ImageJ Basics

ImageJ Basics
- open source public domain Java image processing and analysis program inspired
by NIH Image for the Macintosh.
- can be used everywhere with Java (Linux, Mac OS 9, Mac OS X and Windows),
-handles several data types and file formats, has an IP toolkit to develop new
applets and applications, among other features.
-handles Java plugins and macro language (for more details, see presentation
about Image J plugins)
Advantage: Growing numbers of users participate in writing plug-ins for
ImageJ and Fiji.
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Index
-Installing ImageJ (windows) and plugins p. 3 - 4
-Memory settings p. 5
-Menu and Tools overview p. 6
-File menu: Opening, importing, saving files p. 7 - 8
-Image menu and Image types p. 9 - 11
-Image visualisation/information p. 12
RGB channel splitting/merging/overlays p. 13
Stacks p. 14 – 15
-Image Adjustments
Brightness/Contrast p. 16
Threshold p. 17
Image Crop/Transform/Rotate/Revert p. 18
-Image quantification
Calibrating image size/ Scale bars p. 19
Histogram and line profile p. 20
ImageJ Region measurements p. 21-22
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Installation
Different editions of ImageJ exist:
32bit and 64bit
1. Image J homepage for software and plugin downloads:
http://rsbweb.nih.gov/ij/
Wiki: http://imagejdocu.tudor.lu/
Must have java.exe installed to run ImageJ (available with IJ)
2. For a version with pre-installed plugins:
The MBF ImageJ collection of plugins and macros
http://www.macbiophotonics.ca/imagej/installing_imagej.htm
3. Fiji is a similar software based on ImageJ:
http://pacific.mpi-cbg.de/wiki/index.php/Main_Page
Obtaining Image J Version information and updating the software:
> Help > About ImageJ - version information
> Help > Update ImageJ
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Plugins & Macros
Plugins are additional software modules or code which provide the ability to perform
specific tasks. Writing macros using the ImageJ macro language allows you to
string a series of commands together to perform batch operations. These can also be
converted to plugins.
‣Install plugins if required:
Download plugins and Save them in ImageJ Plugin folder (program files)
Restart ImageJ: Plugins automatically get placed in the Plugins menu
> There is a list of available plugins here:
http://rsb.info.nih.gov/ij/plugins/index.html
> There is a plugins collection preorganised in folders available at:
http://rsb.info.nih.gov/ij/plugins/collection.html
> Many more are available on the website and from other users. Some people have
developed their own collections of plugins and bundled them together. McMaster
Biophotonics Institute has a great collection for microscopy:
http://rsb.info.nih.gov/ij/plugins/mbf-collection.html
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ImageJ memory
‣Set high memory for optimal use
no more than 70% of max Ram
‣Edit > Options > Memory
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ImageJ menus and tool bars
Menu bar
(Biophotonics edition)
Tool bar
Status bar
Region/Measurement tools
Image visualisation
and adjustments
Switch to alternative Tool bar themes.
Very useful in original ImageJ.
Biophotonics edition (shown here) already
starts up with a useful Tool bar.
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File menu: Opening, importing, saving files
‣ Drag and drop file to ImageJ menubar to open file
‣ Open
microscope image formats: .lei .lsm
many image and video formats: .avi .tif
‣ Import
-image sequence…
Open and manage stacks/movies
-many file types and text files/results
‣ Save movies as .avi as compressed
jpeg
Drastically reduces file size without
quality loss (for presentations for ex.)
‣ Save images as .tif
A JPEG image degrades each time it
is opened, edited and resaved.
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Importing OME BIO LOCI & metadata
http://www.loci.wisc.edu/software/bio- formats
informs you on types of compatible formats
‣ Open Bio-formats Importer Plugin
-to import a wide range of formats
-to select meta data to display
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Image Menu
Working with different image types and adjustments can be accessed via the Image Menu
or via image adjustment buttons on the second half of the tool bar
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Image type: 8, 16 bit, RGB
Binary image:
For this type of code, each pixel is either black or white because one bit can
code for one pixel (0 = black, 1 = white).
1 pixel = 1 bit of memory
Image in gray levels: 8bit or 16bit
If we code each pixel on 2 bits we would have 2 2 = 4 possibilities (black, dark
gray, light gray, white). But this image would have very limited levels.
So in general we use 8 or 16bits:
2 8 = 256 possibilities (0 - 255 grey levels) 8 bits = 1 octet (1 byte)
2 16 = 4096 possibilities (0 - 4095 grey levels) 16 bits = 2 octets
Multiply number of pixels by number of bits to get memory:
8 bit image: 1000 pixels = 1000 octets = 1 kbyte
Quantification requires 16bit images (bigger window)
Tif images don’t have 12 bit (usually converted to 16)
Acquisition detectors usually only detect in 12 bit!
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Image type: greyscale LUTs, RGB
‣Pseudocolour in greyscale:
8bit and 12bit greyscale images can be visualised in colour:
Use LookUp tables (LUTs): Assigns a range of colour to the grey levels
> Either use LUT button on startup tool bar or switch to LUT tool bar by
pressing on
(Biophotonics edition)
The following tool bar appears:
‣Colour image (RGB):
an overlap of 3 pixels 8bit (24 bit total)
It’s an additive principle of colours:
We can obtain a certain colour by adding 3 primary colours in various
proportions: 256x256x256 = more than 16 million colours.
Different type of colour coding: RGB, CYMK.
RGB: each colour is coded on 1 octet (8bits). Each pixel is a combination of 3
octets (3 x 8 = 24 bits total with Red = 0-255, Green = 0-255, Blue = 0-255.
