About this manual Please read first

ImageJ is a public domain Java image processing program inspired by NIH Image for the Macintosh. It runs on
any computer with a Java 1.1 or later virtual machine, either as an online applet or as a downloadable application.
The author, Wayne Rasband ([email protected]), is at the Research Services Branch, National Institute of
Mental Health, Bethesda, Maryland, USA.
The best source of information about ImageJ can be found at the ImageJ homepage (http://rsb.info.nih.gov/ij/) and
by subscribing to the ImageJ mailing list (details on the home page). This manual is meant to be an introduction to
ImageJ for light microscopy a small part of ImageJs repertoire.
ImageJ has a large number of native functions supplemented by an ever increasing number of plugins (optional
extras needing installation). The core functions are described in detail on the ImageJ web site (follow the
Documentations link). A plugin is a file (named *.class) which needs to be in the plugins sub-folder of the
ImageJ folder, otherwise ImageJ will not load it.
In this manual, the native functions are referred to in italicised, black text, the plugins by italicised, dark-blue
text. ImageJ functions can be accessed by keyboard shortcuts, or hotkeys. Some hotkeys are hard-wired in to
ImageJ, while others are user-defined. Unlike most other Windows applications, keyboard shortcuts do not
require the control key to be pressed (for example, in Microsoft Word, the hotkey to copy is Ctrl+C); in
ImageJ, it is just C. Unless you have installed ImageJ from the Wright Cell Imaging Facility website (Manuals
and Software link), the plugins referred to in this manual and some of customised the keyboard shortcuts (the
hotkeys) may not work.
This collection of plugins has merely been collated and organised by me; they have all have been obtained
free from the ImageJ website or elsewhere on the Internet. The credit for this work should go to the
authors of the plugins (See Appendix). Please ensure that they are properly acknowledged in any
publication that their work facilitates. I have attempted to include appropriate citations or contact details
for each author. Let me know of any omissions and I will correct them immediately.
If you are a plugin author and you are not happy with the way your plugin is included or described or
cited, please let me know. I will update this manual as necessary.
This manual is intended to be an introductory overview to get you up and running, and as such, is not exhaustive.
I began working on this manual while employed at the Babraham Institute, Cambridge, UK (address below). I
consider it now to be a work in progress. Please send any comments to:
Tony Collins, Ph.D.
Facility Manager
Wright Cell Imaging Facility
Toronto Western Research Institute
MC 13-407
399 Bathurst Street
Toronto, ON, M5T 2S8
Email: [email protected]
Website: www.uhnresearch.ca/wcif

Table of Contents
1

Installing ImageJ ............................................................................................................................1

1.1

Windows ................................................................................................................................................... 1

1.1.1
1.1.2
1.1.3

1.2
1.3
1.4
1.5

Mac and Linux .......................................................................................................................................... 1

OS2 ............................................................................................................................................................ 1
QuickTime functionality........................................................................................................................... 1
Upgrading ................................................................................................................................................. 1

1.5.1
1.5.2

1.6

Importing Zeiss LSM files........................................................................................................................ 4

Importing Noran SGI file .......................................................................................................................... 4
Importing Biorad PIC files ....................................................................................................................... 4
Importing multiple files from folder........................................................................................................ 4
Importing Multi-RAW sequence from folder .......................................................................................... 5
Importing AVI and MOV files ................................................................................................................... 5
Other Import functions ............................................................................................................................ 5

Saving and Exporting Files ...........................................................................................................6

Intensity vs Time analysis .............................................................................................................7
4.1
4.2
4.3
4.4
4.5

Brightness and Contrast ......................................................................................................................... 7

Getting intensity values from single ROI ............................................................................................... 7
Dynamic intensity vs Time analysis........................................................................................................ 7
Getting intensity values from multiple ROIs .......................................................................................... 8
Obtaining timestamp data ....................................................................................................................... 9

4.5.1
4.5.2
4.5.3

4.6

Pseudo-linescan....................................................................................................................................... 9
Automatic Particle counting.................................................................................................................. 10

5.1.1
5.1.2
5.1.3
5.1.4

5.2

Threshold segmentation................................................................................................................................... 10
Watershed segmentation ................................................................................................................................. 11
Analyse Particles ............................................................................................................................................. 11
Particle tracking ............................................................................................................................................... 11

Manual Counting .................................................................................................................................... 12

5.2.1
5.2.2

Cell Counter ..................................................................................................................................................... 12

Point Picker...................................................................................................................................................... 12

Colour analysis .............................................................................................................................13

6.1

Fluorescence Colocalisation Analysis ................................................................................................. 13

6.1.1
6.1.2

Zeiss LSM.......................................................................................................................................................... 9
Noran ................................................................................................................................................................. 9
Biorad ................................................................................................................................................................ 9

Particle Analysis ...........................................................................................................................10

5.1

About ................................................................................................................................................................. 2
JRE version comparison .................................................................................................................................... 2
Installation of JRE .............................................................................................................................................. 2
Compiling ImageJ plugins with a new JRE......................................................................................................... 3

Importing Image Files ....................................................................................................................4

2.1
2.2
2.3
2.4
2.5
2.6
2.7

3
4

ImageJ core program ......................................................................................................................................... 1

Plugins ............................................................................................................................................................... 2

Java Runtime Environment ..................................................................................................................... 2

1.6.1
1.6.2
1.6.3
1.6.4

Install program files............................................................................................................................................ 1

Update core program ......................................................................................................................................... 1
Set memory allocation ....................................................................................................................................... 1

Colocalisation Coefficients ............................................................................................................................... 13

Highlighting colocalised pixels.......................................................................................................................... 14

Image intensity Processing .........................................................................................................15

7.1
7.2
7.3
7.4
7.5
7.6

Brightness and Contrast ....................................................................................................................... 15

Non-linear contrast stretch ................................................................................................................... 15
Gamma .................................................................................................................................................... 16
Filtering ................................................................................................................................................... 17
Background correction.......................................................................................................................... 18
Flat-field correction................................................................................................................................ 19

7.6.1
7.6.2

7.7

Proper correction ............................................................................................................................................. 19

Pseudo-correction ............................................................................................................................................ 19

Masking unwanted regions ................................................................................................................... 20

7.7.1
7.7.2

Simple masking ............................................................................................................................................... 20

Complex masking ............................................................................................................................................ 20

Colour Image processing ............................................................................................................21

8.1

Merging multi-channel images.............................................................................................................. 21

8.1.1
8.1.2
8.1.3
8.1.4

8.2

Pseudocolour ......................................................................................................................................... 22

Stack-slice manipulations ...........................................................................................................24

9.1
9.2

10

Zeiss LSM multi-channel experiments.............................................................................................................. 21

Biorad multi-channel experiments .................................................................................................................... 21
Interleaved multi-channel experiments ............................................................................................................. 21
Colour merging ................................................................................................................................................ 22

Slice shuffling/removing/Adding .......................................................................................................... 24

Stack dimension manipulations ........................................................................................................... 25

z-Functions ...................................................................................................................................26
10.1 Stack-Projections ................................................................................................................................... 26
10.1.1
10.1.2
10.1.3

10.2
10.3
10.4
10.5
10.6

Volume render ........................................................................................................................................ 27

Surface Render....................................................................................................................................... 27
Axial-sectioning...................................................................................................................................... 28
Orthogonal views ................................................................................................................................... 28
Stereo Pairs and Anaglyphs.................................................................................................................. 29

10.6.1
10.6.2

11

Volume rendered-anaglyphs ............................................................................................................................ 29

Surface rendered-anaglyphs ............................................................................................................................ 30

t-Functions ....................................................................................................................................31
11.1
11.2
11.3
11.4

Correcting for bleaching........................................................................................................................ 31

FF0 ......................................................................................................................................................... 32
Delta-F ..................................................................................................................................................... 33
Surface plotting ...................................................................................................................................... 34

11.4.1
11.4.2

12

Maximum intensity Z-projection:....................................................................................................................... 26

Sobel filter based Focussing ............................................................................................................................ 26
Wavelet-transform based focussing ................................................................................................................. 26

Analyze/Surface plot settings......................................................................................................................... 34

SurfaceJ settings ............................................................................................................................................. 34

Annotating images .......................................................................................................................35

12.1 Scale bar ................................................................................................................................................. 35
12.1.1
12.1.2

Spatial calibration............................................................................................................................................. 35
Adding scale-bar .............................................................................................................................................. 35

12.2 Text and lines ......................................................................................................................................... 36

12.2.1
12.2.2
12.2.3
12.2.4

13
14

Appendix 1: Plugin Authors ........................................................................................................38

Appendix II: Citing ImageJ and Plugins .....................................................................................40
14.1
14.2
14.3
14.4

15
16

Issues with adding text..................................................................................................................................... 36

Adding Text/Line/Box ....................................................................................................................................... 36
Adding timestamps .......................................................................................................................................... 37
Adding event marker to movie.......................................................................................................................... 37

ImageJ..................................................................................................................................................... 40
VolumeJ .................................................................................................................................................. 40
TransformJ.............................................................................................................................................. 40
Extended Focus...................................................................................................................................... 40

Appendix III:I Plugins menu and sub-menus.............................................................................41

Appendix IV: Cut out keyboard toolbar......................................................................................42

ii

INSTALLING IMAGEJ

1.1
Windows
If you already have ImageJ installed, it may be worthwhile uninstalling it (rename the folder) and following the
installation instructions below. This will match your copy of ImageJ to this manual. Extra plugins can be easily
added later.
1.1.1 Install program files
1. Download the WCIF_Imagej_setup.exe file from the Wright Cell Imaging Facilitys website
(http://www.uhnresearch.ca/wcif). Follow the Downloads link.
2. Run the program.
3. A shortcut will be installed on your desktop and in your Start menu.
1.1.2 Update core program
Now upgrade the ImageJ core program (file IJ.JAR) immediately from the ImageJ web site (See Section 1.5.1
below) due to the frequency of upgrades, the version in the setup will be out of date. Get in to the habit of doing
this routinely.
1.1.3 Set memory allocation
Unlike other Windows applications ImageJ will only use the memory allocated to it. By default the WCIF ImageJ
installation assumes a PC with 512Mb RAM, so ImageJ has been allocated 380 Mb by default (a recommended
75% of the total 512Mb).
If your PC does not have 512Mb of RAM then change the allocated memory to equal 75% of total RAM via the
menu command: Edit/Options/Memory. Specifying more than 75% of real RAM results in virtual RAM being
used causing ImageJ to become very slow and unstable. See http://rsb.info.nih.gov/ij/docs/install/.
1.2
Mac and Linux
The WCIF ImageJ installation is for Windows. Mac and Linux users can download ImageJ from
http://rsb.info.nih.gov/ij/download.html and then install the WCIF plugins-only package. This will install the
plugins detailed in this manual and the preferences file (IJ_PREFS.TXT), which will match the menu locations
of the plugins and their shortcuts to this manual.

