Particle Analysis - ImageJ

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http://wiki.imagej.net/Particle_Analysis

Particle Analysis
From ImageJ

Fiji Cookbook Topics

Contents
1 Automatic Particle counting
1.1 Setting a threshold
1.2 Watershed separation
1.3 Analyze Particles
1.4 Nucleus Counter
2 Manual Counting
2.1 Cell Counter
2.2 PointPicker
3 Particle tracking

Introduction and Installation

Annotating Images
Colocalization Analysis
Color Image Processing
Deconvolution
Image Intensity Processing
Image Stitching
Importing Image Files
Particle Analysis
Saving and Exporting
Stack-slice Manipulations
T-functions
Z-functions

Automatic Particle counting

Automatic particle counting can be done if the image does not have too many individual particles touching.
Manual particle counting can be done using the Point Picker or Cell counter plugins. The Point Picker plugin
can be found via the menu command Analyze>Tools>PointPicker.
Segmentation, or the ability to distinguish an object from its background, can be a difficult issue to deal with.
Once this has been done, however, the object can then be analyzed.
RAW Threshold Watershed AnalyzeParticles

Setting a threshold
5.1.1.1 Manual thresholding
Automatic particle analysis requires a binary, black and white, image. A threshold range is set to tell the
objects of interest apart from the background. All pixels in the image whose values lie under the threshold are
converted to black and all pixels with values above the threshold are converted to white, or vice-versa.

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There are several ways to set thresholds. Monochrome images are most simply
thresholded via the menu command Image>Adjust>Threshold. The threshold
can be set using the slider bars. The pixels within the threshold range are
displayed in red. When you are satisfied with the threshold settings, you can
then hit Apply. This will permanently apply the threshold settings and convert
the image to binary. You have different options for setting a manual threshold.
The drop-down menu set to Default allows you to choose between Default and
15 other threshold techniques. The drop-down menu set to Red allows you to
choose between a red on white color scheme, a black on white color scheme,
or an over and under color scheme. The Dark Background box will flip the
foreground color with the background color. You can also choose to check the
Stack histogram box to produce a histogram for an entire stack.
For color images, setting the threshold is done with the command sequence
Image>Adjust>Color Threshold.... The Thresholding method option allows
you to choose a thresholding techniqe other than the default. The Threshold
color option allows you to choose between Red, White, Black, or B&W as the
thresholding color. The Color space option allows you to choose between
HSB, RGB, Lab, and YUV. The background of the thresholded image can be
made light or dark. The image can be converted to a binary image via the
menu command Image>Type>8-bit.
Automatic thresholding
There are many algorithms you can use to calculate the threshold without introducing user-bias. An evaluation
of over 40 of these can be found in this paper:
Sezgin & Sankur. 2004. Survey over image thresholding techniques and quantitative performance evaluation.
Journal of Electronic Imaging, 2004. (13:146-165).
Fiji has several plugins found in the menu Image>Adjust>Threshold for automatic calculation of an image
threshold. These include Otsu's thresholding, maximum entropy threshold, and mixture modelling thresholding.
For a complete list of the methods available with Fiji see the Plugins section located in the Documentation
section under the Content tab at the top of this page.

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Watershed separation
Overlapping objects in a binary image can be separated using the menu command Process>Binary>Watershed.
First convert the image to binary by thresholding. The black pixels are then replaced with grey pixels of an
intensity proportional to their distance from a white pixel. Black pixels closer to the edge are lighter than black
pixels that are more central. This is the Euclidian distance map (EDM) of the black area. From this the centers
of the objects are calculated. These are the ultimate eroded points (UEPs) of each black area meaning they are
equidistant from each edge. These points are then dilated until they touch another black pixel. This meeting
point is where a watershed line is drawn.

Analyze Particles
To analyze the particles in a segmented image, use the menu command Analyze>Analyze particles.... This will
provide you with information about each particle in the image..

Set the minimum size and maximum pixel area size to exclude anything that is
not an object of interest in the image. Roundness values between 0.0 and 1.0
can also be selected to help exclude unwanted objects. Select the Show:
Outlines option to display an image of the detected objects. The Show
drop-down menu also allows the user to show Nothing, Bare Outlines,
Ellipses, Masks, Count Masks, Overlay Outlines, and Overlay Masks. The user
can choose whether to Display results, Clear Results, Summarize, Add to
Manager, Exclude on edges, Include holes, Record starts, and/or In situ Show.
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.

Nucleus Counter
This plugin automates many of the steps discussed above.
1. Enter the size range to be counted.
2. Select the automatic thresholding method. This can be either
Current, Otsu, Maximum Entropy, Mixture Modelling or k-means
clustering. Current uses the threshold that has been set manually,
see above.
3. Perform a background correction.
4. Use a Smooth filter.
5. Perform a watershed separation.
6. Add the particles to the ROI manager.
7. Say yes to a summary.
Other options can easily be added on request.
The count, area, and average size are returned as a text window and the
outlined particles are overlaid on a duplicate of the original image.

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Manual Counting
Use the Fiji Crosshair (mark and count) tool to count particles. A greater degree of control can be accessed with
the PointPicker or Cell counter plugins.

Cell Counter
This plugin generates a new image window 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 color of the marking tool. The
results table keeps a tally of the cell types marked. Clicking the Results button will generate a summary of the
data.

PointPicker
The Point Picker plugin will change the Fiji toolbar to the Point Picker toolbar.
Crosses are reversibly overlaid on to the image and incrementally change color. This differs from the Crosshair
tool which irreversibly stamps a point in the image. Crosses can be moved or deleted.
The Point List dialog allows 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 Fiji toolbar by clicking the button.

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Particle tracking
Particle Tracker Particle Tracker is a 2D feature point-tracking plugin for the automated detection and analysis
of particle trajectories as recorded by video imaging in cell biology. The algorithm is decsribed in Sbalzarini
and Koumoutsakos (2005[1]).
TrackMate Use the menu command Plugins>Tracking>TrackMate. This plugin allows you to perform single
particle tracking of spot-like structures. For more in-depth information, see the TrackMate tutorial and
explanation.
Manual Tracking Use the menu command Plugins>Tracking>Manual Tracking. This tool allows you to keep
track of the movement of a cell.
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