define one or more image format options
-density geometry horizontal and vertical density of the image
-depth value image depth
-display server query font from this X server
-dispose method layer disposal method
-dither method apply error diffusion to image
-draw string annotate the image with a graphic primitive
-encoding type text encoding type
-endian type endianness (MSB or LSB) of the image
-extract geometry extract area from image
-fill color color to use when filling a graphic primitive
-filter type use this filter when resizing an image
-font name render text with this font
-format "string" output formatted image characteristics
-gamma value level of gamma correction
-geometry geometry preferred tile and border sizes
-gravity direction which direction to gravitate towards
-green-primary point chromaticity green primary point
-identify identify the format and characteristics of the image
-interlace type type of image interlacing scheme
-interpolate method pixel color interpolation method
-kerning value set the space between two letters
-label string assign a label to an image
-limit type value pixel cache resource limit
-mattecolor color frame color
-mode type framing style
-monitor monitor progress
-origin geometry image origin
-page geometry size and location of an image canvas (setting)
-pointsize value font point size
-profile filename add, delete, or apply an image profile
-quality value JPEG/MIFF/PNG compression level
-quantize colorspace reduce colors in this colorspace
-quiet suppress all warning messages
-red-primary point chromaticity red primary point
-regard-warnings pay attention to warning messages
-respect-parentheses settings remain in effect until parenthesis boundary
-sampling-factor geometry
horizontal and vertical sampling factor
-scenes range image scene range
-seed value seed a new sequence of pseudo-random numbers
-set attribute value set an image attribute
-shadow add a shadow beneath a tile to simulate depth
-size geometry width and height of image
-stroke color color to use when stroking a graphic primitive
-synchronize synchronize image to storage device
-taint declare the image as modified
-texture filename name of texture to tile onto the image background
-thumbnail geometry create a thumbnail of the image
-tile geometry number of tiles per row and column
-title string decorate the montage image with a title
-transparent-color color
transparent color
-treedepth value color tree depth
-trim trim image edges
-units type the units of image resolution
-verbose print detailed information about the image
-virtual-pixel method
virtual pixel access method
-white-point point chromaticity white point
Image Operators:
-adaptive-sharpen geometry
adaptively sharpen pixels; increase effect near edges
annotate geometry text
annotate the image with text
-auto-orient automagically orient image
-blur geometry reduce image noise and reduce detail levels
-border geometry surround image with a border of color
-crop geometry preferred size and location of the cropped image
-extent geometry set the image size
-flatten flatten a sequence of images
-flip flip image in the vertical direction
-flop flop image in the horizontal direction
-frame geometry surround image with an ornamental border
-monochrome transform image to black and white
-polaroid angle simulate a Polaroid picture
-repage geometry size and location of an image canvas (operator)
-resize geometry resize the image
-rotate degrees apply Paeth rotation to the image
-strip strip image of all profiles and comments
-transform affine transform image
-transpose flip image vertically and rotate 90 degrees
-transparent color make this color transparent within the image
-type type image type
-unsharp geometry sharpen the image
Image Sequence Operators:
-coalesce merge a sequence of images -composite composite image
Image Stack Operators:
-clone indexes clone an image
-delete indexes delete the image from the image sequence
-duplicate count,indexes
duplicate an image one or more times
-insert index insert last image into the image sequence
-reverse reverse image sequence
-swap indexes swap two images in the image sequence
Miscellaneous Options:
-debug events display copious debugging information -help print program options -list type print a list of supported option arguments -log format format of debugging information -version print version information
In addition to those listed above, you can specify these standard X resources as command line options: -background, -bordercolor, -borderwidth, -font, -mattecolor, or -title
By default, the image format of `file' is determined by its magic number. To specify a particular image format, precede the filename with an image format name and a colon (i.e. ps:image) or specify the image type as the filename suffix (i.e. image.ps). Specify 'file' as '-' for standard input or output.
play
Usage: % play [options...] file [file2 [file3] ]
file(s) 2D, 3D or 4D image file name (with or without .i2i). FILES MUST BE OF SAME DIMENSIONS!
