Landmark Reference Manual

Landmark Reference Manual

The following description will tell you what how to use the 2D image sample Landmark program to create and edit sets of landmark coordinates between a reference sample (Rsample) and another sample (Sample). For N samples, normally one is defined as the Rsample and the remaining N-1 samples as Samples. These landmark sets are generated in N-1 sessions of using this program. It also discusses the startup options and how to run the program. Figure 1 shows a screen view of the initial Landmark reporting window.

Landmark report window

Figure 1. Screen view of the Landmark program Report Window interface This screen shot shows the initial Landmark Report Window graphical user interface available after the program starts. There are a number of pull-down menus (
File, Edit, View, and Help) as well as pushbuttons at the bottom of the window. Edit Options lets you change the startup options. The Landmark button causes the interactive Landmark Editor window to pop up. When you are finished editing a landmark set, pressing the Done button will save the data set in the landmark database file. You can also do these operations from the menus.

1. Introduction

Landmark is an open source Java 2D sample Landmarking program for creating landmark sets for samples paired with a reference sample (Rsample) into the Open2Dprot database. It is part of the Open2Dprot project ( Landmark is similar to and derived from an earlier landmark program in the GELLAB-II 2D gel spot pairing program landmark described in the GELLAB-II reference manual. Landmark has been generalized to other types of samples besides 2D gels. While the original program was written in C, Landmark is written in Java, uses XML input and output files and has an Java-based graphical user interface. This initial open-source Landmark program code could be used as the basis for more advance spot pairing methods. Currently the Landmark file is landmark.xml and is stored in the project "project"/xml/ directory.

The program is run interactively (-gui). After the spot pairing is finished, the user has the option of interactively examining the paired spot data overlayed on the original sample images. The user may also modify the input switch options and save the new options in a "" file in the current project directory when they exit so that the last used options may be used as the default switch options in subsequent running of Landmark.

Project directory structure for Open2Dprot and Landmark

All Open2Dprot programs assume a project directory structure. This must exist for the program to proceed. You can either create the structure prior to running any of the programs or you can create it on the fly using the -projDir:user-project-directory. It will lookup and/or create the following sub-directories inside of theuser-project-directory.

    batch/ directory holding temporary batch files - [NOT USED by Landmark]
    cache/ directory holding temporary CSD cache files - [NOT USED by Landmark]
    ppx/ directory holding your original gel input files
    rdbms/ directory holding CSD database RDBMS files - [NOT USED by Landmark]
    tmp/ directory holding generated sample image files    
    xml/ directory that holds  Accession DB, landmark DB,
         SSF sample spot-list files, and SPF samples paired-spot-list files

The use of these directories is discussed in the rest of this document.

Landmark database file

The sample and reference samples to be paired is specified by its sample name using the -sample and -rsample command line switches. It can also be specified at run time using the (File | Open Rsample | ...) and (File | Open Sample | ...) menu commands.

The application looks up the sample in the accession database (in xml/accession.xml or as specified using the -accessionFile switch) and gets additional information about the sample. The Open2Dprot pipeline module is used for entering samples into the accession database.

The images associated with the samples can be used with the landmark editor. These images may be in TIFF (.tif, .tiff), JPEG (.jpg), GIF (.gif), or PPX (.ppx GELLAB-II) format. TIFF images may be 8-bits/pixel through 16-bits/pixel, whereas JPEG, GIF, and PPX are 8-bit images. Gray values in the image files have black as 0. This is mapped to 0 for white and the maximum pixel value for black.

The input sample image files, if any, are kept in the user-project-directory/ppx/ sub-directory. This database directory structure is consistent with and is used by the other Open2Dprot analysis pipeline programs.

2. Running Landmark and specifying parameter options via the command line

The program is run interactively (-gui) with a graphical user interface (GUI). The user may also modify the input switch options and save the new options in a "" file in the current project directory so that it may be used as the default switch options in subsequent running of the program. [Status: the file is not enabled.] All options including the input reference sample and other sample to be paired are specified via GNU/Unix style switches on the command line (-switch{optional ':parameters'} and its negation as -noswitch). However, if GUI mode is used, you can interactively specify the switches and their options. It is assigned previously by software that generated the SSF spot lists.

