Particle Analysis - ImageJ- Manual1

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Fiji Cookbook Topics

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 ManipulationsT-functions

Z-functions

Particle Analysis

From ImageJ

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 Counter2.2 PointPicker

3 Particle tracking

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 Pickeror Cell counter plugins. The Point Pickerplugin

can be found via the menu commandAnalyze>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.

le Analysis - ImageJ http://wiki.imagej.net/Particle_

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There are several ways to set thresholds. Monochrome images are most simply

thresholded via the menu commandImage>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 hitApply. 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 toDefaultallows you to choose betweenDefaultand

15 other threshold techniques. The drop-down menu set toRedallows you tochoose between a red on white color scheme, a black on white color scheme,

or an over and under color scheme. TheDark Backgroundbox 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 methodoption allows

you to choose a thresholding techniqe other than the default. The Threshold

coloroption allows you to choose between Red, White, Black, or B&W as the

thresholding color. The Color spaceoption 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 commandImage>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 menuImage>Adjust>Thresholdfor 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 commandAnalyze>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 1.0can also be selected to help exclude unwanted objects. Select the Show:

Outlinesoption 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 toDisplay results, Clear Results, Summarize,Add to

Manager,Exclude on edges,Include holes,Record starts, and/orIn 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.

Enter the size range to be counted.1.

Select the automatic thresholding method. This can be either

Current, Otsu, Maximum Entropy, Mixture Modellingor k-means

clustering. Currentuses the threshold that has been set manually,

see above.

2.

Perform a background correction.3.

Use a Smoothfilter.4.Perform a watershed separation.5.

Add the particles to the ROI manager.6.

Say yes to a summary.7.

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 PointPickeror Cell counterplugins.

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 Pickerplugin will change the Fiji toolbar to the Point Pickertoolbar.

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 Listdialog allows you to Show, Saveor Openthe points' coordinates. Clicking Showdisplays the

coordinates in theResultswindow, 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 TrackerParticle 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]).

TrackMateUse 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 andexplanation.

Manual TrackingUse the menu command Plugins>Tracking>Manual Tracking. This tool allows you to keep

track of the movement of a cell.

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