- Accepts input from stdin("<") or pipe("|") and titles as "<stdin>"
Options:
-sets n Number of 3-D image sets (default=1)
-S n n Image display scale and black-level
-M n Magnification (default=1)
-P n c Map intensity n to color c:{N R Y G C B M W}
-Z n n Range of Z planes (low, high)
-L Limit playback at range of Z planes
-U image values > black-level are displayed > 0
-B n n Scale bar length(microns) and microns/pixel
-project Display maximum intensity projection of Z-planes
DATA/ANALYSIS
-IO inner outer
Set the pixel readout (MIDDLE mouse button) ROI size where:
inner is the radius (+/- center pixel) of the additive ROI (default=0)
outer is the radius (+/- center pixel) of the subtractive ROI (default=0)
-name string
"string" replaces image filename (window title) as root name for creating files
DATA/PSF PREPARATIONS
-psf wavelength NA nD size spaced mask
Visualize the psf extent as a green circle
(wavelength, size and spaced must be in microns)
See preppsf for corresponding options
-min n Displays pixels valued less than "n" as blue
(default is -nomin)
-nomin Disables minimum function
-max n Displays pixels valued greater than or equal to "n" as red
(default is -max 32767)
-nomax Disables maximum function
-nonneg same as -min 0
ANIMATION
-movie {f|r|b}
Sets movie running forward, backword or both
-speed n Sets movie speed n={1,2,....9} (default=2)
-master Makes this a syncronous master
-n n Master(*) divides frame no. by n
-slave Makes this a syncronous slave
-pipe Both a master and a slave
-color Pseudocolor scale (default is gray scale)
-timebar Display time-bar instead of frame number
-hide Do not display the frame number
-o x y Screen origin (x,y) for window
-sched z n
Pause n video fields WHILE displaying plane z
-labels file
file contains one or more lines of x,y,z and labels
formatted |xxxxx yyyyy zzzzz string....
LEFT MOUSE left/right manual movie control MIDDLE MOUSE readout pixel position and value RIGHT MOUSE pops up Main menu
To adjust contrast/brightness (replaces SGI edmap program)`: choose Main menu => Color menu => Help for built-in edmap this will print instructions in the terminal window
To use Z/Time Plot Analysis: A-Key then Hold(!) down the MIDDLE MOUSE button, or press the SHIFT and A-Keys together If you use the first method to enter: release the MIDDLE button to exit If you use the second method to enter: press the A-Key again to exit
To use ROI Analysis: choose Main menu => ROI Analysis (or press the t-key on the keyboard) LEFT MOUSE size or move ROI box MIDDLE MOUSE left/right changes threshold
To use Intensity linescan: choose Main menu => Intensity linescan (or press the l-key on the keyboard) LEFT MOUSE draw scan line through image
To save the current image display(s) as 24-bit color, portable anymap files (.ppm): choose Main menu => Save image(s) => Current image (or > key on keyboard) or choose Main menu => Save image(s) => Entire image set (or = key on keyboard)
To save the entire image set as an MPEG movie:
choose Main menu => Save image(s) => Create MPEG (.mpg) movie from PPM set {+}
or press + key on keyboard. The movie speed will be the same as the current play speed)
To save the entire image set as an Quicktime movie:
choose Main menu => Save image(s) => Create Quicktime (.mov) movie from PPM set {-}
or press - key on keyboard. The movie speed will be the same as the current play speed)
To syncronize 2 movies:
% play -master master.i2i | play -slave slave.i2i
New features:
* "p" key toggles maximum-intensity projection on|off (see -project command line option)
* contrast widget includes multiple colors (beyond gray) for (mono)chroma
* on-image labels are available (see -labels) and can be toggled off|on using the "shift+L" key
* "}" key increases stereo parallax. "{" decreases parallax. Default parallax is 0 (no stereo).
* cycle among multiple images (file1, file2, file3) using END key
play3d
Usage: % play [options...] file
file 2D, 3D or 4D image file name (with or without .i2i).