Local Folders and files created and used by Landmark

When Landmark is first started, it will check for the following folders and files in the installation directory and create them if they can not be found.

Normally the project directory is setup prior to running Landmark and it expects the data to be in the following directories. You can also use the -projDir:user-project-directory switch to specify a (possibly new) project directory.

Landmark command-line arguments switch usage

The command line arguements usage is:

     Landmark -rsample:Reference-sample -sample:sample [< optional switches >]
The complete
list of switches is given later in this manual and as well as some examples of typical sets of switches. The user defined default switches may be specified as a resource string '' file saved in the project directory. For example:

    Landmark -rsample:gel-HM-19 -sample:gel-HM-071 -project:demo/ -gui

Options wizard window for setting the command line switches

If you invoke the Edit options button in the Report window (or from the Edit menu), it will popup an options wizard shown in Figure 2 to let you set or change the switch options and then to save these as the new default switch options. The default is saved in the file when you exit program.

Landmark options window

Figure 2. Screen view of the popup options wizard window for setting the command line switches, parameter and specifying input samples to be paired. All of the switches are available in the scrollable window. Switches are checked if they are enabled and if the switch requires a value, the current value is shown in the data entry window to its right. On the right there may be several threshold sliders for the upper sizing values for several parameters. In the middle, are several Browse buttons to use for specifying a different samples (-rsample: and -sample:), and directories. Clicking on any switch will show a short help message associated with that switch at the top of the window. Pressing the Set new default button will pass the new options values back to Landmark. Note: for this to take effect, you must exit and then restart Landmark. Then to use them, press the Pair spots button in the main Report Window.

Updating Landmark from the Open2Dprot Web server using -update switch

As new versions of Landmark are developed and put on the Web server, a more efficient way of updating your version is to use the -update commands. There are four options:
    -update:program      to update the program jar file
    -update:demo         to update the demonstration files
    -update:doc          to update the documentation files
    -update:all          to update all of the above
After updating the program, it should be exited and restarted for the new program to take effect.

Increasing the allowable memory used by Landmark

If you are working with very large images that require a lot of memory, you might want to increase the memory available at startup.

If you are using the Windows Landmark.exe file or clicking on the Landmark.jar file, you can't change the default startup memory.

If you are using the Landmark.jar in a script using the java interpreter as in the following example which uses the -Xmx256M (specifying using 256Mbytes at startup). Change 256 to a larger size if you want to increase startup memory.

   java -Xmx256M -jar Landmark.jar {additional command line args}

3. Command and Report Window - the command center

Landmark is designed to be used with the graphical user interface (GUI) which creates a Report Window that captures a report of the spot pairing output as well as additional output directed to it by the user. There are a set of pull-down menus as well as a set of buttons for often used functions.

All logged output is sent to the report window in a scrollable text window that may be saved or used for cut and paste operations. A set of command buttons at the bottom of the window are replicates of commands in the menus, but are easier to access. They include the following functions:

4. Pull-down menus in the Landmark Report Window

The menu bar a the top of the Report Window (see Figure 1) contains four menus. There are also some command buttons at the bottom of the window for convenience that are also menu commands.
  1. File menu - to open the samples to be paired and other file operations.
  2. Edit menu - to change invoke various editing and other options including command line switches, computing window, and memory size
  3. View menu - to view Landmark data from the Landmark database.
  4. Help menu - popup Web browser documentation on the Landmark program.

Menu notation

In the following menus, selections that are sub-menus are indicated by a 'Indicates that the
menu has a submenu'. Selections prefaced with a 'Checkbox on' and indicate 'Checkbox off' indicate that the command is a checkbox that is enabled and disabled respectively. Selections prefaced with a 'Radio button on' and indicate 'Radio button off' indicate that the command is a multiple choice "radio button" that is enabled and disabled respectively, and that only one member of the group is allowed to be on at a time. The default values set for an initial database are shown in the menus. Selections that are not currently available will be grayed out in the menus of the running program. The command short-cut notation C-key means to hold the Control key and then press the specified key.

