TIFFSPLIT4.EXE (32bit DOS)

Version 4.12

July 21, 2000

NAME

tiffsplit4.exe - is a utility program intended to extract viewable and editable images from tiff image files saved on the LeicaTCS-NT/SP confocal microscope using the "file -> save as" method.  The "file -> save as" method preserves essential image and parameter information, and is advantageous in that the originally saved images may be reopened on the LeicaTCS-NT system and enough information is retained to create Red/Green/Blue overlays and to calculate geometric calibrations and rescan the sample using identical parameters.  However, on the Leica multichannel fluorescence (e.g. double labeling), z-series and/or time lapse images are saved as multipaged tiff format files.  Most commercially available software can only display the first image in a multipage tiff format file.  The remaining images, although present in the file, are not displayed.  Tiffsplit4.exe fulfills several functions: 1. splits multipaged tiff format files into individual tiff or bitmap files, 2. generates a contrast enhanced extended focus image for each channel along with an overlayed RGB image of the extended focus channels,  3.  generates a maximum projection image for each channel along with the overlayed  RGB image,  4. linearizes the palette of the output files to grayscale using photometrically correct weights from the red, green and blue channels (i.e. 1:1:1 in contrast to 0.3:0.59:0.11 used by Photoshop when converting color to grayscale modes) and removes green/blue under/overload indicator pixels by converting them into black or white.

Tiffsplit4 has several command line options that direct its operation and enhance its function.  By default tiffsplit4.exe splits multi-page tiff image files into separate bitmap (.bmp) files for each channel and creates an information file (.inf) which contains the original text information embedded in the Leica tiff image file.  This program is intended to split multi-channel and/or multi-paged z-series or time series files created on the LeicaTCS-NT version 1.x.xxx  software, which have been saved using "save as".  The images are split into a series of simple tiff (.tif) or bitmap (.bmp) format image files.  tiffsplit4.exe can be run in a Windows 32 bit Dos box (Windows 95, 98, XP, NT, 2000, 2003, etc. command prompt).  By default images are split into individual bitmap files (.bmp).  The -t option directs the creation of separate files in tiff format (.tif).  The -e option generates intensity normalized (expansion) extended focus images (average) of each channel's image series along with a RGB overlay view of up to 3 channels.  The -m option generates maximum intensity projections for each channel's image series.  Extended focus and maximum projection images are always created as bitmap (.bmp) format files and by default contrast is expanded to fit the available 8 bit range.  Contrast normalization may be suppressed by adding -n to the command line.  The -b, -t, -n, -m and -e options may be combined.  In all operations of tiffsplit4.exe the original tiff format file is left intact, and output files will overwrite existing files of the same name without warning.

SYNOPSIS

tiffsplit4 file1 [ file2 [ file3 ... ] ] [ -g ] [ -c ]  [ -b ]  [ -e ]   [ -m ] [ -n ]
                [ - r {RGBN} {RGBN} [RGBN] ]  [ -p path ]  [ -t ]  [ -T ] [ -l ]  [ -i ]  [ -v ]

Where: file1 ... is(are) the filename(s) of an Intel type tiff format image file.  Dos style wild cards (*or ?) are permitted and the extension .tif should be supplied.  Multiple tiff files may be given and tiffsplit4 will split each one independently into respective file series.   Thus an entire directory of multipaged tiff files may be split with one command.

-g  changes images to gray scale.  Without this option images retain the color look up palette supplied by LeicaTCS-NT (except for overlayed images, which are always created in RGB mode).

-c  leaves green/blue under/over load indicator colors intact from output tif and bitmap files including the extended focus images of each channel.  Normally glow over/under indicator colours are removed.

-b  outputs files in bitmap format (.bmp) (default output)

-t  outputs files in tiff format (.tif)

-e  generate an extended focus image for each channel along with an overlayed RGB image of up to three of the extended focus images.  Created extended focus files use the same name as the source file with an underscore and 'xf', and another underscore and letter denoting the channel. e.g. myosin_xf_a.  Overlayed stacks would be called myosin_xf_ovr.bmp.  Extended focus files are always created in the bitmap image file format.  By default contrast is expanded to fill the intensity range for each channel.

-m  generate a maximum projection image for each channel and an overlayed RGB image of up to three channels.  Created maximum projection image files use the same name as the source file with an underscore and 'mp', and another underscore and letter denoting the channel. e.g. myosin_mp_a.  Overlayed stacks would be called myosin_mp_ovr.bmp.  Maximum projection image files are always created in the bitmap image file format.  By default contrast is expanded to fill the intensity range for each channel.

-n  supresses contrast expansion when generating extend focus and/or maximum intensity projections with the -e and -m options.

-r {RGBN}{RGBN}[RGBN] reorders the colors assigned to the channels in the overlayed image.  In -r grb 1st channel will be green, 2nd red & 3rd blue.  This is the default order and is the order used by the LeicaTCS-NT software.  With -r grn the 1st channel is green, 2nd channel is red and 3rd is suppressed.   Note: can not assign a channel to more than one color, and only one channel may be suppressed.  Overlayed images are generated when generating extended focus or maximum intensity projections from multiple channel stacks.

-p path specify path (directory and/or drive) for output files.  i.e. split, extended focus and maximum projection files.

-l  lists files found which match the specifications of file name.   This is useful for displaying files found without outputting any images.

-i  displays the color index, palette, information stored in the original file for each channel of the original tiff image file.

-T  displays embedded text lines in the tiff file.

-?  or no command line parameters will display a terse command line help message.

-v verbose mode, extra information is displayed on the command line while processing.