- Accepts input from stdin("<") or pipe("|") and titles as "<stdin>"
Options:
-sets n Number of 3-D image sets (default=1)
-S n n Image display scale and black-level
-M n Magnification (default=1)
-P n c Map intensity n to color c:{N R Y G C B M W}
-Z n n Range of Z planes (low, high)
-L Limit playback at range of Z planes
-U image values > black-level are displayed > 0
-B n n Scale bar length(microns) and microns/pixel
-project Display maximum intensity projection of Z-planes
DATA/ANALYSIS
-IO inner outer
Set the pixel readout (MIDDLE mouse button) ROI size where:
inner is the radius (+/- center pixel) of the additive ROI (default=0)
outer is the radius (+/- center pixel) of the subtractive ROI (default=0)
-name string
"string" replaces image filename (window title) as root name for creating files
DATA/PSF PREPARATIONS
-psf wavelength NA nD size spaced mask
Visualize the psf extent as a green circle
(wavelength, size and spaced must be in microns)
See preppsf for corresponding options
-min n Displays pixels valued less than "n" as blue
(default is -nomin)
-nomin Disables minimum function
-max n Displays pixels valued greater than or equal to "n" as red
(default is -max 32767)
-nomax Disables maximum function
-nonneg same as -min 0
ANIMATION
-movie {f|r|b}
Sets movie running forward, backword or both
-speed n Sets movie speed n={1,2,....9} (default=2)
-master Makes this a syncronous master
-n n Master(*) divides frame no. by n
-slave Makes this a syncronous slave
-pipe Both a master and a slave
-color Pseudocolor scale (default is gray scale)
-timebar Display time-bar instead of frame number
-hide Do not display the frame number
-o x y Screen origin (x,y) for window
-sched z n
Pause n video fields WHILE displaying plane z
-labels file
file contains one or more lines of x,y,z and labels
formatted |xxxxx yyyyy zzzzz string....
LEFT MOUSE left/right manual movie control MIDDLE MOUSE readout pixel position and value RIGHT MOUSE pops up Main menu
To adjust contrast/brightness (replaces SGI edmap program)`: choose Main menu => Color menu => Help for built-in edmap this will print instructions in the terminal window
To use Z/Time Plot Analysis: A-Key then Hold(!) down the MIDDLE MOUSE button, or press the SHIFT and A-Keys together If you use the first method to enter: release the MIDDLE button to exit If you use the second method to enter: press the A-Key again to exit
To use ROI Analysis: choose Main menu => ROI Analysis (or press the t-key on the keyboard) LEFT MOUSE size or move ROI box MIDDLE MOUSE left/right changes threshold
To use Intensity linescan: choose Main menu => Intensity linescan (or press the l-key on the keyboard) LEFT MOUSE draw scan line through image
To save the current image display(s) as 24-bit color, portable anymap files (.ppm): choose Main menu => Save image(s) => Current image (or > key on keyboard) or choose Main menu => Save image(s) => Entire image set (or = key on keyboard)
To save the entire image set as an MPEG movie: choose Main menu => Save image(s) => Create MPEG movie from PPM set (or + key on keyboard) (note the MPEG speed will be the same as the current play movie speed)
To syncronize 2 movies:
% play -master master.i2i | play -slave slave.i2i
New features: 1) "p" key toggles maximum-intensity projection on|off (see -project command line option) 2) contrast widget includes multiple colors (beyond gray) for (mono)chroma 3) on-image labels are available (see -labels) and can be toggled off|on using the "shift+L" key 4) "]" key increases stereo parallax. "[" decreases parallax. Default parallax is 0 (no stereo).
rgbmerge
Takes one to three 2D grayscale images and merges them into a singel RGB image.'
Syntax:
rgbmerge R|G|B:filename.ext [R|G|B:filename.ext [R|G|B:filename.ext] ] merged.ext
Where:
R:, G:, or B: indicate the desired color channel for that image. Omitted color channels are black by default. The case of the color channel (R|r, G|g, B|g) does not matter. The order of the images on the command line does not matter.
Examples:
Merge a green JPEG image and a blue PNG image into one color TIFF image rgbmerge G:one.jpg B:two.png result.tif is the same as rgbmerge b:two.png g:one.jpg result.tif
Valid image file types are those recognized by ImageMagick.