1.3
OS2
ImageJ will run on OS2; however, for Biorad users running OS2, ImageJ installation is complicated by the 8.3
filename limitation required by the LaserSharp software. Contact me (email tonyc) for help with this.

1.4
QuickTime functionality
When QuickTime is installed with its default settings, Java functionality is not installed with it; this renders the
QuickTime functions of ImageJ non-functional. QuickTime needs to be installed with a custom installation, with
the QuickTime for Java option checked. This option is unchecked by default and is some way down the
installation options list, so keep scrolling until you find it. This requires you to be logged in with administrative
privileges. See http://rsb.info.nih.gov/ij/plugins/qt-install.html.

1.5
Upgrading
1.5.1 ImageJ core program
You do not need to be logged in as an administrator to do this, but ImageJ must be closed. ImageJ upgrades are
very frequent, and major bugs addressed within days so ImageJ itself should be upgraded routinely. You can
check your version of ImageJ by either noting the version that appears below the toolbar at start up, or by finding
the version at the bottom of the ImageJ property list which can be called via the menu command
Plugins/Utilities/ImageJ
properties.
The
latest
version
of
ImageJ
is
available
at:
http://rsb.info.nih.gov/ij/upgrade/.

The file needed is called IJ.JAR. Download the new IJ.JAR to the C:\ImageJ directory and over-write the
existing IJ.JAR. Upgrades are currently being released almost on a weekly basis. Details of the upgrade features
can be found on the News page link on the ImageJ homepage site.
1.5.2 Plugins
The ImageJ/News page also has details about new plugins. These *.class files need to be downloaded to
C:\ImageJ\plugins. ImageJ will load all the plugins in the plugins folder at start up. Plugins saved to the
plugins folder after ImageJ will not be available until ImageJ has been restarted. Other plugins can be found on
the ImageJ/Plugins web-page.

1.6
Java Runtime Environment
1.6.1 About
ImageJ runs in a Java virtual machine which is part of the Java runtime environment (JRE) from Sun. So
long as the computer has the correct JRE, ImageJ will work, independent of the operating system (MAC, Win,
OS2, Linux, SGI, etc.).
The latest and older versions of JRE can be found by following the links on the ImageJ/Links web-page.
Periodically, check to see if the Windows JRE has been upgraded by Sun. Sometimes the upgrade is worthwhile;
sometimes it is not. The Software Development Kit (SDK) also available from Sun alongside the JRE has JRE as
a component as well as other development resources.
1.6.2 JRE version comparison
The ImageJ Windows installation from the ImageJ website contains JRE1.3; the ImageJ from the WCIF website
has JRE1.5 Beta 1.
1.
2.
3.
4.

JRE1.3 is the most stable of the JREs to date and is distributed with the ImageJ Windows installation from the
ImageJ website.
JRE1.4.0 allowed the use of the System-clipboard plugin to copy and paste between ImageJ and other applications
(PowerPoint, CorelDRAW etc.).
JRE1.4.2 introduced a bug that caused ImageJ to crash whenever a measurement was made.
JRE1.5 Beta 1 has larger window-paned Open file dialog boxes making it much easier to find your images. (You
may at this point see strange effects on dialog fonts in Win98.)

JRE1.4 File/Open dialog

JRE1.5 Beta 1 File/Open dialog

1.6.3 Installation of JRE

1. Log in as Administrator for WinXP/NT/2000.
2. Uninstall any existing version of Java Runtime
(Start/Settings/Control Panel/Add-Remove programs or
something like this).
3. Run the new installation file.
4. Select Custom installation.
5. When prompted for the installation directory, click the Change
button and
change it to
C:\IMAGEJ\JRE
6. Then OK all dialogs.
2

Installing SDK rather than JRE will mean that javaw.exe will not be in the expected place. You need move the
contents of the JRE folder installed by JDK to C:\IMAGEJ\JRE.
1.6.4 Compiling ImageJ plugins with a new JRE
The JRE from Sun does not include the tools.jar file needed for
the menu command "Plugins/Compile and Run" to work.
Without this file you will receive the error message right.

The WCIF installation can be used to compile plugins. If you upgrade the JRE the files required for compiling
plugins may also require upgrading. When you try to compile a plugin with an old version of tools.jar you receive
an error message in the Errors log along like this:
Note: sun.tools.javac.Main has been deprecated.
error: Invalid class file format in C:\ImageJ\jre\lib\rt.jar(java/lang/Object.class). The major.minor version '49.0'
is too recent for this tool to understand.
The tools.jar file is distributed in the Sun SDK, not the JRE.
1.
2.
3.
4.
5.

Download and install the SDK. Note the location of the destination folder.
Delete or rename Image/jre.
Locate the jre sub-folder in the SDK folder.
Copy it to the ImageJ folder.
Copy sdk/lib/tools.jar to ImageJ/jre/lib/ext.

IMPORTING IMAGE FILES

ImageJ primarily uses TIFF as the image file format. The menu command File/Save will save in TIFF format.
The menu command File/Open will open TIFF files and import a number of other common file formats (e.g.
JPEG, GIF, BMP, PGM, PNG). These natively supported files can also be opened by drag-and-dropping the file
on to the ImageJ toolbar.
Several more file formats can be imported via ImageJ plugins (e.g. Biorad, Noran, Zeiss, Leica). When you
subsequently save these files within ImageJ they will no longer be in their native format. Bear this in mind; ensure
you do not overwrite original data.
There are further file formats such as PNG, PSD (Photoshop), ICO (Windows icon), PICT, which can be
imported via the menu command File/Import/Jimi Reader.

2.1
Importing Zeiss LSM files
Files acquired on the Zeiss confocal are can be opened directly (with the Handle Extra
File Types plugin installed) via the File/Open menu command, or by dropping them on
the ImageJ toolbar. They can also be imported via the Zeiss LSM Import Panel which is
activated by the menu command File/Import/Zeiss LSM Import panel. This plugin has
the advantage of being able to access extra image information stored with the LSM file, but
it is an extra mouse click.
Images are opened as 8-bit colour images with the no-palette pseudocolour (!) from the
LSM acquisition software. Each channel is imported as a separate image/stack. Lambda
stacks are therefore imported as multiple images, not a single stack. They can be converted
to a stack with the menu command: Image/Stacks/Covert Images to stack.
Once opened, the file information can be accessed and the z/t/lambda information can be irreversibly stamped in
to the images or exported to a text file.

2.2
Importing Noran SGI file
Noran movies can be opened in several ways:
File/Import/Noran movie opens the entire movie as an image stack.
File/Import/Noran Selection allows you to specify a range of frames to be opened as a stack.
The Noran SGI plugins are not bundled with the ImageJ package. To receive them, please contact
[email protected] or their author, Greg Joss, so he can keep track of users. Greg Joss [email protected]
is in the Dept of Biology, Macquarie University, Sydney, Australia.

2.3
Importing Biorad PIC files
Biorad PIC files can be now be imported directly via the menu command File/Open. Just the first frame of a
Biorad stack can be opened via File/Import/Biorad First frame. Experimental information, calibration, and
other useful information can be accessed via Image/Show Info. Biorad PIC files can also be opened by drag-anddropping the file on to the ImageJ toolbar. The PIC file is opened with the same LUT with which it was saved in
the original acquisition software.

2.4
Importing multiple files from folder
Each time point of an experiment acquired with software such as Perkin Elmers UltraVIEW or Scion Images
time lapse macro is saved by the acquisition software as a single TIF file. The experimental sequence can be
imported to ImageJ via the menu command File/Import/Image Sequence.
Locate the directory, click on the first image in the sequence and OK all dialogs. (You may get a couple of error
messages while ImageJ tries to open any non-image files in the experimental directory.) The stack will
interleave the multiple channels you recorded, and can be de-interleaved via Plugins/Stacks Shuffling/Deinterleave.
4

Selected images that are not the same size can be imported as individual images windows using
File/Import/Selected files to open or as a stack with the File/Import/Selected files for stack. Unlike the
File/Import/Image Sequence function, the images need not be of the same dimensions. If memory is limited,
stacks can be opened as Virtual-Stacks with most of the stack remaining on the disk until it is required
File/Import/Disk based stack.

2.5
Importing Multi-RAW sequence from folder
To form an image, ImageJ needs to know the image dimensions, bit-depth, slice number per file and any
extraneous information in the file format (offset and header size). All you really need to tell it is the image
dimension in x and y. These values should be obtainable from the software in which the images were acquired.
Armed with this information follow these steps:
1.
2.
3.

File/Import/Raw
Select experimental directory.
Typical values for the dialog box are:
Image type = 16-bit unsigned
(or 8 bit typically)
width and height as determined earlier
offset = 0, number of image = 1, gap = 0, white is zero = off
Little-endian byte order = on, open all files in folder = on to open all files in folder.
Non-image files will also be opened and may appear as blank images and need deleting: Image/Stacks/Delete
slice. The stack will interleave the multiple channels you recorded, and can be de-interleaved via
Plugins/Stacks Shuffling/Deinterleave.
2.6
Importing AVI and MOV files
There are two plugins which can open uncompressed AVIs and some types of MOV file.
For opening (and writing) QuickTime you need a custom installation of QuickTime to include QT for Java (see
section 1.3). QuickTime movies are then opened via File/Import/QuickTime .
Uncompressed AVIs can be opened via File/Import/AVI.

2.7
Other Import functions
Leica SP- Leica users, please contact me if you could supply a z- and t-series to test SP files with ImageJ.
Olympus Fluoview - available from http://rsb.info.nih.gov/ij/plugins/ucsd.html. Not bundled with the current
download.
Animated GIF - This plugin opens an animated GIF file as an RGB stack. Also opens single GIF images.
IPLab. Allows Windows IPLab files to be opened directly with the File/Open menu command, or drag-anddrop.