4.1 File menu

These commands are used to open the samples to be paired and other file operations. The current menus and the menu commands (non-working commands have a '*' prefix) are listed below. You can use either the "Edit options" button to popup the Options Window editor to change the input samples or the (File menu | Open Rsample) and (File menu | Open Sample) commands.

4.2 Edit menu

These commands are used to change various defaults. These are saved when you save the state and when you exit the program.

4.3 View menu

This menu contains commands to inspect Landmark database data.

4.4 Help menu

These commands are used to invoke popup Web browser documentation on Landmark. Some of the commands will load local documentation in the the GUI report window.

The documentation is kept on the Internet at Normally, these help commands should pop up a Web browser that directly points to the Landmark Web page. If your browser is not configured correctly, it may not be able to be launched directly from the Landmark program. Instead, just go to the Web site with your Web browser and look up the information there.

5. The popup Landmark Editor Window

Landmark popup editor window

Figure 3. Screen view of the popup Landmark Editor Window. This lets you create or edit landmarks defined to be common spot positions between the two samples. One of the samples is the Rsample (on the left) and the sample to be landmarked with it on the right (Sample). You select the (Rsample, Sample) in the main Landmark Report Window (see Figure 1). The editor window shows four images. The unmagnified view of Rsample and Sample are on the lower row of images and the magnified corresponding images are on the top row. There is a text area at the top for messages to the user. There is a set of pull-down menus at the top
File, View, Edit, and Help. A row of controls on the bottom helps speed up landmarking. There is a 1X to 6X zoom on the left that only zooms the upper images. After you have clicked on corresponding spots in the Rsample and Sample (lower images), select the Flicker checkbox (with (C-F) short-cut) to alternately display the upper Rsample and Sample images in the upper right image. You can use this to verify the spots are probably the same between the two samples. The flicker rate is determined by the delay scrollers at the bottom. The Add LM button (with (C-A) short-cut) will add these two selected coordinates as a new landmark spot. Similarly, by first selecting a landmark spot with the mouse and then pressing the Delete LM button (with (C-D) short-cut) it will delete this landmark from the list. See the detailed menu commands below for other information on landmarking usage.

The menu bar a the top of the Report Window (see Figure 1) contains four menus. There are also some command buttons at the bottom of the window for convenience that are also menu commands.

  1. File menu - to save the landmark set and other related operations.
  2. View menu - to change Landmark editor viewing options.
  3. Edit menu - to edit landmarks
  4. Help menu - popup Web browser documentation on the Landmark Editor window

5.1 Editor File menu

5.2 Editor View menu

5.3 Editor Edit menu

5.4 Editor Help menu

6. Downloading, installing and running Landmark

The installation packages are available for download from the SourceForge Files mirror. Look for the most recent release named "". These releases include the program (both as Windows .exe file and a .jar file), required jar libraries, demo data, Windows batch and Unix shell scripts. Download the zip file and put the contents where you want to install the program. Note that there is a Landmark.exe (for Windows program). You might make a short-cut to this to use in more easily starting the program. Alternatively, you can use the sample .bat and .sh scripts to run the program explicitly via the java interpreter. Note that this method assumes that you have Java installed on your computer and that it is at least JDK (Java Development Kit) or JRE (Java Runtime Environment) version 1.5.0. If you don't have this, you can download the latest version free from the Website.

There are several ways to run the program. On Windows, you can start Landmark by clicking on the startup icon shown in Figure 6 below. For Unix systems including MacOS-X, you can start Landmark from the command line by running the Landmark.jar file. If your computer is setup to execute jar files, just type the jar file. In both systems, you can specify additional command line arguments in Windows .bat and unix .sh scripts (see demo examples below.

Landmark startup icon

Figure 6. Startup icon for Landmark. Clicking on the icon starts Landmark. To start Landmark, click on the startup icon shown in Figure 6 below - or you can run the demo-Landmark.bat script. For Unix systems including MacOS-X, you can start Landmark from the command line by clicking on the Landmark.jar file or using the script. These two scripts run the program in batch but use a GUI. You could make short-cuts (Windows) or symbolic-links in Unix to make it easier to start.