== Image-Processing ==
A collection of various and sundry programs for image processing.
Like many of the programs listed abouve, these routines are designed to work with image piplines. Substituting a dash "-" for an image name routes input from <stdin> and/or output to <stdout> Routines can be chained together with the comandline pipe character "|" for easy prototyping of complex functions.
----
bleachfit
bleachfit: Command not found.
derivima
derivima [-{diff|lr|exp,scale,max}] inputfile outputfile
dFoverF
Compute a fluorescence time-ratio image.
Usage:
dFoverF [-{h,sets,norm,range,noratio,AC,median,threshold,all,mask,scale,(no)flags}] input_file output_file
Use -h to get further help
diffima
derivima [-diff f|c|b] inputfile outputfile
dilateima
Usage: % dilateima [options] input_file output_file
Performs 2D dilation of given pixel value. Pixels not dilated are left intact.
Options: -P n Pixel value to dilate (def=1) -A also dilate all pixels > n
edges
Copyright 2010 University of Massachusetts Medical School and the Biomedical Imaging Group All rights reserved. Explicit permission to use this program must be received prior to use. Revised - May, 2014
Syntax: edges [options] input_file output_file
input_file The image to be filtered.
output_file The filtered image.
Options:
-LAP x y Laplacian kernel
size in x (pixels)
size in y (pixels)
-DOG s l Difference of gaussians kernel:
std.dev. of smaller gaussian (pixels)
std.dev. of larger gaussian (pixels)
-E n Extent (+/-no. of sd's) of DOG kernel
-M n image mean to preserve [0,1]
-S n image scale factor.
-I invert filtered image
-V verbose
erodeima
Usage: % erodeima [options] input_file output_file
Performs 2D erosion of given pixel value.
Options: -P n Pixel value to erode (def=1) -F n Value to fill eroded pixels (def=0)
FRETratio
FRETratio file [threshold [dx dy] ]
the threshold is applied to the YFP(upper) image. default is 0. the shifts (dx,dy) are applied to the CFP(lower) image. defaults are 0 and 0.
histima
Usage: histima [options] imagefile >textfile
Imagefile An image file name (extension optional)
Options:
-bins n Number of bins in histogram (default=256)
-size n Number of grey levels per bin
(default = max-intensity / number-of-bins)
-bg n Value for lowest bin (default=0)
Notes:
Histogram is output to stdout as ASCII compatable
with xmgr. (see /usr/local/xmgr)
Example:
% histima -bins 100 my_image | xmgr -pipe &
kinetic-analysis
Syntax: kinetic-analysis numerator[.i2i] denominator[.i2i] filtered-denominator[.i2i] threshold results[.txt]
or kinetic-analysis help
where:
numerator the ratio numerator (dependent) time series image denominator the ratio denominator (independent) time series image filtered-denominator corresponding image used for identifying 2-D maxima > threshold for ratio calculation threshold 2-D maxima intensity threshold (> or =) applied to filtered-denominator image results name of a text file for the kinetic time course results (see help)
help provides more information
lsr_align
Least Square Residual based image alignment.
Usage: LSRalign [options] in-image out-image in-image images to align out-image aligned images Options: -sets n Number of 3-D images sets -range dx dy +-range to align -first always align to 1st Z plane
maskedsmul
maskedsmul rescales image intensities in outlined regions
Usage: % maskedsmul {options] image1 image2 image3
image1 image to correct
image2 image with mask outline
image3 output image
Options:
-I n scale factor for pixels inside mask (def=1)
-O n scale factor for pixels outside mask (def=1)
-P n value for boundary pixels (def= -1)
-silent redirects stderr to /dev/null
Notes:
1) The outline pixels are considered outside the line
2) Use planimeter and addlines to create mask image
maski2i
Usage: maski2i [options] image1 outimage options:
-T # <= # = 0, > # = 1 [Default = 0]
OR -C # Complement, >= # = 0, < # = 1 [Default = 0]
mathi2i
mathi2i (Sep 20, 3013)
Basic pixel operations (+,-,*,/,max,min) between images
Syntax:
mathi2i [options] input-image-name input-image-name output-image-name
Options:
-a|s|m|d Performs image addtion, subtraction, multiplication or division respectively
-max|min Minimum or maximum of the two values at each pixel
-and|or Boolean operation: zero -> 0, non-zero -> 1, result is always 0 or 1
-S n1 n2 Scale(multiply) 1st and 2nd images by n before performing operation
Notes:
All calculations are done as floating point. Output images is integer truncated to [-32767:+32767].