SAVING AND EXPORTING FILES

The File/Save (hotkey: S) menu command will save the image as a TIF file. Other
formats are available (see menu image on the right) and can be accessed by
File/Save As.... When the Save or Save as dialog is opened, ImageJ will enter
the image windows name, plus the appropriate file suffix, as the File Name.
ImageJ will not add the suffix back if you delete it while changing the file name. This
may make your file inaccessible from other programs, which require the suffix to
identify the file format.
You can tell this from the name in the image window it should display a suffix after
it has been saved. If you have deleted the file suffix then resaving the file in the
desired format usually restores it.
Animated GIF This plugin saves a stack as an animated GIF. It works on RGB or 8
bit images.
PNG, PICT, XBM, TGA, Photoshop (PSD), XPM or PCX can be saved with
File/Save As/Jimi Writer. Run the plugin and select the desired format from the
drop-down box.
Uncompressed AVI files are exported via either File/Save as/AVI and opened
via File/Save as/AVI... The frame-rate of the exported AVI movie is determined in
the Image/Stacks/Animation Options settings and can be between 0.1 and 100
frames per second (fps).
The AVI format that ImageJ saves is uncompressed. Uncompressed files are large,
but should be playable on any PC/Mac without any decoder issues. There is freeware
available that will convert between AVI types (e.g. RAD Video Tools) but once compressed, ImageJ will no
longer be able to import them (unless you use the RAD video tools to resave them as uncompressed AVIs).
QuickTime MOV files are exported via the menu command File/Save As/QuickTime Export and opened via the
menu command File/Import/QuickTime.
The frame-rate of the final QuickTime movie is set in the dialog that pops up after youve named you *.MOV file.
Compression settings are also set here. Despite this greater flexibility compared to AVI files, AVIs tend to be
more MSWindows friendly and do not require a custom installation of QuickTime (see section 1.4).
Flash MX will import uncompressed AVI files. The frame-rate will then be determined by Flash, not the AVI
movie file.

INTENSITY VS TIME ANALYSIS

4.1
Brightness and Contrast
To improve the visualisation of the image, the displayed brightness and contrast can be
adjusted with Image/Adjust/Brightness-Contrast (hotkey: Shift+C).
The Auto applies an intelligent contrast stretch to the way in which the image is displayed.
The brightness and contrast is adjusted based on an analysis of the image's (or selections)
histogram. If pressed repeatedly, it allows a progressively increasing percentage of pixels to
become saturated.
Reset changes the maximum and minimum back to 0 and 255 for 8-bit images and back
to the maximum and minimum of the images histogram for 16-bit images.
If Auto does not produce a desirable result, select a region of the cell plus some background
with a region-of-interest (ROI) tool, then hit the Auto button again. It will then do a stretch
based on the intensities within the ROI.
Pressing the Apply button permanently changes the actual grey values of the image.
Dont press this button during while analysing image intensity!
If you prefer the image to be displayed as black on white rather than white on black, the
image display can be inverted by the command Image/Color Tables/Invert-LUT. The command
Edit/Invert inverts the pixel values not just the way the image is displayed.

4.2
Getting intensity values from single ROI
If the movie has been opened as a stack, the ROI selected can be analysed with the command: Plugins/Intensity
vs Time Plot (hotkey: 1). This generates a single column of numbers - one slice intensity per row.
The top 4 rows of the column are details of the ROI. This is useful to ensure the same ROI isnt analysed twice
and allow relocation of any interesting ROIs. The details are comprised of x-coordinate, y-coordinate, width and
height of the ROI. If the ROI is a polyline/freehand ROI rather than a square/oval, the details are given as if the
ROI was an oval/square. The (oval)ROI can be restored by entering the details prompted by the Plugins/Restore
ROI command.
The results are displayed in a plot-window with the ROI details in the plot window title. The plot contains the
buttons List, Save, Copy. The Copy button copies the data to the clipboard where it can be pasted into a waiting
Excel sheet. The settings for the copy button can be found under Edit/Options/Plot profile options.
Recommended settings are: Do not save x-values (prevents slice number data being pasted into Excel) and
Autoclose (prevents you having to close analysed plot each time.

4.3
Dynamic intensity vs Time analysis
The plugin Plugins/Stacks Z&T projections/Dynamic ZProfile will update the plot window each time the ROI
selection is moved. This may help locate initiation sites of calcium signals for example.

4.4
Getting intensity values from multiple ROIs
Multiple ROIs can be analysed at once using Bob Doughertys Multi Measure plugin. There is a native ROI
manager function which does a similar job except the results generated are not sorted in to columns. Check
Bobs website for updates: http://www.optinav.com/ImageJplugins/list.htm
The Multi Measure plugin that comes with the installation is v2.
1.
2.

Open t-series.
Its worthwhile generating a reference stack to add the ROIs to. Use the
Image/Stacks/Z-project function (hotkey: Shift+Z). Select the Average option.
3. Rename this image Ref expt name or something memorable.
4. Open Multi-measure plugin (Plugins/ROI/Multi Measure).
5. Select background ROI first of all and to list with the Multi Measure dialog button
Add+Draw <CR> (hotkey: Enter a.k.a. Carriage return). Continue adding cell
ROIs.
6. Once finished selecting ROIs to be analysed in the reference image, save the
reference image to the experiments data folder and then select the stack to be
analysed.
7. Click Multi Measure dialog Multi (not the Measure) button to measure all the
ROIs. Click Yes on Process stack? dialog.
8. Go to Results window. Select menu item Edit/Select All. Then Edit/Copy All.
9. Go to Excel and paste data. With large data sets this can take some time so check youre pasting new data in
and not the last dataset copied from Multi Measure.
10. To copy ROI co-ordinates in to the Excel spreadsheet, ensure there is an empty
row above the intensity data. Got to Multi Measure dialog and click Copy list
button.
11. Go to Excel, select the empty cell above the first data column and then paste the
ROI co-ordinates.
The ROIs can be stored and re-opened later using the Multi Measure dialog button
Save. Save them in the experimental data folder. The ROIs can be opened later
either individually (Multi Measure dialog button Open) or all at once (Multi
Measure dialog button Open All) which opens all the ROIs in a folder.
Oval and rectangular ROIs can also be restored individually from x, y, l, h values using
Plugins/ROI/SpecifyROI, , (hotkey: Shift + G).

ImageJ assumes stacks to be z-series rather than t-series so many functions related to the third-dimension of an
image stack are called z- something e.g. z-axis profile is intensity over time plot.

4.5

Obtaining timestamp data

4.5.1 Zeiss LSM

Once imported via the Zeiss LSM panel, the timestamp data can be extracted with the panels Apply t-stamp
button. This will then ask you if you want the timestamp to be added to the image, or displayed in a text file for
saving/pasting to excel etc.
4.5.2 Noran
In most instances the x-axis data, i.e. time of each frame, can be calculated from acquisition rates and frame
number (e.g. frame 301 acquired with the acquisition rate set to 1 frame per 0.5 seconds was acquired at 25
minutes). However, the acquisition rate is non-linear, in the above experiment frame 301 was actually acquired at
25 minutes 12 seconds. Each frame has stored with it a timestamp, the precise time (in nanoseconds!) that it was
acquired. This information can be extracted from an opened movie.
The movie must be opened as a stack and the timestamps can be extracted with the command: Import/Noran
timestamp (msec) while the Noran Movie is open and selected. The timestamp data appears in the Results
window. To copy data, click with the right mouse button on the windows, select Select All, then right-click
again and select Copy. The timestamp data (accompanied by the movie filename) can then be pasted into Excel.
The Noran SGI plugins are not bundled with the ImageJ package. To receive them, please contact
[email protected] or their author, Greg Joss <[email protected]>, Dept of Biology, Macquarie
University, Sydney, Australia.
4.5.3 Biorad
This can be accessed via the menu command Image/Show Info. Scroll down and it should give the time at
which each slice was acquired. This can be selected, copied in to Excel and the time number obtained by
searching and replacing (Excel menu command Edit/Replace) the text, leaving only the time data. The
elapsed time can then be calculated by subtracting row 1 from all subsequent rows.

4.6
Pseudo-linescan
Linescanning is a mode of acquisition common to many confocal microscopes where a single pixel wide line is
acquired over a period of time instead of the norma1 2-D, x-y image. Usually this allows faster acquisition. The
single pixel wide images over the time course are stacked from left to right to generate a 2-D image (i,e, x-t).
A pseudo-linescan is the generation of a linescan-type x-t plot from a 3-D (x, y, t) timecourse and can be useful
in displaying 3-D data (x, y, t) in 2 dimensions.
A line of interest must be drawn followed by the command:
Image/Stacks/Reslice or keyboard /. It will prompt for the line width.
Enter the width of line you wish to be averaged. A pseudo-linescan stack
will be generated, each slice representing the pseudo-linescan of a singlepixel wide line along the line of interest. To average the pseudo-linescan
stack, select Image/Stacks/Z-Project and select the Average
command. A polyline can be used although this will only allow a single
pixel slice to be made.
This example shows the elementary calcium events preceding a calcium
wave. HeLa cell loaded with the calcium-sensitive fluorophore, Fluo-3 and
imaged whilst responding to application of histamine. A. Frame taken from time-course at the peak of the
calcium-release response. (B) The line of pixels along X-Y was taken and stacked side by side from right-to left
to generate a "pseudo-line scan". This allows visualisation of the progression of the wave from its initiation site.

PARTICLE ANALYSIS

Particle counting can be done automatically if the specimen lends itself to it, i.e. the individual particles can touch
but not too much! If automatic particle counting cannot be done, ImageJ can facilitate manual counting with the
Point Picker or Cell counter plugin.

5.1
Automatic Particle counting
The biggest issue is one referred to as segmentation which is to distinguish the object from the background.
Once the objects has been successfully segmented, they can then be analysed.
RAW

Threshold

Watershed

Analyze Particles

Merge

5.1.1 Threshold segmentation

Automatic particle analysis requires the image to be a binary image i.e. black or white. The software needs to
know exactly where the edges are to perform morphology measurements. A threshold range is set and pixels in
the image whose value lies in this range are converted to black; pixels with values outside this range are converted
to white (or vice-versa depending on what the user requests).
There are several ways to set thresholds. For monochrome images
it is most simply done via thee menu command
Image/Adjust/Threshold. While the threshold is being set, the
pixels within the threshold range are displayed in red. The user
then changes then Apply the threshold and the image will be
converted to a binary image.

For colour images, setting the threshold is a bit more

complicated as each of the red, green and blue channels
require different threshold ranges. The plugin
Plugins/Colour functions/Threshold Colour can be
used to do this most easily. This opens a fairly
complicated and extensive control window. The
simplest way to perfume a colour threshold is to use
one of the selection tools to select one of the objects of
interest, and then click the Sample button in the
Threshold Colour control window. This should
remove the pixels in the image that do not have pixels
of the same colour as those in the selection. The
thresholding can be manually tweaked with scroll bars
in the histogram panes. It may be easier to
conceptualise what you are doing at this point by
switching the Control window from the default Hue,
Saturation and Brightness colour model to red, green
blue model by switching the option buttons at the
bottom of the control window. The image can be
displayed as a binary image with the Threshold
button, and converted to a binary image via the menu command Image/Type/8-bit (?!).
10

5.1.2 Watershed segmentation

Objects in a binary image that are slightly overlapping may be separated using the menu command
Process/Binary/Watershed. This segmentation process is nicely illustrated with this figure from the ImageJ
documentation web pages (http://rsb.info.nih.gov/ij/docs/index.html).

