Short Description

This segmentation method performs a N-class thresholding based on a K-Means classification of the image histogram, then extracts objects in a bottom-up manner using user-defined minimum and maximum object sizes. Very useful to detect clustered objects in fluorescence microscopy.

This book chapter proposes a video tutorial on the HK-Means plugin (see tutorial 2). Images used in the tutorial are available on this Zenodo repository.

Team: Bio Image Analysis Unit
Institution: Institut Pasteur
Website: https://icy.bioimageanalysis.org

Documentation

To cite this plugin, please refer to this paper: A. Dufour, V. Meas-Yedid, A. Grassart, and J.-C. Olivo-Marin, “Automated Quantification of Cell Endocytosis Using Active Contours and Wavelet”, Proc. ICPR 2008, Tampa, FL, USA.

Getting started

Input: Sequence to process.
Gaussian pre-filter: To pre-process the sequence with a Gaussian filter give an integer value >0 (sigma of the Gaussian blur in x,y for a 2D sequence and x,y,z for a 3D sequence)
Intensity classes: 10 by default. Increase this value if the objects of interest have different intensitye values.
Min object size (px): minimum size of clusters, in pixels. To estimate the size of objects (for instance nuclei) on a 2D sequence, draw a ROI around an object and check the value of its interior. To estimate object size on a 3D sequence, draw a 2D ROI on one slice (one Z position), and multiply the interior value of this ROI by the number of slices spanned by the object.
Max object size (px): maximum size of clusters, in pixels. See above how to estimate objects size.
Min object intensity: at least one pixel of the object must be of this intensity. Mouse over objects and background to evaluate the minimum objet intensity.
Export ROIs: outputs objects as region of interest overlayed on the original image
Export Labels: outputs a labelled image

Tracking

When working with a timelapse, you’ll see the option “Prepare for tracking”. If you tick it, ROIs detected by HK-Means will be sent to the Swimming Pool, a location that can be accessed by several plugins, including the Spot tracking plugin.

At any time, you can see the content of the Swimming Pool by opening the Swimming Pool viewer.

To further process your timelapse with the Spot Tracking plugin, click on “Select detection results here” to select the ROIs you just created with the HK-Means plugin. Then click on Run the Spot Detector plugin if you have a simple tracking case or go to Interface → Advanced interface to set advanced tracking options.

Block HK-Means

A few parameters in the block are different from the HK-Means plugin interface:

Frame: to apply HK-Means on all or only one frame.
binary sequence: output a binary sequence witth two labels (background and objects)
list of ROI: output objects as region of interest.

Note that, if the image has several channels and you would like to apply the HK-Means only on one channel, you need to use the block Extract channel first and input the extracted channel.

Resources needing this

One review on “HK-Means

  1. IcyLyd
    Very usefull to set threshold manually or automatically using protocols. The export in color coded ROI is great and visual.

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Changelog

  • Version 2.9.1.0 • Released on: 2020-03-18 18:00:00
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    Description:

    Updated for Icy 2.1 changes (dependency and exception throwing)

  • Version 2.9.0.0 • Released on: 2019-01-31 18:53:25
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    Description:

    Updated to be compatible with volatile Sequence (Icy 2.0)

  • Version 2.8.2.0 • Released on: 2016-08-30 17:22:09
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    Description:

    Fixed an issue when filtering by minimum intensity

  • Version 2.8.1.0 • Released on: 2016-02-03 17:55:29
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    Description:

    Fixed an issue when processing multi-channel images

  • Version 2.8.0.0 • Released on: 2016-02-02 19:59:00
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    Description:

    * Switched to the ROI-based implementation (connected components must die!)
    * Fixed an issue with the color map of the labeled sequence
    * ROIs are now coloured based on the color map of the channel they were detected in
    * Added status bar and console output
    * A bit more documentation in the GUI
    * Some cleaning and fixes

  • Version 2.7.4.0 • Released on: 2015-07-31 11:57:06
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    Description:

    Added the frame parameter in block mode

  • Version 2.7.3.0 • Released on: 2015-06-26 13:06:27
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    Description:

    * Added an option in standalone mode to process only a single frame
    * Use a channel selector instead of a dimension picker
    * Fixed the removal of 3D ROIs produced by the plugin before running again

  • Version 2.7.2.0 • Released on: 2015-05-28 17:17:11
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    Description:

    Fixed an issue causing where the labeled output was missing in Protocol mode.