If either image image value is -32768 ("no data") then output is always -32768.
Division by zero is undefined and result is -32768 ("no data") at that pixel.
(you can make "no data" pixels show up as red(R) with the command: play -P -32768 R ...)
maxima
Usage: % maxima [options] input_file output_file
Identifies 3D image maxima (peaks). The all non-maximal pixels are set to zero, unless the -F options is used. Then, non-maximal pixels are preserved and maximal pixels are set to the specified value.
Options: -T n maximum threshold value n -7 maximum if > 7 of 8 neighbors -E define maximum as > or = (mutally exclusive with -7) -F n flag maximum pixels with value n -Z z-axis maximum only -R input image is 4-byte floating point
If the output is omitted, the output is an ASCII list of maxima positions as (x,y,z,intensity)i sent to stdout.
normbg
Usage: % tranima [options] inputfile outputfile Input file image file name Output file image file name. Options: -roi xleft ybottom xright ytop -smooth nplanes (+/-planes to smooth, default=0) -zref zplane (default=1) -skip zstop zstart (repeat as needed) -corrbg (correct planes for dark errors, default is no)
nv-coloc-analysis
Syntax: nv-coloc-anal.csh results[.txt] folder1 [folder2 [ folder3 [...]]]
1) All the folders named on the command line will be analyzed. 2) Results of the colocalization analysis are appended to the named results textfile.
If the results textfile does not exists it will be created.
openclose2d
openclose 2D gray scale erosion(min) and dilation(max) Input file The image to be filtered. Output file The filtered image. Options: -open r erosion->dialtion over radius r (pixels) -close r dilation->erosion over radius r (pixels)
OTFz
Syntax: OTFz psf[.i2i] z-spacing(um) wavelength(um) NA(objective) nD bead(um)
OTFx
Syntax: OTFx psf[.i2i] pixel-size(um) wavelength(um) NA(objective) bead(um)
OTFy
Syntax: OTFx psf[.i2i] pixel-size(um) wavelength(um) NA(objective) bead(um)
padima
Usage: % padima [options] input_file output_file
Pads an image (on both sides) in X, Y and/or Z
Options: -B n Extent of padding in XY -X n Extent of padding in X -Y n Extent of padding in Y -Z n Extent of padding in Z -V n Value of padding pixels (def=0)
reduceima
REDUCEIMA reduces image resolution and size by binning Input file Image file name. Output file Image file name. Options: -sets n Specifies the number of 3-D image sets. set the binning factors: start(1 is first pixel) end(-1 is last pixel) incr(-1 is last-first+1) -X n n n X axis start, end and increment in pixels -Y n n n Y axis start, end and increment in pixels -bin bx by Equivalent to: -X 1 -1 bx -Y 1 -1 by -Z n n n Z axis start, end and increment in pixels -P Same as -Z 1 -1 -1 -N n n n Time axis start, end and increment in pixels -S Eliminate axes of size 1 on output set the binning mode -T Keep total(sum) of pixels -A Keep average of pixels -L Keep lowest(minimum) pixel -H Keep highest(maximum) pixel (DEFAULT) -M Keep median (not mean!) pixel -when Keep Z position of lowest|highest|mean pixel (combine with -L|-H|-M)
radcolpsf
RADCOLPSF: Radially averages psf images Input file An image file to be radially averaged. Output file The averaged image file. Options: -C n n Center of rotation (x,y)
ratio2wl
syntax: ratio2wl fura[.i2i] threshold(WL-1) [dark[.i2i]]
register
Usage: register [options] in-image out-image in-image images to register out-image registered images Options: -SSC Stochastic Sign Change method (default) -range dx dy +-range to register -first always register to 1st Z plane
RITS-fusion-analysis
Syntax:
RITS-fusion-analysis filename-root event_no.