The image first needs to be converted to binary (set via thresholding). The black pixels are then replaced with
grey pixels of an intensity proportional to their distance from a white pixel (i.e. black pixels close to the edge are
light grey, those closer to the middle) are nearer black. This is the Euclidian distance map (EDM). From this it
calculated the centres of the objects the ultimate eroded points (UEPs), i.e. points that are equidistance from the
edges. These points ere then dilated until the meet another black pixel, then a watershed line is drawn.
5.1.3 Analyse Particles
Once the image has been segmented, the menu command Analyze/Analyze particles
can be used to obtain various information regarding particle size and numbers.
Set the minimum size and maximum size to exclude objects that appear in the binary
image that are clearly not objects of interest. Select the Show: Outlines option to
display an image of the detected objects. This can then be merged with the original for
presentation purposes. Invert the outline image and use the menu command
Plugins/Colour functions/Colour merge to add the original and outline image.
The particle analysis can be automated via plugins or macros once the correct
threshold value and particle size range has been determined for your objects of interest.
See the Plugins/Particle Analysis/ Nucleus counter plugin and its source code to
customise it to your images.

5.1.4 Particle tracking

The MTrack2 plugin (Plugins/Particle counting/MTrack2) tracks particles and reports their coordinates at
each time-point and their total path length.

Raw

Background corrected
Smoothed, Binary

MultiTracker
result

Motion paths
of two objects

11

5.2
Manual Counting
You can use the ImageJ toolbar Crosshair (mark and count) tool to count particles. However, a greater degree of
control can be accessed with the Point Picker or Cell counter plugin.
5.2.1 Cell Counter
When then Plugins/Particle counting/Cell
Counter plugin) is run, a new Image
window is generated with the original image
plus four buttons along the bottom (Red type,
Green type, Blue type and Yellow type).
Clicking on a button changes the colour of
the marking tool. The results table keeps a
tally of the number of each of the cell types
marked. Clicking the Results button will
generate a summary of the data.

5.2.2 Point Picker

Plugins/Particle counting/PointPicker. Running this plugin will change the ImageJ toolbar to the PointPicker
toolbar (below).
Apply to single slice/all slices

Add
Crosses

Move
Crosses

Return to ImageJ toolbar

Delete Import/Export
Crosses point coordinates

Image Zoom

Crosses are reversibly overlaid on to the image, incrementally changing colour (this differs from the Crosshair
tool which irreversible stamps a point in the image). With the Point Picker tool, crosses can be moved and
deleted. Once marking is complete, the cross coordinates can be accessed via the
button.
This produces the Point List dialog allowing you to Show, Save or Open the points coordinates.
Clicking Show displays the coordinates in the Results window, where they can be saved or copied to
the system clipboard. Once finished, you can return to the regular ImageJ toolbar by clicking the
button.

12

COLOUR ANALYSIS

6.1
Fluorescence Colocalisation Analysis
One key issue that can confound colocalisation analysis is bleed through. Colocalisation typically involves
determining how much the green and red colours overlap. Therefore it is essential that the green emitting dye does
not contribute to the red signal (typically, red dyes do not emit green fluorescence but this needs to be
experimentally verified). One possible way to avoid bleed-through is to acquire the red and green images
sequentially, rather than simultaneously (as with normal dual channel confocal imaging) and the use of narrow
band emission filters. Single and unlabelled controls must be used to assess bleed-through.
6.1.1 Colocalisation Coefficients
6.1.1.1
Image Correlator plus and Red-Green Correlator plugins
These two plugins are effectively the same, except Image Correlator plus requires two grey-scale images to be
selected and the Red Green Correlator plugin works on the current RGB image.
The plugins generate a 256256 scatter plots plus correlation coefficients. In each scatter plot, the first (typically
red) image component is represented along the x-axis, the second image (typically green) along the y-axis. The
intensity of a given pixel in the first image is used as the x-coordinate of the scatter-plot point and the intensity of
the corresponding pixel in the second image as the y-coordinate.
The intensities of each pixel in the Correlation Plot image represent the frequency of pixels that display those
particular red/green values. Since most of you image will probably be background, the highest frequency of pixels
will have low intensities so the brightest pixels in the scatter plot are in the bottom left hand corner i.e. x~ zero,
y ~ zero. The intensities in the Red-Green correlation plot image represent the actual colour of the pixels in the
image.
Mito-DsRed; ER-EGFP
Pearson's correlation (R)
Overlap coefficient (R)
Nred Ngreen pixels
Colocalisation coefficient for red (Mred)
Colocalisation coefficient for green (Mgreen)

0.34
0.40
0.66
0.96
0.49

TMRE (red) plus Mito-pericam (Green)

Pearson's correlation Rr
Overlap coefficient R
Nred Ngreen pixels
Colocalisation coefficient (red) Mred
Colocalisation coefficient (green) Mgreen

0.93
0.94
0.93
0.99
0.98

13

The Plugins/Stacks-RGB/Red-Green Correlator plugin will take an RGB image, split it and then generate the
correlation plots and data. It will work on the ROI in the original image or the whole image if no ROI is selected.
Both plugins generate various colocalisation coefficients: Pearsons (Rr), Overlap (R) and Colocalisation (M1,
M2) See Manders, E.E.M., Verbeek, F.J. & Aten, J.A. Measurement of co-localisation of objects in dual-colour
confocal images, J. Microscopy, 169, 375-382, (1993). See tutorial sheet Colocalisation for details.
6.1.2 Highlighting colocalised pixels
6.1.2.1
Colocalization plugin
Plugins/Colour functions/Colocalization. This plugin will generate an image of colocalised pixels and also an
image of those colocalised pixels superimposed on a RGB-merge of two 8-bit images. This type of colocalisation
requires users to decide a threshold
value which can introduce bias.
This plugin highlights the colocalised
points of two 8-bits images (or stacks).
The colocalised points will appear white
by default (Display value = 255). The
plugin merges the red and green 8-bit
channels to an RGB image and highlights
the colocalised pixels in white. Pixels are
considered colocalised if their intensities
are higher than the threshold of their
channels (set at 50 by default): and if the
ratio of their intensity is higher than the
ratio setting value (set at 50% by default).
A second image of just the white
colocalised pixels is generated if the
Also show colocalised pixels alone
option is checked.

6.1.2.2
Colocalization finder plugin
Menu command Plugins/Stacks RGB functions/Colocalization finder
This plugin will prompt you for two greyscale images and creates a scatter plot and
a red-green merged image. The pixels
represented by the scatter plot point can
be highlighted by selecting the points with
the rectangular selection tool only. The
32-bit scatter plot is best visualised after
having had its contrast stretched
(Image/Adjust/Brightness&Contrast).

14

IMAGE INTENSITY PROCESSING

7.1
Brightness and Contrast
The Brightness and Contrast window (Hotkey: Shift+C; menu command
Image/Adjust/Brightness and Contrast) will allow you to change the way the image is
displayed. The x-axis of the plot represents the pixel intensities in the image; the y-axis
represents the intensity that they are displayed (bottom of y-axis is display black; top is
display white). Initially, there is a linear relationship between the image intensity and the
display intensity. Increase the minimum value to display your image background as black
(i.e.) zero and decrease the maximum value to display the brightest objects in your image as
white. This is best achieved in a greyscale image by loading the HiLo LUT
(Plugins/LUT/Hi Lo Indicator; hotkey: F1).
The Auto button applies a linear histogram stretch (a.k.a. Normalisation), i.e. it sets the
displayed value to match the maximum and minimum intensity in the image. Clicking the
Auto button again will allow increasing amounts of saturation. By clicking the Apply button
the values in the image are changed to match their displayed value. Do not do this prior to
quantifying intensity values! If you are processing a stack, you will be given the option to
apply this adjustment to the entire stack.
If Auto does not produce a desirable result, select a region of the cell plus some background with an ROI tool,
then hit the Auto button again. It will then do a stretch based on the intensities within the ROI.
If you prefer the movie to be displayed as White and Black rather than Black and White, the image display
can be inverted by the command Image/Color Tables/Invert-LUT. The command Edit/Invert inverts the
pixel values not just the way the image is displayed.

7.2
Non-linear contrast stretch
More control of the brightness and contrast adjustment can be achieved with the
Process/Enhance contrast menu command. Here, when applied to a stack, it applies
the adjustment based on each slices histogram, not just the one currently displayed as is
done with the Brightness and Contrast window.
Equalisation applies a non-linear stretch of the histogram based on the square root of
the intensity (see online guide to image processing:
http://www.dai.ed.ac.uk/HIPR2/histeq.htm).
The Normalize option performs a simple linear stretch on the image in a similar way to the Auto option in the
Brightness and Contrast window, except that when applied to a stack each slice in stack is adjusted differently
based on the slices histogram.

15

7.3
Gamma
This can be though of as a non-linear histogram adjustment. Faint objects can be made more intense without
saturating bright objects (gamma <1). Similarly, medium-intensity objects can be made fainter without dimming
the bright objects (gamma > 1). The intensity of each pixel is raised to the power of the gamma value and then
scaled to 8-bits or the min and max of 16-bit images.
For 8 bit images; New intensity = 255 [(old intensity255) gamma]
Gamma can be adjusted via the Process/Math/Gamma command or the Plugins/Utilities/Gamma Scroll-bar
plugin. The latter will open up a new window copy of your image and you can adjust the gamma with the scroll
bar. Click on Done when you are finished. This is a bit flakey and doesnt react well if you change images in
mid-adjust! This does not work on stacks. You can use the Scroll-bar to determine the desired gamma value on
one slice of your stack, and then apply this gamma value to the stack via the Process/Math/Gamma command.

16

7.4
Filtering
See the online reference: http://www.dai.ed.ac.uk/HIPR2/filtops.htm for a simple explanation of digital filtering.
Filters can be found under the menu item Process/Filters.... Typically use a Radius (pixels) of 1 which
equates to a 33 kernel see online reference.
Mean filter: the pixel is replaced with the average of it and its neighbours within the radius. The menu item
Process/Smooth is a 33 mean filter.
Gaussian filter: The is similar to smoothing but replaces the pixel with a pixel of value proportional to a normal
distribution of its neighbours not explained well, I know, but youve probably skipped the online reference and
you need to read that to understand the way the filter works properly.
Median filter: the pixel is replaced with the median of it and its adjacent neighbours. This removes noise and
preserves boundaries better than simple mean filtering, but can look odd. (The menu item
Process/Noise/Despeckle is a 33 median filter).
Kalman filter: Sophisticated filtering for time-course experiments a sort of weighted running average. Best
used if the sample frequency is higher than the event frequency i.e. slowly occurring events. Otherwise you get
odd, blurry results. Process/Filter/Kalman Stack.
Sigma filter: A modification on the standard mean filter but preserves edges better can be though of as a gentle
smooth. The user specifies the kernel size, the Sigma width and the minimum number of pixels to include. A
Sigma value for the kernel is calculated (based on the variance and mean of the intensities) and only pixels within
this Sigma range (= Sigma the user defined Sigma Width scaling factor) are used to calculate the mean. If there
are too few pixels (exact number set in the user dialog: Minimum number of pixels) in the kernel that are within
the Sigma range then the central pixel which is assumed to be spuriously low or high and the mean of the rest of
the kernel is taken. Increasing the Sigma width and the minimum number of pixels results in increased smoothing
and loss of edges. This plugin is under development, please send me any feedback..
J.S. Lee, Digital Image Smoothing and the Sigma Filter, Computer Vision, Graphics, and Image Processing 24, (1983) p. 255-269.

