October 19, 1999


tiffsplit3.exe - is a utility program that extracts viewable and editable images from tiff images 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 my 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, z-series and 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.  Tiffsplit3.exe fulfills several functions: 1. splits multipaged tiff format files into individual tiff or bitmap files. 2. generates an extended focus image for each channel along with an overlayed RGB image of the extended focus channels.  3. 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) or removes green/blue under/overload indicator pixels by converting them into black or white.

Tiffsplit3 has several command line options that direct its operation and enhance its function.  By default tiffsplit3.exe splits multi-page tiff image files into separate tiff files for each channel and creates an information file 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", into a series of tiff (.tif) or bitmap (.bmp) format image files.  Tiffsplit3.exe can be run in a Windows 95, 98 or WindowsNT dos box.  By default images are split into individual tiff files (.tif).  The -b option directs the creation of separate files in the bitmap format (.bmp).  The -e option generates normalized extended focus images for each channel along with a RGB overlay view of up to 3 channels.  Extended focus images are always created as bitmap format files.  The -b and -e options may be combined.  In all operation of tiffsplit3.exe the original tiff format file is left intact, and output files overwrite existing files of the same name without warning.


tiffsplit3 file1 [ file2 [ file3 ... ] ] [ -g ] [ -c ]  [ -b ]  [ -e ] 
                [ - r {RGBN}{RGBN}[RGBN] ]  [ -p path ]  [ -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 tiffsplit3 will split each one independently into respective series of files.   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  remove green/blue under/over load indicator colors from output tif and bitmap files including the extended focus images of each channel.

-b  outputs files in bitmap format (.bmp) instead of 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 save the same name as the source file with an underscore and letter denoting the channel, another underscore and 'xf', e.g. myosin_a_xf.  Overlayed stacks would be called myosin_ovr.bmp.  Extended focus files are always created in the bitmap image file format.

-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.

-p path specify path (directory and/or drive) for output files.

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

-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.


tiffsplit3 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) are 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 index number is also added.  The number of digits in the index number depends on the number of pages in the tiff file per channel.  Leading zeros are added to the index number to aid natural 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).

tiffsplit3 will also, optionally, generate extended focus images from z-series stacks.  An extended focus 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, channel letter (a=channel 1, b=channel 2, etc.), underscore and xf.  e.g. myosin.tif will create the extended focus files myosin_a_xf.bmp & myosin_b_xf.bmp.  An RGB overlayed image of the extended focus channels 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 "_ovr.bmp".  E.g. mysosin_ovr.bmp.  Single section images will also generate an RGB overlay file along with intensity normalized images from each channel.

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. Please note that "save as" with the export option does save parametric information as an ".exp" file (readable as text) along with each image channel as separate tif files.


tiffsplit3 myosin.tif: generates individual tiff files for each image creating myosin_a01.tif, myosin_a02.tif ... and myosin.inf.

tiffsplit3 *.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.

tiffsplit3 *.tif  -b -g  : splits all valid tiff files into individual grayscale bitmap files.

tiffsplit3 *.tif  -b -c  : splits all valid tiff files into individual grayscale bitmap files and suppresses green/blue under/over load colors.

tiffsplit3 *.tif  -l  : lists matching TIFF image files found in the current directory.  No split file is generated.


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
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


This program uses the arcane DOS prompt style 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!

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.  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.

tiffsplit3.exe is a 32 bit command line program and will not run on DOS 6.2x and earlier or Windows 3.11 and earlier.

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

Be careful when applying tiffspit3.exe with wild card expansion more than once in a directory.  It will create split files with long index numbers from the already split 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 late in 1999.  We will not upgrade until the first half of 2000.

Please report bugs to mchuaEXPUNGETHIS@med.unc.edu


A future version of tiffsplit3.exe may make separate RGB overlays of each individual z-series level.  But for now use tiffsplit3 file -b & bmovrlay.exe for making red/green overlays of each level in a z-series.