Version 4.10

August 17, 2000


tiffsplit4.exe - is a utility program that extracts 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 multi-channel fluorescence (e.g. double labeling), z-series and/or time lapse images are saved as multi-paged tiff format files.  Most commercially available software can only display the first image in a multi-page tiff format file.  The remaining images, although present in the file, are not displayed.  Tiffsplit4.exe fulfills several functions: 

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 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 tiff (.tif) or bitmap (.bmp) format image files.  tiffsplit4.exe can be run in a Windows 95, 98, NT or 2000 Dos box (DOS 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 normalized extended focus images for each channel along with a RGB overlay view of up to 3 channels.  The -m option generates maximum intensity projections for each channel of a z-series.  Both extended focus and maximum intensity projections reduce the 3-D image into a 2_d overview.  Depth information is lost but a single glance overview of the features in to 3-D data set is produced.  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 (256 levels of intensity).  Contrast expansion can 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.


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

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 multi-paged tiff files may be split with one command.  N.B. items inside [  ] are optional.  Choose one item inside each of the { } pairs.  Command line switches may be supplied in any order.  Supplying not command line arguments will list terse synopsis of the command line switches.

-g  changes images to gray scale.  Without this option images retain the color look up palette supplied by LeicaTCS-NT (except for overlaid 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 colors 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 overlaid 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.  Overlaid 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 overlaid 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.  Overlaid 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  suppresses 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 overlaid 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.  Overlaid 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.

-h  lists a terse help message in the DOS box.  Same as providing no command line arguments.  This is a very useful way to briefly list the function of the command line switches as well as display the version of tiffsplit4.exe.


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, c=channel 3 & d=channel 4). 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 followed by another underscore and the channel letter.  An RGB overlaid 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 normalization can be suppressed by adding -n to the command line.

It should be noted that maximum intensity projections enhance what is brightest in a z-series along the z-axis.  A structure of interest will be obscured be anything brighter along the z-axis (i.e. at the same x-y position) in the z-series stack.   This limitation can be overcome be performing a maximum projection on selected layers of the z-series.  This is better done using a free program called Confocal Assistant.  Maximum projections along any orientation can be made by the general 3-d rendering program VoxBlast.

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.


Starting with a z-series data set file sim.tif   which contains 10 z-levels of two channels of fluorescent images, hence 20 images in total.


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.


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!

Tiffsplit4.exe has existed as many version as well as older version called tiffsplit3.exe, tiffsplit2.exe & tiffsplit.exe.  To avoid running an older version please ensure that all other copies of this program are removed from your computer.   Use the Windows search function (windows-key + F) to help find and delete tiffsplit.exe, tiffsplit4.exe, etc..  To determine which version you invoke type tiffsplit4 without any files or commands.  The terse help output will display the version number at the last line.

Large numbers of files are created duplicating what is already in the original tiff file.  Large volumes of redundant image file may end up choking the available disk space, especially if the original data is large time lapse or z-series files.  Since the original image is stored as a tiff format file one could delete all .bmp files in order to clean up a directory.

Problems may occur when reassigning colors in more creative orders when making overlaid files.  Colors actually assigned may not be the order specified in the -r BGR and similar command line switch.  This is a plain old simple programming bug, which may be eliminated in the future!

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 previously 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. Please let me know about any problems so that they can be suitably dealt with. 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.

Leica will be changing the image format used on the TCS-NT/SP confocal systems in 2000.  We will not upgrade until Summer of 2000.

Please report bugs to mchua<EXPUNGETHIS>@med.unc.edu


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.

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