Raw

Smooth

Gaussian

Median

Sigma

Kalman

17

7.5
Background correction
Background correction can be done in several ways and is facilitated if the grey image has the Plugins/LUT/Hi
Lo indicator (Hotkey: F1) LUT loaded. This displays the zero values blue and the 255 white values red.
If the background is relatively even across the image, it is most simply remove with the Brightness&Contrast
command slowly raise the Minimum value until most of the background is displayed blue. The press the Apply
button to change the grey-values and remove the background.
For uneven background the menu command Process/Subtract background can be used. This menu command
removes uneven background from images using a rolling ball algorithm. The radius should be set to at least the
size of the largest object that is not part of the background. It can also be used to remove background from gels
where the background is white. Running the command several times may produce better results.
RAW

Process/Subtract Background

Once the background has been evened, final

adjustments
can
be
made
with
the
Brightness&Contrast control.

18

7.6

Flat-field correction

7.6.1 Proper correction

This technique is applied to brightfield images. Uneven illumination, dirt/dust on lenses can result in a poor
quality image. This can be corrected by acquiring a flat-field reference image with the same intensity
illumination as the experiment. The flat field image should, ideally, be a field of view of the coverslip without any
cells/debris. This is often not possible with the experimental coverslip, so a fresh coverslip may be used with
approximately the same amount of buffer as the experiment. With fixed-specimens try removing the slide
completely

RAW

Flat field

Corrected

1.
2.

Open both experimental image and flat-field image.

Select all of the flat-field image (hotkey: A) and measure the average intensity (hotkey: M). This value will
appear in the results window and represents your k1 value below.
3. Use the Image Calculator plus plugin (Analyse/Tools/Calculator plus).
4. i1 = experimental image; i2 = flat-field image; k1 = mean flat-field intensity; k2 = 0.
This can also be done using the Process/Image Calculator function and ensuring the 32-bit Result option is
checked. You will then need to adjust the brightness and contrast and change the image to 8-bit.
7.6.2
RAW

Pseudo-correction
Pseudo-flat field

Corrected

Often it is not possible to obtain a flat-field reference image.

However, it is still possible to correct for illumination intensity
(although not small defects such as dust) by making a pseudoflat field image by performing a large-kernel filter on the image
to be corrected. This is particularly useful for DIC images where
there is an intrinsic, and distracting, gradient in illumination.
The menu command Process/Filters/Pseudo-flat field
automates this process. You are prompted to enter the kernel
size for the mean filter; try the default value of 50 first. You can
opt to keep the flat field image open to check whether the kernel
is large enough. The object should not be visible in the flat field
image.

RAW

Corrected

The first RAW image (top) pseudo-flat field

corrected. Here the pseudo-flat field corrects for
the uneven illumination but does not correct the
dust specks. Compare this with the result of a
proper flat-field correction above.

19

7.7
Masking unwanted regions
7.7.1 Simple masking
Draw around the area you want with one of the ROI tools then use: Edit/Clear outside. This will change area
outside the selected region to the background value.

7.7.2 Complex masking

A more sophisticated masking can be done by thresholding the image and subtracting this new binary image
from the original.
1.
2.
3.
4.

5.
6.

Duplicate the image (if its a stack its worthwhile generating a average projection of a few frames).
Threshold this image using the menu command Image/Adjust/Threshold (hotkey Shift+T).
Hit the Auto button and then adjust the sliders until cells are all highlighted red.
Then click Apply. Check the tick box: black foreground, white background. You should now have a
white and black image with your cells black and background white. If you have white cells and black
background, invert the image with Edit/Invert.
This can be smoothed (Process/Smooth) and the black area enlarged slightly with Process/Binary/Dilate
to give a better mask
Using the regular Image calculator Process/Image calculator subtract this black and white mask image
from your original image/stack.

20

COLOUR IMAGE PROCESSING

Images can have colour in two ways: pseudocolour or RGB. A pseudo-coloured image is a single channel, (i.e.
grey) image that has colour ascribed to it via a look up table or LUT (a.k.a. palette, colour table). This is
literally a table of grey values (zero to 256 or 4095 whether 8-bit or 12-bit grey) with accompanying red, green
and blue values. So instead of displaying a grey, the image displays a pixel with a defined amount of each colour.
Differences in colour in the pseudo-coloured image reflect differences in intensity of the object rather than
differences in colour of the specimen that has been imaged. For pseudocolour functions see later.
The colours in RGB images (24-bit, 8-bits for each of the red, green and blue channels) and used to show multichannel images. The colours are designed to reflect genuine colours, i.e. the green in an RGB image reflects green
colour in the specimen, the differences in intensity of the green reflects differences in intensity of green in the
specimen. There are several RGB functions in ImageJ. Native functions can be found in Image/Color and
additional RGB functions can be found in Plugins/Colour functions.
Fluorescence and transmitted light brightfield images can be merged with the Plugins/Colour functions/RGBGrey Merge plugin.

8.1
Merging multi-channel images
Currently, only 24-bit colour images are supported, i.e. 8-bit red, green and blue channels. 48-bit colour image
(i.e. 16-bit red, green and blue) processing is under development, but currently 16-bit grey images need to be
converted to 8-bits before merging. This can be done by adjusting the Brightness&Contrast to the desired level
and then using the menu command Image/Type/8-bit. Using the Plugins/LUT/HiLo indicator LUT can
facilitate setting the brightness and contrast: zero is displayed as blue; saturated-white (255) as red.
The brightness and contrast of each channel is best adjusted prior to merging, however, each channel can be
individually adjusted after merging with the Image/Adjust/Color function. This will not open the control panel
if the Brightness & contrast control is already open.
8.1.1 Zeiss LSM multi-channel experiments
Each channel in a Zeiss LSM file is imported as separate stacks. For single slice/time point, triple channel
experiments this will produce 3 images one for each of red, green and blue. Dual channel z- or t- series are
imported as two separate stacks. To merge the red, green and blue components select Image/Color/RGB merge
and put the appropriate image in the appropriate channel.
8.1.2 Biorad multi-channel experiments
Dual channel, 3-D (i.e. xyz or xyt) experiments are saved as two separate files, one for each channel. Each file
needs to be imported and then the stacks merged with the menu item: Image/Color/RGB merge. Typically, the red
channel should be ***01.pic and the green ***02.pic.
Dual-channel, single frame experiments are saved as single PIC files with two slices. These can be RGB merged
with ImageJ menu: Image/Color/convert stack to RGB. ImageJ assumes that if its a two slice stack, slice 1 is
red and two is green and there is no blue channel.
Biorad dual-channel images acquired with CoMOS software have both channels displayed side by side in a single
slice. The plugin Plugins/Colour Functions/Biorad Aligner will allow these images to be merged. The user can
designate the colour for each half of the image from the drop-down option box. Brightness and crontrast of each
half of the image can be adjusted separately by selecting each half of the image, adjusting and then applying the
Brightness and Contrast change. To facilitate this, the keyboard function key F4 selects the left hand image
panel when pressed once, then the right hand image panel the next time it is pressed.
8.1.3 Interleaved multi-channel experiments
Multi-channel experiments acquired on Noran and Perkin-Elmer systems are imported with the different channels
interleaved, i.e. slice 1 is timepoint1-channel1, slice2 is timepoint1-channel2. The stack needs to be Deinterleaved before it can be RGB-merged. This can be done with Plugins/Stacks-Shuffling/DeInterleave and
entering the number of channels in the dialog (typically 2). The two stacks can then be merged via:
Image/Color/RGB merge.

21

8.1.4 Colour merging

An alternative to the normal Red-Green merge
is to merge the images based on Cyan and
Magenta, or Cyan-Yellow or any other colour
combination.
This can aid visualisation of colocalisation due
to our poor perception of red and green
colours.
The
Plugins/Colour
functions/Colour Merge function will
perform a difference arithmetic processing
on the image stacks you select. This is not
strictly a merge (when cyan and magenta
merge they produce white, not yellow) but
facilitates visualisation of the separate
channels (See Demandolx and Davoust, J. Microscopy, 1997 v185. p21). You can perform a true merge if you
turn off the Difference option.
Run the plugin and select the two images to be merged. Select the desired colours from the drop-down options.
<Current> uses the LUT that the image currently has (this is often the desired LUT). The Difference option
performs a difference arithmetic operation rather than a addition. If the Pre-sub 2 from 1 option is checked
the second image is subtracted from the first prior to merging. This may be useful for merging grey brightfield
images with fluorescence where simple addition can result in washed-out look t the fluorescence image. It can
look unnatural though. Another option may be to darken the brightfield image prior to merging (use menu
command Process/Math/Multiply and use the value 0.5-0.75).

Normal merge

Subtraction of red from grey prior to

merge

Grey image multiplied by 0.75 prior to

normal merge

8.2
Pseudocolour
Judicious use of LUTs can be very useful in highlighting the desired features of an image. The human eye can
only perceive relatively few shades in one scene. Pseudo-colouring images can make the data more visible

Traditional Green LUT

Enhanced Green Hot LUT

Microtubules under nucleus now more apparent

22

Have a play and see which LUT helps illustrates the features in your image.

Montage compiled from a stack generated using the plugin Plugins/LUT/List LUTs.
Different LUTs are available via the menu commands Image/Lookup table and also Plugins/LUT. Custom
LUTs can be made with the Plugins/LUT/LUT panel. Extra LUTs can be found in C:\ImageJ\LUT and can be
applied by highlighting your image and selecting Plugins/LUT/OpenLUT then selecting the required *.lut file.
Opening LUTs via this command rather than other ways (i.e. File/Open or File/Import/LUT) prevents the
default folder from changing to C:\ImageJ\LUT. Custom LUTs can be made and edited using the
Plugins/LUT/LUT panel plugin.