6.1 Requirements: minimum hardware and software requirements

A Windows PC, MacIntosh with MacOS-X, a Linux computer or a Sun Solaris computer having a display resolution of at least 1024x768. We find that a 1024x768 is adequate, but a 1280x1024 screen size much better since you can also see the Popup Report window, Options window, and Landmark, ROI or Calibration window at the same time. At least 30 Mb of memory available for the application is required and more is desirable for comparing large images or performing transforms. If there is not enough memory, it will be unable to load the images, the transforms may crash the program or other problems may occur.

An Internet connection is required to download the program from the Open2Dprot Landmark Web site. New versions of the program and associated demo data will become available on this Web site and can be uploaded to your computer using the various (File | Update | ...) menu commands. If you have obtained the installer software that someone else downloaded and gave to you, then you do not need the Internet connection to install the program. We currently distribute Landmark so that it uses up to 256Mb. See discussion on increasing memory.

6.2 Files included in the download

The following files are packaged in the distribution you install. you can periodically a (File | Update from Web server | ... program) menu command to update the files from the Web server.

7. List of the command line switches

The command line usage is:

    Landmark -rsample:reference-sample -sample:sample [< optional switches >]
where the order of arguments is not relevant. In the following list, items in bold are specific values which must be used (e.g., for -inputFormat:{X | T}, whereas variable values in italics indicate that a numeric value for that variable should be used. Some switches have several alternate fixed choices in which case this indicated as a list of bolded items inside of a set of '{...}' with '|' separating the items. You must pick one of the items and do not include the '{}' brackets. Also, do NOT include any extra spaces in the arguments of the switch - it will be counted as if it were another switch.

Command line switches

-accessionFile:accFile to overide the default accession
      database file.  (Default is -noaccessionFile:accession.xml).

-backupDatabases makes backups of the Landmark and landmark databases "+
      "if they are edited. (Default is -commutativeLMS).

-commutativeLMS lets you search the landmark database for sample pairs
      where the Rsample and Sample are swapped. (Default is

-debugBits:bits,optLandmarkNbr dumps various conditional
      debugging parameters onto the report window.  The 'bits' are the
      debug bits specified as either octal or decimal and enable
      particular debugging output if the program was compiled with
      debugging enabled.  (Default is -nodebug).

-default sets the default switches to a specific configuration:+
         -inputFormat:X -projDir:demo/,
      This disables -demo if it was set. (Default -nodefault).

-demo sets the default switches and sample input sample to a specific 
      configuration. This may be overriden by turning off the -demo
      switch in the Options Wizard.

-dtd adds the XML DTD file (Open2Dprot-SPF.dtd) in the output XML
      if -spfFormat:X is set. (Default is -nodtd).
-gui to start the spot pairing with a popup Graphical User Interface
      rather than in batch mode.  Landmark does not run in batch
      mode. This captures messages from Landmark.  You can then cut
      and paste the results or save it to a text file.  The GUI is
      also used to change the switch options, re-run the spot pairing
      and view images after each analysis.  (Default is -gui).

-inputFormat: defines the input formats for 
      the Landmark database file accMode). Where mode is: 'F'
      for full tab-delimited data that includes 2 files: the list of
      spots, parameters and statistics. The 'T' for tab-delimited
      (.txt) data, and 'X' for XML (.xml) data. (Default is 

-landmarkFile:lmsFile to overide the default landmark
      database file.  (Default is -nolandmarkFile:landmark.xml).

-projDir:alternate project directory path to specify the
       project directory to use instead of the default 'demo/' file in
       the installation directory. (Default is -noprojDir).

-propertiesFile:alternate '' file to specify
       the alternate startup properties file to use instead of the
       default '' file. (Default is

-rsample: is name of the reference sample.

-sample: is the name of the Sample to be paired with the

-timer enables a timer to capture processing times for each step.
       (Default is -notimer).

-update:{all | program | demo | doc} specifies that all of the
       Landmark files, the program jar files, the documentation files
       or the demonstration files should be updated from the
       Open2Dprot Web server.  The program should be exited and
       restarted after updating the program for this to take
       effect. (Default is -noupdate).