RITS-process
This is the main application to fit RITS fusion model to TIRF time-series images
Syntax: RITS-process RFP[.i2i] GFP[.i2i] rootname
1) the coordinate textfile must be rootname.txt 2) images are named rootname_r|g_###.i2i 3) individual output graphs are named rootname_###.ps 4) the files of collected graphs are named rootname.ps and rootname.pdf
Necessary files:
RITS-select-event RITS-fusion-analysis RITS-2D-fusion-analysis-exe RITS-2Dto3D-fusion-analysis-exe RITS-fusion-analysis.par
RITS-select-event
RITS-select-event RFP[.i2i] GFP[.i2i] x0 y0 radius z0 z-before z-after rootname id
rotateima
Usage: % rotateima input_image outputimage Input file 3D An image file to be rotated. Output file 3D The rotated image file. Options: -F file File containing 3D transformation matrix -C n n n Center of rotation (x,y,z). -S n n n Scaling applied to X, Y, and Z axes. -R n n n Rotation about X, Y, and Z axes (degrees). -P n Perspective (distance to eye in pixels) -D nx ny nz Fix the output image size as nx,ny,nz
-arrow x0 y0 x1 y1
Defines an arrow from tail (x0,y0) to tip (x1,y1).
Image is rotated within the XY plane such that the
arrow is pointing up (increasing Y).
rotproj
ROTATEIMA: Produces a sequence of 3D image rotations and
projections of varying image attributes
Input file An image file to be rotated.
Output file The rotated image file.
Options:
-M n Projection mode: n=1 voxel SUM,
n=2 voxel MAX,
n=3 SUM w/opacity.
n=4 RANGE shading
-K n Optical Density factor: OD = n * intensity
(when mode=3) opacity = OD/(1+OD)
-T n Intensity threshold
-I c Interpolation N|L (Near-neighbor | Linear)
-C n n n Center of rotation (x,y,z).
-S n n n Scaling applied to X, Y, and Z axes.
-R n n n Rotation about X, Y, and Z axes (degrees).
-P n Perspective (distance to eye in pixels)
-V n n Viewport size (X, Y)
-Z n n Z-dimension clipping planes (zmin,zmax)
-A n n n Start, stop, delta rotation angles (deg)
-F Flag pixels outside data bounds with -32768
scaleima
Usage: % scaleima [options] input_file output_file
Options:
-Z z1 z2 dz scale Re-scale intensities of Z-planes
z1 to z2 stepping dz by scale
-V Verify -- list scale factors
Notes: Multiple Z ranges may be specified. Z ranges may be specified in arbitrary order. Any Z-plane not specified is scale by one(1.0) Maximum of 32767 Z-planes allowed.
segment
Usage: % segment [options] input_image output_image
Options:
-sets n Specifies the number of 3-D image sets.
-X n n n X axis start, end and increment in pixels
-Y n n n Y axis start, end and increment in pixels
-Z n n n Z axis start, end and increment in pixels
-N n n n Image set start, end and increment in pixels
-I n Include n'th Z plane in repeat series given by -Z option
(planes in repeat series are number from 1 to z-increment)
-ROI x0 y0 x1 y1 Corners of a rectangular region to segment
(alternative to -X and -Y options with increments of one)
-flag n Flag padding pixels with intensity n (default = -32768)
-S Eliminate axes of size 1 on output
Notes: -I option can be used to reorder planes in series.
If the region of interest extends outside of XY plane, the segmented image is padded with the flag value(-flag)
signalmass
During a Ca2+ "spark", free Ca2+ (diffusion coefficient, D = 250 μm2/s) and Ca2+ bound to fluo-3 (D = 22 μm2/s; Smith et al. 1998) quickly diffuse away from the spark release site as Ca2+ continues to be discharged. To quantify the total fluorescence arising from the binding of fluo-3 to the discharged Ca2+(i.e., the Ca2+ signal mass), the increase in fluo3/fluo4 fluorescence ) must be collected from a sufficiently large volume to provide a measure of the total quantity of Ca2+ released. Custom software was used to process the images and extract signal mass information for each spark event. The signal mass time course for each spark was computed from the two-dimensional, widefield fluorescence images of fluo-3 according to the following equations.