23

STACK-SLICE MANIPULATIONS

9.1
Slice shuffling/removing/Adding
Once imported, the slices in each stack can be manipulated in many ways. See images below for explanation of
some of these functions
Deleting single slice: Image/Stacks/Delete Slice
Deleting a number of slices: Plugins/Stacks - Reducing/Slice remover
Select the slices to remove: Plugins/Stacks- Reducing/Substack maker
Stack to images/Images to stack: Image/Stacks/Stack to images (Images to Stack).
Images to Stack requires images to be the same size Plugins/Stack building/Stack Builder works on images
of different sizes. The sequence in the stack is determined by the order in which they were opened/created.
Montage: Image/Stack/montage Many settings are self explanatory. Ideal for generating a montage of a stack
for a lab book.
Reversing stack: Plugins/Stacks Stack Shuffling/Stack reverser
Concatenate: Plugins/Stacks Building/Concatenator
Stack Combine: Plugins/Stacks Building/Stack Combiner
(De)Interleave: Plugins/Stacks Shuffling/DeInterleave and /Interleave
Stack Inserter: Plugins/Stacks Building/Stack Inserter
Stack Sorter: Plugins/Stacks Shuffling/Stack Sorter. Control the position of individual slices
or groups of slice. Advanced Insert functionality.

B
Stack
Combine

Concatenate

De-Interleave

Interleave

7
6

Images to Stack
Or Stack
Builder

87
65

Stack Inserter
Destination

Stack to Images
Sub-stack
maker

1
Stack reverser

32

23
45
6
78

3
4

Stack Maker

Montage
Montage

Source

24

9.2
Stack dimension manipulations
Images and stacks can be resized and rotated with native functions or the more sophisticated TransformJ set of
plugins from Erik Meijering. These are found in the Plugins/TransformJ menu list.
E. H. W. Meijering, W. J. Niessen, M. A. Viergever, Quantitative Evaluation of Convolution-Based Methods for Medical Image
Interpolation, Medical Image Analysis, vol. 5, no. 2, June 2001, pp. 111-126.

More details can be found on this web-site: http://www.imagescience.org/meijering/software/transformj/

Spatial calibrations are lost so see the section on how to copy calibrations from one image to another with scaling
(12.1.1.1)

25

10

Z-FUNCTIONS

10.1 Stack-Projections
A z-series is generally difficult to represent as a 2-D image for publication purposes. A montage will allow the 3D dataset to be visualised in 2-D, but results in each frame being very small. There are several ways to flatten
the 3D stack.
10.1.1 Maximum intensity Z-projection:
Menu command Image/Stacks/Z-project (hotkey: Shift+Z). Select Maximum Intensity from the drop-down
box. Also look at Plugins/Stacks Z&T projections/Grouped ZProjector which can be used to perform a
frame average.
10.1.2 Sobel filter based Focussing
Menu command: Plugins/Stacks Z&T projections/Stack Focuser plugin uses a Sobel edge filter to calculate
best focus. Try 3 for kernel value in the first instance. Only works on 8- or 16-bit images. It can result in a
pixelly image.
10.1.3 Wavelet-transform based focussing
Daniel Sage, Jesse Berent, Brigitte Forster, Dimitri Van De Ville, Biomedical Imaging Group,
Swiss
Federal
Institute
of
Technology
Lausanne
(EPFL),
Switzerland,
http://bigwww.epfl.ch/demo/edf/index.html.

RAW

Superior, but slower, algorithm compared to the Sobel-filter focussing. Menu command
Plugins/Stacks Z&T projections/ExtendedFocus plugin uses a wavelet transform (a more
sophisticated transform compared to the Fourier transform) to calculate to best focus. Run
plugin and click Fusion button. Works with RGB images. A full description of the plugin and
the can be found at the Authors website. A topology image is also generated. Please cite the
authors papers below if you publish work using this plugin.

For more information see the following papers:

B. Forster, D. Van De Ville, J. Berent, D. Sage, M. Unser , "Extended Depth-of-Focus for MultiChannel Microscopy Images: A Complex Wavelet Approach" Proceedings of the Second 2004 IEEE
International Symposium on Biomedical Imaging: From Nano to Macro (ISBI'04), Arlington VA, USA,
April 15-18, 2004, in press.
J. Berent, B. Forster, D. Van De Ville, D. Sage, M. Unser, "Extended depth-of-focus for color images in
light microscopy using the wavelet transform," Poster at the Autumn Meeting of the Swiss Society of
Extended
Pharmacology and Toxicology, October
27-28, 2003, Basel, Switzerland.

Focus
26

10.2 Volume render

This is a 3D reconstruction method where the object will appear semitransparent.
Select the z-series stack then run the menu command: Image/Stack/3D-Project.
Try these initial settings:
Volume render

1.
2.

3.

4.

Projection Method: Use Brightest point method.

Axis of Rotation: Select y-axis. Theres a bug which causes errors in x-axis
rotation. If you want an x-axis rotation, prior to running the 3D-project
command rotate the stack by 90 (Image/Rotate/Flip 90 right), 3D-project, the
rotate the projection stack back 90 (Image/Rotate/Flip 90 left). Keep an eye
out in the ImageJ/News web page to see if this bug is corrected.
Slice Spacing: This determines you aspect ratio of the stack. Biorad stacks are
internally calibrated and this value should be correct unless you set the wrong
objective in the Biorad software during acquisition.
Interpolate when slice spacing > 0: check this option, although it will slow
down the render so for a large data set it may be worthwhile having this off
while youre sorting out the settings.

10.3 Surface Render

Plugins/VolumeJ/VolumeJ. This is a 3D reconstruction method where the object
surface will appear opaque, giving a more solid look to the object. See:
http://bij.isi.uu.nl/vj_manual.htm and http://bij.isi.uu.nl/vr.htm for updates.

Surface render

6. Set Rotation angle

(try -20 first box to "tip
the volume backwards
slightly).

1. Select Volume stack that

is to be rendered.

7. Ensure Aspect ratio is

correct VolumeJ should
pick up the spatial
calibration of the stack if
it is present.

2. Select
Classifier
(i.e.
render algorithm). Choose
Gradient no index for
greyscale
stacks;
chose
Ramp + index for RGB
stacks.

8. Set Scale as 0.5 for

faster preliminary
renderings. Set it to 1 or
2 for final rendering.

3. Set Classifier threshold as

the intensity of the surface
of the object. This can be
determined
using
the
Image/Adjust/Threshold
command.

9. Click on Render button

to start rendering.
(10.) Click on Stop
renderer if youve made a
mistake!

4. Set classifier deviation.

High values result in blobby
look, low values result in
sharp
edges.
A
good
compromise is 1-2.
Please cite: M.D. Abrmoff, and M.A. Viergever.
Computation and Visualization of Three Dimensional
Motion in the Orbit. IEEE Trans Med Imag., 21 (4), 2002.

27

10.4 Axial-sectioning
This generates a side-view of a stack along a user defined line. Select part of stack to be axially sectioned using
the line ROI tool from the toolbar. Select menu item:
Plugins/Stacks Z&T projections/ReslicePlus.
You will be prompted for the line width. Enter the width of line you wish
to be averaged. An axial-section stack will be generated, each slice
representing the axial section of a single-pixel wide line along the line of
interest. The title of the new image contains the line co-ordinates and the
slice width. The line can be restored using the menu item
Plugins/ROI/SpecifyLine.
To average the pseudo-linescan stack, select Image/Stack/Z projection
and select the Average command. A polyline can be used although this
will only allow a single pixel slice to be made.
You can also reslice your stack using the native function
Image/Stacks/Reslice but this will not return the line coordinates.

10.5 Orthogonal views

This plugin comes from the field of medical
imaging and as such use a lot of terminology that
maybe unfamiliar to light-microscopists. The
plugin:
Plugins/Stacks Z&T projections/OrtView will
allow users to display the xy, x,z and yz views of
a selected point in the image (click on desired
point). After selecting a point a Volume View can
be generated with the Ortview plugins menu
command Options/Volume View. This will
open an ImageJ window with the volume image.
The stack can be smoothed with various
algorithms selected from the Process menu.
One of the algorithms is a 2x2x2 smooth. This
will take in to account adjacent slices and may be
the best way to gently smooth stacks. The
smoothed stack can be sent to an ImageJ window
for subsequent processing, analysis or export via the OrtView menu command File/ExportImages/Export
Transaxial Stack
A fuller description of the plugins capabilities can be found in the OrtView help file (found in the folder:
C:\ImageJ\Plugins\Stacks Z&T functions\OrtViewHelp.txt). The plugin is easy to use but limited to grey-scale
images (8- or 16-bit) due to its nuclear medicine heritage.

28

10.6 Stereo Pairs and Anaglyphs

10.6.1 Volume rendered-anaglyphs
These plugins are under development and are at an early stage please send feedback to
[email protected].
This is an adaptation of the methods described at: http://rsb.info.nih.gov/nih-image/more-docs/confocals.html.
The menu command Plugins/Stacks Z&T projections/Stereo pair plugin will create a side by side stereo pair
from your stack and also a red-green anaglyph. The plugin will let you specify which of the stereo pair projections
you want and also the angle of rotation between the pairs. Typical values would be 6-9.

Stereo Pair

Red-Green Anaglyph

Red-Cyan Anaglyph

29

10.6.2
Surface rendered-anaglyphs
Surface stereo pairs are simply made with VolumeJs stereo
pair button. This generates a stereo pair with a 5 difference.

These two images can be merged to form an anaglyph using

either the menu command Plugin/Colour Functions/Colour
merge or Image/Color/RGB merge.
The left eye needs to be red; the right eye cyan or green.

Surface rendered anaglyph movies can be constructed using

VolumeJ following an approach from Harvey Karten and Joel
Sheffield.
1.

Surface render your z-series (see Section 10.3 Surface

Render) with a Cine frame increment of 6-9.

2.

Duplicate the Surface rendered movie.

3.

Delete the top slice from the original movie.

4.

Delete the last slice from the duplicate movie.

5.

Merge the stacks using the menu command Image/Color/RGB merge.

6.

Assign the duplicate stack as red and the original stack as green. For Red-Green anaglyphs set blue to be
none; for red-cyan anaglyphs set the blue channel to be the same as the green.