-usage prints all of the switches. (Default is -nousage).

7.1 Examples of some typical sets of switches

The following shows a few examples of useful combinations of command
line switches.

Any case-independent switch may be negated by preceeding it with
a 'no' eg. '-notimer'.

The command line syntax used to invoke it is:

     Landmark [< opt.-switches >]

The following examples using switches might be useful:     

     Landmark  -gui -project:demo/
  # Start Landmark and load the Landmark database from the demo project.

     Landmark -rsample:gel-HM-019 -sample:gel-HM-071 -gui -project:demo/
  # Start Landmark with two samples already in the Landmark database

8. Demonstrations


8.1 Examples - samples of screen shots

To give the flavor of running the spot pairing program, we provide a few screen shots of the graphical user interfaces and some images generated by the program.

You can these images in the list below or view all of the screen shots in a single Web page.

8.2 Example - output of the Report Window for ...

The following Report Window output was generate for the images in the above example.

8.3 Examples - using command line processing for batch

It is possible to run the Landmark from the command line in your operating system. We give two examples doing this. The first example shows a script for the Microsoft Windows batch (.bat) file for processing 3 images with the same reference image demo-Landmark.bat file (available on the Files Mirror. The second example shows the same commands in a shell script for a Unix operating system (Linux, MacOS, Solaris, etc.) is available at from the Files mirror at

The data for these scripts is in the demo/ directory available with the installation which also includes the scripts. Alternatively, you can download the demo data from the Files Mirror as Demo.Z.

The installation packages are available from the Files mirror under the Landmark releases.

8.3.1 Examples - batch processing under Microsoft Windows

REM File: demo-Landmark.bat - interactively define landmark spots for each of 3 samples 
REM when paired with the reference sample.
REM This example assumes that all .jar files listed below and demo/ directory are
REM in the current directory. Modify for other situations.
REM The JDK should be installed and version 1.4 or later is required.
REM You can download the latest JDK from
REM The files needed are listed below:
REM JAR files required and mentioned in manifest:
REM      xml-apis.jar xercesImpl.jar jai_codec.jar jai_core.jar O2Plib.jar
REM demo image files:
REM      demo/ppx/gel-HM-019 (Reference sample)
REM      demo/ppx/gel-HM-071
REM      demo/ppx/gel-HM-087
REM      demo/ppx/gel-HM-096
REM Accession database file is in:
REM      demo/xml/accession.xml
REM Landmark database file is in:
REM      demo/xml/landmark.xml
REM Sample Spot-list files (SSF) are in:
REM      demo/xml/gel-HM-019-SSF.xml
REM      demo/xml/gel-HM-071-SSF.xml
REM      demo/xml/gel-HM-087-SSF.xml
REM      demo/xml/gel-HM-096-SSF.xml
REM Generated images are saved in:
REM      demo/tmp/
REM P. Lemkin $Date$ 
echo "demo-Landmark.bat"

date /T 
java -Xmx256M -jar Landmark.jar -demo -project:demo\ -sample:gel-HM-071 -rsample:gel-HM-019
java -Xmx256M -jar Landmark.jar -demo -project:demo\ -sample:gel-HM-087 -rsample:gel-HM-019
java -Xmx256M -jar Landmark.jar -demo -project:demo\ -sample:gel-HM-096 -rsample:gel-HM-019

echo "-- Finished landmarking the samples ---"
date /T 

8.3.2 Examples - batch processing under Unix (or MacOS-X)

Because java is relatively operating system independent, the same java command lines are used with the "\" changed to "/", "REM" changed to "#", and "DATE/T" to "date" from Windows to Unix script and file path conventions.