Total fluorescence,
Ftotal(t)= SUM[F(x+∆x, y + ∆y, t)].
The fluorescence F is summed over a 13.7-μm square region (41 pixels on a side in the x-y plane) surrounding the spark epicenter pixel (x,y) as determined from the ΔF /F0 images from Formula 1.
Signal mass,
sm(t) = G*(Ftotal(t)-Ftotal(t0))
The signal mass sm(t) is the change in total fluorescence FT(t) over the baseline fluorescence FT(t0) times the detector gain G (see below). The time t0 corresponds to the image immediately preceding the beginning of the spark. The beginning of the spark event was identified as the first image having a flux, i.e., an increase in total fluorescence (FT) relative to the preceding image, exceeding 2 SDs of the noise(1).
(1) Dynamics of signaling between Ca(2+) sparks and Ca(2+)- activated K(+) channels studied with a novel image-based method for direct intracellular measurement of ryanodine receptor Ca(2+) current.
ZhuGe R, Fogarty KE, Tuft RA, Lifshitz LM, Sayar K, Walsh JV Jr. J Gen Physiol. 2000 Dec;116(6):845-64.
Syntax:
signalmass radius [SF] < listfile > outfile
where:
radius is the halfedge size of bounding box SF is the [optional] scale factor for scalnge the dF/Fo traces (default=1) listfile is the spark|puff|syntilla description textfile |filename x y z | x y z |filename x y z | x y z outfile is the table of signalmass statistics
SLMrecon
SLMrecon phase0[.i2i] phase120[.i2i] phase240[.i2i] output-image[.i2i] Structure illumination optical sectioning according to T.Wilson Options: -S n SL reconstruction image scale factor (default: 1.0) -WF filename[.i2i] Save sum of three phases as WF image
SLMreconS.csh
syntax: SLMreconS root{-0.i2i,-1.i2i,-2.i2i}
root-0 is phase 0, root-1 is phase 120, root-2 is phase 240
spectrum
Usage: % spectrum [options] input_file output_file
Computes 2D FFT of image. The output file is complex FFT (.cxi file) unless -M or -E option used(.i2i file).
Options:
-A Apodize image with raised cosine window
-Z Pad image with zeros (default)
-P Pad image with mean-gray-level
-M Compute magnitude spectrum(not -E)
-E Compute energy spectrum(not -M)
-C Center spectrum at N/2(with -M or -E)
-LOG Log transform of magnitude or energy spectrum
-DC Zero the DC component of FFT
-N x y FFT size (Nx by Ny)
-S x y Pixel size in X and Y(def=1,1)
-F image Filter input_file using image as Fourier mask
-xlines dist order(s) width
Filter input_file using X layer lines where:
dist is no. pixels to layer line
orders are the line(s) to include
terminated with a zero.
width is of number of lines about this line
-ylines dist order(s) width
Filter input_file using Y layer lines
-R x y Filter out components within radius n of DC
Notes:
-A option scales spectrum by 1/mean-squared-value of
apodization window function
-S option scales spectrum by pixel area
-F Alternative to creating a convolution kernel
sqrtima
sqrtima [-S n] inputfile outputfile
statima
Copyright 2010 University of Massachusetts Medical School and the Biomedical Imaging Group All rights reserved. Explicit permission to use this program must be received prior to use.
Usage: statima [options] imagefile [textfile]
Imagefile An image file name (extension optional)
Textfile Optionally write statistics to file
Options:
-sets n Specifies the number of 3-D image sets.
-X n n left and right X coord for Region-Of-Interest
-Y n n bottom and top Y coord for R.O.I.
-Z n Statistics on single Z plane = n
-E Statistics on each Z plane
-P Mean and variance of positive(>0) pixels.