30

11

T-FUNCTIONS

11.1 Correcting for bleaching

Often, during acquisition of a time-course, the fluorophore may bleach and the
intensity of the image be reduced. This can make it harder to discern events at the
end of the sequence. A stretch in contrast at time point 1 may not be adequate at time
point 100. The process of bleaching and decrease in image intensity can be fitted
with a mono-exponential decay, although it often follows a bi-exponential. A monoexponential decay is described by the equation:
Corrected intensity = (Intensity at time t) exp

-kt

k = decay constant

If you know the decay constant (k), you can use the Process/Filters/Bleach Correction plugin. Run the plugin
and enter the decay constant. It may be worth performing a background subtraction prior to running the plugin.
Note that the plugin is expecting the k value to be per-slice rather than per-second, per-minute etc.
RAW time course

Bleach corrected time course

The k value can be calculated in ImageJ by:

1. Make sure the Edit/Options/Plot profile options setting Do not save X-values is off.
2. Select a region of the cell or the whole image and plot the decay using the menu command Image/Stack/Plot
Z-axis profile
3. Click the Copy button of the plot window.
4. Open the curve fitting dialog: Analyze/Tools/Curve fitting.
5. Delete the default data and paste in your copied data.
6. Select Exponential from the option list and click the Fit button.
7. In the Log window, scroll down to find the value labelled as -b, this is your k value.
Since bleaching is often not mono-exponential, quantification of fluorescence intensities after bleach correction is
not possible. This plugin should really only be used to enhance time-course movies for presentation rather than
quantification.
Another way to compensate for bleaching is to use the menu item Process/Enhance
Contrast. This method is quicker to implement than the proper bleach correction above
and can be useful for correcting for fluorophore bleaching during a movie if the intensity
of the fluorophore is changing only because of bleaching. Check the Process Entire
Stack option and the plugin will scan through the stack applying Brightness and the
contrast adjustment selected on each slice based on each slices histogram (see section
7.2). The intensity values are adjusted so that quantitative intensity measurements are no
longer possible.
Again, use this function to enhance movies for presentation, not quantification.

31

11.2 FF0

There are several drawbacks with the use of single wavelength fluorescent probes; some include uneven
fluorescence intensity (F) due to cell thickness and cell to cell variation in loading. These can be largely corrected
for by normalising fluorescence against resting fluorescence i.e. F0. This does not correct for bleaching and dye
loss during the experiment.
First ensure the image is properly background corrected (7.5 Background correction).
1.

Generate an F0 image by averaging the first few frames of the stack. This can be done with the
Image/Stacks/Z-project menu item, using the Average drop-down box option. Select Start slice as 1
and Stop Slice as 5-10 depending on how many frames you wish to average. Rename the new z-projected
image (Image/Rename) Fzero.
2. Open the Image Calculator Plus via Process/Image Calculator.
3. Image 1 = Original stack
4. Image 2 = image Fzero
5. Operation = Divide
6. Select 32-bit Result and Create New Window
7. Rename this result window (Image/Rename) FdivF0.
8. The maximum and minimum intensity in the stack can be determined with the Analyze/Tools/Stack
Histogram plugin.
9. Using the Brightness and Contrast (See section 4.1) dialog set the maximum and minimum to these values.
10. Convert the 32-bit stack to 8-bit (Image/Type/8-bit).
11. The Images in FdivF0 will probably have a noisy background between the cells and need to be cleaned up.
Using your original stack, create a mask of the cells. This is done by first thresholding the original; stack
(Image/Adjust/Threshold). Hit the Auto button and then adjust the sliders until cells are all highlighted red.
Then click Apply. Check the tick box: black foreground, white background. You should now have a
white and black image with your cells black and background white. If you have white cells and black
background, invert the image with Edit/Invert.
12. Using the Image calculator Process/Image calculator to subtract this black and white stack from your
FdivF0 stack.
13. Pseudocolour to taste.
This process could relatively easily be automated via a plugin or macro. However, due to multiple user-defined
steps (i.e. how many frames for the Z-projection, the thresholding of the original stack for the mask, whether its
better to correct for bleaching before thresholding the stack) I have not done so.

ImageJ assumes stacks to be z-series rather than t-series so many functions related to the third-dimension of an
image stack are called z- something e.g. z-axis profile is an intensity vs time plot.
32

11.3 Delta-F

Raw

Delta F (up)

Rapid frame-to-frame changes in intensity (e.g. calcium puffs or TMRE depolarisations) can best be
illustrated by performing subtracting each frame from the previous/next.
Plugins/ Stacks - Z&T projections/Delta F up
For increases in intensity (e.g. a calcium puff) the calculation is [Frame(n+1)-Framen].
Plugins/ Stacks - Z&T projections/Delta F down
For drops in intensity (e.g. TMRE plus irradiation induced mitochondrial depolarisations) the calculation is
[Framen - Frame(n+1)].
Note: The plugin generates a second result stack. For large memory consuming stacks, run the plugin with the
Alt-key down. If the plugin is run with the Alt-key down, the calculation is made on the original stack.
Plugin may also be useful to clean up time courses prior to motion tracking.

33

11.4 Surface plotting

Surface plots can be generated in two ways: via the menu command Analyze/Surface plot or via the plugin
Plugins/VolumeJ/SurfaceJ. These functions will surface-plot movies as well as single frame images. Ensure the
features youre interested in are Contrast stretched optimally. This can be done using a Max intensity
projection on the stack. Getting the max and min pixel intensities and applying these to the stack. Remember, do
not perform intensity analysis on images that have had their contrast stretched.
11.4.1 Analyze/Surface plot settings
When this function is selected, a dialog will appear. Try the settings below first and play
with them to optimise the surface plot. The LUT of the final surface plot is taken from the
LUT of the image.
11.4.2 SurfaceJ settings
You can surface plot either a single frame or a movie. Surface rendering is a slow process
so it is best to pick a frame from the movie that shows the features youre trying to
demonstrate. Duplicate this (Ctrl+D) and use it as a test image to get the best settings for surface plotting your
movie.
Select the image to be rendered with the Source image(s) drop down box.
The options with three text-entry boxes along side represent x, y and z values.
Rotate(): value of -20 in the first (x-axis) box gives a good 3D effect. Play with this.
Scale. Set to 0.5 speeds things along also when adjusting the settings and set to 2 or more for the final surface
plotting.
Aspect /: Surface plot is can be stretched in height by increasing the z-axis aspect (i.e. the third) box.
Although 1 is usually OK if youve stretched the contrast properly. This will not affect the
pseudocolour of the peaks which is determined by the pixel intensity.
Index LUT: by default the plot will have the spectrum LUT. Change this drop-down box to Load custom
and youll be prompted to locate a desired LUT when you start the surface-plot
Gaussian Smoothing value of 2-4. This slows rendering down but gives smoother surface plot.
Rendering surface plot: starts surface plotting.

34

12 ANNOTATING IMAGES
12.1 Scale bar
Scale bars should be present on all publication/presentation images/movies. Its worth putting them in sooner
rather than later. Choose a standard scale bar size for all your images if possible to avoid confusion.
12.1.1 Spatial calibration
The spatial calibration of an image can be found in ImageJ via the menu item Image/Properties. It can be
manually (re)applied here also. The units um are automatically changed to m.
Biorad PIC, Noran MOV and Zeiss LSM confocal image files (Leica and Olympus?) are internally calibrated
based on the zoom and objective magnification settings. Some experimental file formats are not calibrated (e.g.
Perkin Elmer) and some exported image files (e.g. Zeiss zvis exported as TIFFs) lose their calibration
information. This is sloppy programming on part of the microscope manufacturers and it should be the pointedly
drawn to their attention.
If the files have no calibration, you need acquire images of a stage micrometer with the same settings (camera
binning, objective, confocal zoom and frame size etc.) as your experiment. The calibration can be applied using
the Plugins/Spatial calibration/Set scale command (see below). The spatial calibration can be found (and set)
in the Image/Properties dialog. Also look at the Microscope scale plugin which can be customised to apply
spatial calibrations from a drop-down box of objectives (http://rsb.info.nih.gov/ij/plugins/microscope-scale.html).
Many image processing functions can cause the spatial calibration to be lost let me or Wayne Rasband
know if you find one and well try to fix it. If the final image has lost its calibration open original file and access
the spatial calibration via: Image/Properties. Copy these values to the Image/Properties of your final,
processed image. Replacing the word micron with um will automatically convert to m.
The spatial calibration can be reapplied in three ways: manually via the Image/Properties dialog; via the
Plugins/Spatial calibration/Copy Pixel Size plugin; or via the Plugins/Spatial calibration/Set scale command
12.1.1.1

Copy-calibration
1.
2.
3.

4.
5.

12.1.1.2

Re-open the original image with the calibration

Run the Plugins/Spatial Calibration/Copy
Pixel Size plugin.
In the from drop-down box select the
original image; in the to box, select your
processed image.
Click the OK button.
Another dialog will open giving you the
opportunity to enter a value by which the
receiving image has been scaled.

Set Scale
If you know the size of a feature (previously applied scale bar for instance) you
can use this command to apply a calibration.
1. Using the line selection tool, draw a ling along the length of the
feature/scale bar.
2. Run the menu command Plugins/Spatial calibration/Set scale1.
3. Enter the dimensions of the object/scale bar in the known distance box
and set the units in the Unit of length box.
4. Do not check Global unless you wish all your images to have this
calibration! Click OK.
12.1.2 Adding scale-bar
1

This simple plugin simply points to the native menu command Analyze/Set scale and is
included here to keep the spatial calibration functions in one place.

Use the line ROI tool to draw a line of approximately the desired length and
position.
Run Scale bar dialog via Plugins/Spatial calibration/Add Scale bar2. Change the
Width in *** value to something sensible i.e. 5, 10, 50 etc. Stick to something
consistent. Height will determine how many pixels fat your bar will be. If youve
used the Green LUT then the white (i.e. grey level 255) scale-bar will appear
green. Convert the image from 8-bit colour to RGB with Image/Type/RGB color
before adding the scale bar to get a white scale-bar on a pseudocoloured image (see
section 6.9.1). Check the Label all slices box to add bar to the whole stack.

12.2 Text and lines

12.2.1 Issues with adding text
Adding text to single frame images should be done in CorelDRAW or some such application so the text does not
become part of the image and can therefore be edited/moved/removed at any time. Adding text to an image/movie
is irreversible so should not be done on RAW data. Images can be copied to and pasted from the system clipboard
between programs using the Plugins/Utilities/Copy-Paste to clipboard panel (hot key: Shift+V) only available
using JRE 1.4 and above. Whole images are copied, not selections. To copy a selection, crop the original image
before copying, or duplicate the selection..
If you find the image is not pasting accurately, try using the receiving applications Edit/Paste Special function
and select Device independent bitmap.
Firstly, save your images before adding text. Many things can go wrong here and often cannot be corrected so its
best to have a saved version ready to reopen.
Text colour is set via the colour picker button on the tool bar. Double-click on it to pull up the palette and select a
colour usually black or white.
Pseudocoloured images should be converted to RGB prior to adding
time stamps. The text in the counter is anti-aliased. This means that
instead of being completely white in colour, some pixels on the curved
edges of letters are averaged with neighbouring pixels to be grey, giving a smoother appearance to the text e.g.
The text looks better, however, it means that some of the texts pixels are not grey-scale value 255 and so some
LUTs will have them displayed as yellow/orange/green thereby loosing the nice smoothing
effect of the antialiasing e.g. with the Hot Green Blue LUT the counter on frame 1 looks like:
If you use the red or blue LUT then the text will appear red or blue even though you selected
the text to be white via the colour picker.
To avoid this, convert the pseudocoloured image from its current format (i.e. 8-bit color) to
an RGB image with Image/Type/RGB color before adding the text. e.g. in the example
above:
12.2.2 Adding Text/Line/Box
12.2.2.1 Adding to a single image
Adding a text line or box to a single frame should be done in a package such as CorelDRAW, Adobe Illustrator,
Deneba Canvas etc. (see 6.7.1). However, there are times when lines and boxes are required, e.g. to reference an
analysed/sectioned region.