# File: - interactively define landmark spots for each of 3 
# samples when paired with the reference sample.
# This example assumes that all .jar files listed below and demo/ directory are
# in the current directory. Modify for other situations.
# JAR files required and mentioned in manifest:
#     xml-apis.jar xercesImpl.jar jai_codec.jar jai_core.jar O2Plib.jar
# The files needed are listed below:
# JAR files required and mentioned in manifest:
#     xml-apis.jar xercesImpl.jar jai_codec.jar jai_core.jar O2Plib.jar
# demo image files:
#      demo/ppx/gel-HM-019 (Reference sample)
#      demo/ppx/gel-HM-071
#      demo/ppx/gel-HM-087
#      demo/ppx/gel-HM-096
# Accession database file is in:
#      demo/xml/accession.xml
# Landmark database file is in:
#      demo/xml/landmark.xml
# Sample Spot-list files (SSF) are in:
#      demo/xml/gel-HM-019-SSF.xml
#      demo/xml/gel-HM-071-SSF.xml
#      demo/xml/gel-HM-087-SSF.xml
#      demo/xml/gel-HM-096-SSF.xml
# Generated images are saved in:
#      demo/tmp/
# P. Lemkin $Date$ 

echo ""


java -Xmx256M -jar Landmark.jar -demo -project:demo/ -sample:gel-HM-071 -rsample:gel-HM-019
java -Xmx256M -jar Landmark.jar -demo -project:demo/ -sample:gel-HM-087 -rsample:gel-HM-019
java -Xmx256M -jar Landmark.jar -demo -project:demo/ -sample:gel-HM-096 -rsample:gel-HM-019

echo "-- Finished landmarking the samples ---"


9. Landmark References

These papers (a subset of the GELLAB-II papers), reference the GELLAB-II spot pairing program. The Open2Dprot Java-language Landmark program was other Open2Dprot programs and concepts were used from the old GELLAB-II C-language program as well as from code from the MAExplorer and Flicker projects. This program will be replaced with a full XML editor that will make use of the new MIAPE standard. New Java code was added as well. Although Landmark has been enhanced in many ways, the basic algorithm is similar so these papers may be useful for more details on the algorithm.

  1. Lipkin, L.E., Lemkin, P.F. (1980) Database techniques for multiple PAGE (2D gel) analysis. Clinical Chemistry 26, 1403-1413.

  2. Lemkin, P., Lipkin, L. (1981) GELLAB: A computer system for 2D gel electrophoresis analysis. I. Segmentation and preliminaries. Computers in Biomedical Research 14, 272-297.

  3. Lemkin, P., Lipkin, L. (1981) GELLAB: A computer system for 2D gel electrophoresis analysis. II. Pairing Spots. Computers in Biomedical Research 14, 355-380.

  4. Lester, E.P., Lemkin, P.F., Lipkin, L.E. (1981) New Dimensions in Protein Analysis - 2D Gels Coming of Age Through Image Processing, Invited paper, Analytical Chemistry 53, 390A-397A.

  5. Lemkin, P.F., Lipkin, L.E. (1983) Database Techniques for 2D Electrophoretic Gel Analysis, in Computing in Biological Science, Elsevier/North-Holland, M. Geisow, A. Barrett (eds), 181-226.

  6. Lemkin, P.F., Lipkin, L.E., Lester, E.P. (1982) Extensions to the GELLAB 2D Electrophoresis Gel Analysis System. Paper given at "Clinical Applications of 2D Electrophoresis", Mayo Clinic, Nov. 15-18, 1981. Clinical Chemistry 840-849.

  7. Lemkin, P.F., Lester, E.P. (1989) Database and Search Techniques for 2D Gel Protein Data: A Comparison of Paradigms For Exploratory Data Analysis and Prospects for Biological Modeling, Electrophoresis 10(2): 122-140.

  8. Lemkin, P.F. (1989) GELLAB-II, A workstation based 2D electrophoresis gel analysis system, in proceedings of Two-Dimensional Electrophoresis T. Endler, S.Hanash (Eds), Vienna Austria, Nov 8-11, 1988, VCH Press, W.Germany. pp 53-57.

  9. Lemkin, P.F., Rogan, P. (1991) Automatic Detection of noisy spots in two-dimensional Southern Blots, Applied and Theoretical Electrophoresis 2, 141-149.

  10. Myrick, J.E., Lemkin, P.F., Robinson, M.K., Upton, K.M. (1993) Comparison of the BioImage Visage(TM) 2,000 and the GELLAB-II two-dimensional electrophoretic analysis systems. Applied and Theoretical Electrophoresis 3, 335-346.

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         Revised: 05/19/2006