1 pixel = 3 octets = 24 bits
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Image information & Visualisation
Status Bar = information for cursor
position (x,y) and intensity value.
Or memory usage
Top of Image = information:
Channels
Image size (um or pixels)
Image type (bits)
Image memory
Depending on image dimensions,
Scroll bars appear at the bottom
to scroll across
c: channels
z: stack slices
t: time frames
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RGB channel splitting/merging/overlays
‣Image > Color > Merge (overlay)/Split channels
Press once: Merges channels of an RGB stack to one RGB file
Press twice: Splits into 3 individual channels
Press again: Merges channels back with options as to which channel goes where. Overlay
possible with transmitted light images by using the Gray channel. Do not create
composite.
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Stacks…
‣Image > Stacks
You need to have a series (time, z, x-y) of images which can be built into a stack. Opeining
images as a numbered sequence will automatically create a stack.
Compatible with Metamorph stacks.
Measuring and analysis tools can be often used to apply to all images in a stack
Convert Images to Stack: converts a set of 2D images that you have opened into a stack.
Animate: animates the images in a stack at a rate up to 100 frames per second.
Convert Stack to Images: splits the stack into individual images.
Next Slice/Previous Slice: browsing images can be done using the > and < keys. The
number of the current slice and the total number of slices are displayed in the title bar.
You can also use the slider bar in the stack window.
Z Project: simple projection algorithms designed to render 3D images into 2D
projections, allows volume rendering, useful for visualizing the internal structures of 3D
images.
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…Stacks
For immediate maximum intensity projection (MIP) use this button on the tool bar:
‣Image > Stack > 3D Project: Allows you to project the stack and then rotate it.
‣Orthogonal view: provides an orthogonal (or section) view.
‣Image > Duplicate allows you to save the slice you are viewing as an individual file
‣Image > Stacks > Movies
Time stamper: allows you to add the time information onto your movie.
Zoomify: allows you to make a movie where you zoom in on a particular region.
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Image Adjustments: Brightness/Contrast
‣Image Adjust > Brightness/Contrast
Or B/C Button on tool bar
Contrast and brightness: use to enhance
images by dynamically changing the lookup
table mapping.
Click on the brightness slider and drag from
side to side.
You can also adjust the contrast setting
independently.
Brightness - adds or subtracts a constant to
each pixel – shift in histogram along x axis
but doesn’t change the
distribution.
Contrast – lower level set (e.g. to 0) and
higher level set (e.g. to 255) and rest of pixel
values adjusted
proportionally.
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DO NOT CLICK on APPLY. This stretches
the histogram to fit new min and max values
and is not reversible. This affects future
Measurements!
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Image Adjustments: Threshold
‣Image > Adjust >Threshold
is the simplest method of image
segmentation. From a grayscale
image, thresholding can be used to
create binary images.
Threshold above/below: Individual
pixels in an image are marked as
“object” pixels if their value is
greater/lower than some threshold
value (assuming an object to be
brighter than the background) and
as “background” pixels otherwise.
Threshold inside/outside, when a
pixel value is between two
thresholds/vice-versa.
Typically, an object pixel is given a value of “1” while a background pixel is given a value of
“0.” Finally, a binary image is created by coloring each pixel white or black, depending on a
pixel's label.
Wikipedia
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Image Crop/Transform/Rotate/Revert
‣Image >
Crop - You can make selection areas to determine the area to be cropped
Rotate - You can also Rotate the image into different angles
All this can be applied to a single image or to all slices of a stack
‣File > Revert
And revert to the original file (File > Revert or Control-R)
To be safe, always click on Image > Duplicate and work on the copy of your image
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Calibrating image size units
Calibrating image size is important
for further image processing:
-measurements
-scale bar
‣ Image > Properties
Enter unit of length ( m or pixels)
Image now shows image size in m
‣ Press scale bar button in tool menu
to add scale bar to single image or all
slices of a stack.
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Image histogram and line profile
Histogram:
Shows the number of pixels of
each value, regardless of location, and the mean
and max values. The log display allows for the
visualization of minor components.
‣ Analyse > Histogram
‣ Generate list of values by
clicking on List.
Line plot: Shows the intensity profile of
pixels along a selection.
Select line tool, draw line for profiling
‣ Analyse > Plot profile
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ImageJ Region measurements
‣ Area SelectionTools
-Use these tools to create area selections -Location, width and height are displayed in
the status bar. (Change properties of selection regions in Edit > Selection > Properties
-The contents of an region can be copied, cleared (to white), filled with the current
drawing color, outlined (using Edit/Draw), filtered, or measured.
Backspace
- Edit/Clear
> Image > Colors -Set the drawing color
Double click any line tool
- Change the line width used by Edit/Draw
Arrow keys with ALT
- to "nudge" a selection one pixel at a time
Use the small "handles"
- to resize
Holding down Alt
- forces ellipse circles or rectangles squares
-To create the polygonal selection, click repeatedly with the mouse to create line
segments, double click when finish.
-Use the wand tool to reposition selections drawn with the rectangle capture tool, oval
capture tool, etc.
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ImageJ Region measurements
‣Measurements can be
managed and logged through
the Region of Interest (ROI)
Manager available in the
Biophotonics ImageJ
Version tool bar, or from
‣Image > Analysis > Tools
Create regions with the selection tools
Click on Add or type t
Select Show All so that the regions appear visible
Go to More > and choose Measure all
‣In Analyse > Set Measurements: you can decide
What type of measurements will appear in the list.
You can save the region measurements (More > Save)
And apply them to all stacks or to new images
You can also save the results as an excel file.
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