This plugin simply points to the native menu command Analyze/Tools/Scale bar. It is included here just to
keep the spatial calibration functions in one place.
36

Drawing of lines/boxes is achieved by first selecting the desired line/box in the image with the line/Rectangle ROI
tool from the toolbar or defining the line/box via Plugins/Specify ROI or Plugins/Specify Line. The
line/box can be resized here to the desired size/shape with the small square handles on the ROI. The line/box can
then be stamped in to the image by right clicking on the image and selecting Draw. This will add the box/line to
a single frame, the colour determined by the by the color picker toolbar button and the width in pixels defined in
Edit/Options/Line width.
Adding text is done by the toolbars text button. Click the toolbar; click the image; write the desired text. The font
size is set by double clicking the toolbar-text button, the colour determined by the by the color picker toolbar
button. Antialiasing can be turned on/off via the menu item Edit/Options/Miscellaneous (although this option
does not affect the `Stamper plugins which have antialiasing on permanently). The text is stamped in to the image
by right clicking and selecting Draw.
12.2.2.2 Adding to movie-stack
Drawing/writing on a stack only adds the drawn object to the current slice*. If you want the text/box/line to be
added to an entire movie stack you need to create a new single frame image add the text/box/line to this and
then add this single frame to the movie stack. This is best done by:
1. Duplicate a frame from the movie stack (Image/Duplicate and disable Duplicate entire stack option).
2. Rename (Image/Rename) text/box/line image
3. Select the whole image (Edit/Selection/Select all or Shift-A)
4. Edit/Clear
5. Go to the movie stack and select the desired text/box/line (see 6.7.2.1 above).
6. Go to the text/box/line image
7. Restore the selection by Edit/Selection/Restore or Shift-E.
8. Draw the selection (right click the selection and select Draw)
9. Use the Image Calculator (Image/Calculator) to add the text/box/line image to the movie image.
*This may become redundant as ImageJ develops. Keep a look out in the ImageJ/News web page.
12.2.3 Adding timestamps
1. Draw a rectangular ROI at the location and of the approximate size of the desired counter. The text will, by
default be of the same height as the ROI and aligned to its left edge. Ensure it is not too close to the edge of
the image. This will result in a bar underneath the counter e.g.
2. Run the Plugins/Movies/Time Stamper.
3. Enter appropriate settings. The X and Y location and font size are taken from the ROI and can be changed at
this point.
Remember, the bigger the text, the smoother it will look and the easier the timestamp will be to read. Bigger is
better. It may be worth changing it to a consistent value for all your movies.
Also, bear in mind that the interval neednt be time". The Time Stamper plugin also can be used to add z-axis
depths to z-stacks and angle of rotation to rotating volume movies ( = Alt+247; = Alt+230).
12.2.4 Adding event marker to movie
The Plugins/Movies/Event Stamper plugin can be used to add text to a movie for a designated number of
frames, e.g. Agonist ON. This is simply done by running the Event Stamper plugin setting the desired text in the
Text box, enter start frame and end frame and click OK. The text size and location is taken from the current
ROI and can be changed once the dialog is opened.

37

13 APPENDIX 1: PLUGIN AUTHORS

Animated GIF
Kevin Weiner, FM Software, W. Rasband., 5

Image Correlator plus

Wayne Rasband, Tony Collins, 13

Animated Gif
Ryan Raz (http://rraz.ca)

Interleave
J. Anthony Parker, MD PhD
([email protected]), 24

AVI
Daniel Marsh ([email protected]), 5
Calculator plus
Wayne Rasdband, 19
Cell Counter
Kurt De Vos ([email protected]), 12
Colocalization
Pierre Bourdoncle ([email protected]),
Institut Jacques Monod, Service Imagerie, Paris, 14
Colocalization finder
Christophe Laummonerie, Jerome Mutterer, Institut
de Biologie Moleculaire des Plantes, Strasbourg,
France., 14
Concatenator
Wayne Rasband, 24
Copy Pixel Size
J. Anthony Parker, MD PhD
([email protected]), 35
Copy-Paste to clipboard
Amrit Aneja and Wayne Rasband, 36
Cyan Magenta Merge
Tony Collins, Wayne Rasband, 22
Deinterleave
Russell Kincaid ([email protected]), Tony Collins, 4
DeInterleave
Tony Collins, Russel Kincaid, 24
Delta F down
Tony Collins, 33
Delta F up
Tony Collins, 33
Disk based stack
Wayne Rasband, 5
Dynamic ZProfile
Kevin (Gali) Baler (gliblr at yahoo.com) and Wayne
Rasband, 7
ExtendedFocus
Jesse Berent, Brigitte Forster, Dimitri Van De Ville,
Daniel Sage & Michael Unser, 26
Grouped ZProjector
Charlie Holly, caholly at colby.edu, 26

IPLab reader
Wayne Rasband (wsr at nih.gov), 5
Jimi Reader
Wayne Rasband and Ulf Dittmer (udittmer at
mac.com), 4
Jimi Writer
Jeffrey Kuhn ([email protected]) The
University of Texas at Austin, 6
Kalman filter
Christopher Philip Mauer
([email protected]), 17
Leica SP
Nico Stuurman ([email protected])
and Wayne Rasband, 5
List LUTs
G. Landini.
http://www.dentistry.bham.ac.uk/landinig/software/s
oftware.html
LUT panel
Patrick Pirrotte & Jerome Mutterer (jerome.mutterer
at ibmp-ulp.u-strasbg.fr, http://www-ibmp.ustrasbg.fr/sg/microscopie/methods/lut/lut.htm )
Microscope scale
Paul Baggethun ([email protected]),
35
MTrack2
Nico Stuurman ([email protected]), 11
Multi Measure
Wayne Rasband, Bob Dougherty, 8
Noran movie
Greg Joss ([email protected]), 4
Noran Selection
Greg Joss ([email protected]), 4
Noran timestamp (msec)
Greg Joss <[email protected]>, 9
OpenLUT, 23
OrtView
Paulo Bonetto ([email protected]), 28
PointPicker
Philippe Thvenaz ([email protected]), 12

Handle Extra File Types

Gregory Jefferis (jefferis at Stanford.edu), 4
38

Process/Filters/Bleach Correction
Tony Collins, Wayne Rasband, 31

Stack Builder
Nicolas Roggli, Wayne Rasband, 24

Process/Filters/Pseudo-flat field
Tony Collins, 19

Stack Combiner
Wayne Rasband, 24

QuickTime Export
Wayne Rasband, 6

Stack Focuser
Michael Umorin ([email protected]),
26

QuickTime
Wayne Rasband (wsr at nih.gov), Robert Dougherty
(www.optinav.com) Jeff Hardin (jdhardin at
facstaff.wisc.edu), 5
Wayne Rasband (wsr at nih.gov), Robert Dougherty
(www.optinav.com), Jeff Hardin (jdhardin at
facstaff.wisc.edu), 6

Stack Histogram
Wayne Rasband, 32
Stack Inserter
Wayne Rasband, 24
Stack Maker, 24

Red Green Correlator

Wayne Rasband, Tony Collins, 13

Stack reverser
Wayne Rasband, 24

Red-Green Correlator
Wayne Rasband, Tony Collins, 14

Stack Sorter
Bob Dougherty ([email protected]), 24

ReslicePlus
Tony Collins, Wayne Rasband, 28

Stereo pair
Tony Collins, 29

RGB Grey Merge

Eugene Tkachenko ([email protected]) &
Justin D. Pearlman, 21

Substack maker
Anthony Padua (padua001 at mc.duke.edu) Daniel
Barboriak, MD (barbo013 at mc.duke.edu)
Neuroradiology Duke University Medical Center,
24

Selected files for stack

Albert Cardona
[email protected], 5
Selected files to open
Wayne Rasband, 5

SurfaceJ
Michael Abrmoff, http://bij.isi.uu.nl/

Slice remover
Wayne Rasband, 24

Threshold Colour
G. Landini.
http://www.dentistry.bham.ac.uk/landinig/software/s
oftware.html

Specify Line
Wayne Rasband, 36

Time Stamper
Wayne Rasband, Tony Collins, 37

Specify ROI
Jeffrey Kuhn ([email protected]), The
University of Texas at Austin, Anthony Padua
([email protected]), Duke University
Medical Center, Department of Radiology, 36

TransformJ
Erik Meijering, 25

SpecifyLine
Wayne Rasband, 28
SpecifyROI
Jeffrey Kuhn ([email protected]), The
University of Texas at Austin, Anthony Padua
([email protected]), Duke University
Medical Center, Department of Radiology, 8

VolumeJ
M. Abrmoff, http://bij.isi.uu.nl/
Zeiss LSM Import panel
Patrick Pirrotte, Yannick Krempp, and Jerome
Mutterer ([email protected]),
4

39

14 APPENDIX II: CITING IMAGEJ AND PLUGINS

14.1 ImageJ
Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 19972004.

14.2 VolumeJ
Abramoff, M. D. and Viergever, M. A. (2002). Computation and visualization of three-dimensional soft tissue
motion in the orbit. IEEE Trans.Med.Imaging 21, 296-304. [PUBMED].

14.3 TransformJ
Meijering, E. H., Niessen, W. J., and Viergever, M. A. (2001). Quantitative evaluation of convolution-based
methods for medical image interpolation. Med.Image Anal. 5, 111-126. [PUBMED].

14.4 Extended Focus

Extended Depth-of-Focus for Multi-Channel Microscopy Images: A Complex Wavelet Approach B. Forster, D.
Van De Ville, J. Berent, D. Sage, M. Unser Proceedings of the Second 2004 IEEE International Symposium on
Biomedical Imaging: From Nano to Macro (ISBI'04), Arlington VA, USA, in press.

40

15 APPENDIX III:I PLUGINS MENU AND SUB-MENUS

41

16

APPENDIX IV: CUT OUT KEYBOARD TOOLBAR

42