Sets threshold equivalent to -T 0
-T n Pixels with intensities less than or equal (< or =) n are ignored
Default is no threshold unless -P is invoked (see above)
Combine with -P to change threshold
-SE Standard error instead of standard deviation
-S Show image statistics (default)
-H Show image gray level histogram in portrait format (default)
Combine -H -S in this order to show both statistics and histogram
-L Show image gray level histogram in landscape format (not implemented)
-silent Redirects sdterr to /dev/null
Example: % statima -X 101 150 -Y 41 70 -T 10 -E -S my_image shows just the image statistics of those pixels greater than ten(10) in a 50 by 30 pixel rectangular region, Z-plane by Zplane, in the image in file my_image.i2i
thresholdima
Biomedical Imaging Group University of MA Medical School Copyright 2010
Syntax: %thresholdima [options] input_file output_file
input_file Image file name.
output_file Image file name.
Options:
-T n Threshold (<) intensity (default=0)
-V n Replacement value (default=0)
-S Subtract rather than threshold
-R low mid high
Triangular ramp function
low < low -> 0
mid = mid -> mid
high > high -> 0
-P n Power of ramp function (default=1)
TIRFex
Usage: % scaleima [options] input_file output_file
Options: -z planes the mean excitation distance for TIRF (default=0) -e fraction the fraction of maximal excitation due to epi background (default=0) -zref plane the z-plane where the relative TIRF excitation equals 1.0 (default=0)
Notes:
TIRF_WF
TIRF_WF [options, -h] inputfile outputfile
tranima
Usage: % tranima [options] inputfile outputfile
Input file Image file name.
Output file Translated image file name.
Options:
-T x y z Translation in X, Y and Z
(Positive translation is right, up, rear)
-rotate Translations are circular (DEFAULT)
- what goes off the edge wraps back on the opposite edge.
-shift Translations are not circular
- what goes off the edge is lost
- image is padded with -32768
Translations are rounded to the nearest integer value.
transp
Usage: % transp [option] inimage outimage Options: -sets n Number of image sets (4-D: default=1) -XY Transpose X and Y axes -XZ Transpose X and Z axes -YZ Transpose Y and Z axes
Signal-Processing
These programs work in the time domain on simple 1-D or 2-D (x|t,y) data
digfilt
NAME
digfilt - abstract
SYNOPSIS
digfilt [options] <stdin >stdout
DESCRIPTION
digfilt...
digfilt has the following options:
-lowpass Hz -3dB frequency
-highpass Hz -3dB frequency
-timestep seconds Sampling interval
EXAMPLES
SEE ALSO
NOTES
CAVEATS
LAST REVISION
--/--/--
BUGS
pdcr
Prints out columns of file Usage: pdcr [options] {file|-} options:
-c n # of columns -p n # of points to print out
straighten
Computes average pixel intensity along a single curve traced using planimeter program by mapping pixels to closest(euclidian distance) point along the line. Only points within a given distance from the line (-s option) are used and ONLY positive pixels are mapped.
Usage:
% straighten [options] image rptsfile [image]
where
image input image file name
rptsfile points along cell center line (planimeter)
image optional output image file name where output
image pixels contain average intensity at
position on line to which pixel was mapped
(ALL pixels are mapped for this operation)
Options:
-S n Maximum dist from centerline (def=nearest)
-N Normalize the center position [0..1]
-Z n Do only Z plane = n
-P Output image(optional) pixels replaced with
line position instead of average intensity
-A n Normalize average along line to n
-J Replace pixel intensities in output image
JUST under the curve.
-NR Do not rewind points file each time
-V Verbose mode
-xmgr Xmgr compatable output
-neg Allow negative values
line position (x) and average intensity (y) are sent to stdout and can be piped to graph|plot or Xmgr
Examples:
% straighten -S 10 image1 image1.rpts | graph | plot plots the average of those pixels 10 pixels or less from the centerline given in image1.rpts
% straighten -S 10 image1 image1.rpts > image1.graph sends the output to the file named image1.graph
% straighten -S 10 image1 image1.rpts image2 creates the pixel classification map instead