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- 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
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Index
-Installing ImageJ (windows) and plugins -Memory settings -Menu and Tools overview p. 3 - 4 p. 5 p. 6
p. 7 - 8
p. 9 - 11 p. 12 p. 13 p. 14 15 p. 16 p. 17 p. 18 p. 19 p. 20 p. 21-22 Imaging
-Image quantification
Calibrating image size/ Scale bars Histogram and line profile ImageJ Region measurements Biology
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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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
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Region/Measurement tools
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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Open and manage stacks/movies -many file types and text files/results
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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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If we code each pixel on 2 bits we would have 22 = 4 possibilities (black, dark gray, light gray, white). But this image would have very limited levels.
28 = 256 possibilities (0 - 255 grey levels) 216= 4096 possibilities (0 - 4095 grey levels)
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 dont have 12 bit (usually converted to 16) Acquisition detectors usually only detect in 12 bit!
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Its 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 Biology Imaging
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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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.
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
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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.
DO NOT CLICK on APPLY. This stretches the histogram to fit new min and max values Contrast lower level set (e.g. to 0) and and is not reversible. This affects future higher level set (e.g. to 255) and rest of pixel Measurements!
values adjusted proportionally.
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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.
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 Biology Imaging
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
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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. > Image > Colors Double click any line tool
Backspace
Arrow keys with ALT Use the small "handles" Holding down Alt
- Edit/Clear -Set the drawing color - Change the line width used by Edit/Draw - to "nudge" a selection one pixel at a time - to resize - 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. Biology Imaging
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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