DESCRIPTION

tiffsplit4 extracts text information embedded in TIFF image files and stores it in text file(s) with the same name stem as the original TIFF image file with the added extension of ".inf" in the current directory.  The individual images (pages) can be extracted and stored as separate TIFF image files or bitmap image files with an appended underscore and letter to denote the channel.  A further two or more digit decimal number is also added as an index denoting the image position in the sequence for each channel.  Leading zeros are added to the index number to aid computer sorting in windows directory listings.  This program is specifically for splitting multi-channels images saved with the LeicaTCS-NT confocal system, and is not a generalized program (contrary to TIFF standards).

tiffsplit4 will also, optionally, generate extended focus and/or maximum intensity projection images from z-series stacks.  An extended focus and/or maximum intensity projection image is generated for each channel in the original z-series stack.  These images are intensity normalized 8 bit bitmap files, i.e. the intensity histogram is linearly scaled to fill to full 8 bit range (256 intensity levels).  Extended focus file names are derived from the original tiff file name stem with an appended underscore and xf and underscore channel letter (a=channel 1, b=channel 2, etc.). e.g. myosin.tif will create the extended focus files myosin_xf_a.bmp & myosin_xf_b.bmp.  Similarly maximum projection images will have the file name stem appended with _mp flollowed by another underscore and the channel letter.  An RGB overlayed image of the extended focus and/or maximum projection  for each channel is also created if the number of channels is less than 4 and greater than 1.  The output file will also be a bitmap with a name formed from the tiff file stem appended with "_xf_ovr.bmp or "_mp_ovr.bmp".  E.g. mysosin_xf_ovr.bmp and myosin_mp_ovr.bmp.  Single section images will also generate an RGB overlay file along with intensity normalized images from each channel.  Intensity mormalization can be supressed by adding -n to the command line.

The LeicaTCS-NT program saves much useful information inside the TIFF image file when one uses "save as". Please note that "save selected" saves no parametric information with the TIFF file.  Also note that "save as" with the export option does save parametric information as an ".exp" file (readable as text) along with each image section and channel as separate tiff files.

UTILIZATION EXAMPLES

tiffsplit4 myosin.tif: generates individual bitmap files for each image creating myosin_a01.bmp, myosin_a02.bmp ... and myosin.inf.

tiffsplit4 *.tif -e: creates extended focus images for each channel and extracts the parametric information of all the TIFF image files in the current directory and saves them as readable text in respective ".inf" files.  Note that individual image files are not created.

tiffsplit4 *.tif  -t -g  : splits all valid tiff files into individual grayscale tiff image format files.

tiffsplit4 *.tif  -b -c  : splits all valid tiff files into individual grayscale bitmap files and leaves green/blue under/over load colors, if any.

tiffsplit4 *.tif  -l  : lists matching TIFF image files found in the current directory.  No split file is generated.

tiffsplit4 *.tif  -b -t -e -g  : generates gray scale split  images on both bitmap & tiff format from all matching TIFF image files found in the current directory.  Split images are gray, which is handy for measuring intensities or creating customized overlays in PhotoShop (Adobe).

SEE ALSO

TiffTags.exe - dump tiff image tags
TiffText.exe - output embedded text in tiff files as an ".inf" text file
Tiffsplit2.exe - old version of tiffsplit3.exe - can not make extended focus images or overlays
Tiffsplit3.exe - old version of tiffsplit4.exe - extended focus (xf) images are named slightly differently - split file format is tiff (not bitmap) by default
Tiffsplit.exe - old version of tiffsplit2.exe - can not make bitmap files or specify output file path
bmovrlay.exe - overlay matching pairs of images as red/green images
bmp2pic.exe - converts a series of bitmap files into a Biorad .pic format file, which can be opened by Confocal Assistant.

BUGS

The newer Leica LCS software Tiff images are not handled.  Extended focus image generation is handle in the LCS & the free LCS-lite software and only single page Tiff files are generated by this software. 

The multipage .LSM files produced by the Zeiss 510 software are not handle by tiffsplit4.exe

This program uses the arcane DOS prompt and has a complex set of command line options, some of which interact with each other.  However this program does function effectively when used with thought and experience i.e. give it a go!

Large numbers of files are created duplicating what is already in the original tiff file.  Large volumes of informationally redundant image files may end up chocking the available disk space, especially if the original data is large time lapse or z-series files.

Problems may occur when reassigning colors in overlayed files.  Colors assigned may not be what is specified in the -r RGB command line switch.

Using file* in the command line to match a wide range of files can run into problems if careful consideration of names used is not made.  e.g. trying to re-split already split files, which were created in the .tif format.  It is up to the user to     name files and place files appropriately to avoid this situation.

Notice: This program is intended to split TIFF images created by the LeicaTCS-NT confocal system only.  It is at a nascent level of development. Be mindful with its use. This program does not handle RGB type tiff files saved using the "save selected" approach on the LeicaTCS-NT system.

Existing ".inf" or ".tif" or ".bmp" files with the same name as generated files are overwritten without warning.

tiffsplit4.exe is a 32 bit command line program and will not run on DOS 6.2x, DOS 7 and earlier or Windows 3.11 and earlier.

Can not split tiff files with more than 9999 image pages.

Be careful when applying tiffsplit4.exe with wild card expansion more than once in a directory.  It can create split files with long index numbers from tiff files already derived from tiff files.

Help section is written in Unix manual help style.

Please report bugs to mchuaEXPUNGETHIS@med.unc.edu

FUTURE ENHANCEMENTS

A future program derived from tiffsplit4.exe may make separate RGB overlays of each individual z-series level.  But for now use tiffsplit4 file -b & bmovrlay.exe for making red/green overlays of each level in a z-series.


Copyright 1997-2001  Michael Chua