  • Version 2.7.1.0 • Released on: 2015-05-22 14:08:13
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    Description:

    * Fixed extraction on single channel sequences
    * Fixed swimmingpool output

  • Version 2.7.0.0 • Released on: 2015-05-22 13:58:19
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    Description:

    * Extracted core methods into class HKMeans with static access (useful for scripting)
    * Export to swimming pool now includes full mask information (useful for intensity measurement after tracking)

  • Version 2.6.0.0 • Released on: 2013-10-30 18:24:23
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    Description:

    Updated to Icy 1.4.0.0 (new support for 3D ROI)

  • Version 2.5.0.0 • Released on: 2013-10-30 15:47:18
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    Description:

    Added final threshold parameter in block version

  • Version 2.4.0.0 • Released on: 2013-06-12 16:52:22
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    Description:

    3D ROI export

  • Version 2.3.1.0 • Released on: 2012-11-16 16:53:52
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    Description:

    Display a message dialog indicating that ROIs cannot be exported in 3D

  • Version 2.3.0.0 • Released on: 2012-10-31 17:28:21
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    Description:

    New: output the extracted objects as a list of ROI in block mode

  • Version 2.2.0.0 • Released on: 2012-09-11 09:42:24
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    Description:

    HK-Means is channel aware

  • Version 2.1.2.0 • Released on: 2012-08-03 10:42:00
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    Description:

    Fixed the output file name generation

  • Version 2.1.1.0 • Released on: 2012-08-02 17:02:14
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    Description:

    * Show appropriate error when given an incorrect class number
    * Adjust the output file name to the input name + a suffix (and a incremental number if used via EzPlug)

  • Version 2.1.0.0 • Released on: 2012-06-28 17:57:52
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    Description:

    * fix: throw handled exception if input is null (obviously…)
    * blocks: updated output to use VarGenericArray

  • Version 2.0.0.0 • Released on: 2012-06-21 13:35:29
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    Description:

    Support for Blocks programming.

  • Version 1.4.2.0 • Released on: 2012-05-31 16:30:48
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    Description:

    fixed: trying to access an EzGUI when running within Blocks

  • Version 1.4.1.0 • Released on: 2012-05-31 15:18:02
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    Description:

    adjusted classpath and field visibility

  • Version 1.4.0.0 • Released on: 2012-05-11 18:58:29
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    Description:

    new: changed internal structures and output to use integers instead of shorts
    new: updated to ConnectedComponents v.3.2.0.0 (components sorting)

  • Version 1.3.3.0 • Released on: 2012-04-12 13:56:01
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    Description:

    Updated to Filter toolbox 2.1.0.0

  • Version 1.3.2.0 • Released on: 2012-04-06 18:50:43
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    Description:

    Results names are incremental for easier differentiation.

  • Version 1.3.1.1 • Released on: 2012-02-20 11:54:00
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    Description:

    Updated to EzPlug 1.6.4.1 (no logic change)

  • Version 1.3.1.0 • Released on: 2011-11-29 17:15:16
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    Description:

    * added label on the interface displaying the total number of detected objects

  • Version 1.3.0.0 • Released on: 2011-11-19 16:52:45
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    Description:

    * new: export detections to the swimming pool for tracking purposes
    * new: export detections as ROIs on the input sequence

  • Version 1.2.0.1 • Released on: 2011-11-09 13:27:26
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    Description:

    updated to icy.type.DataType

  • Version 1.2.0.0 • Released on: 2011-09-09 19:12:39
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    Description:

    New: added optional parameter to discard objects below a certain intensity threshold within the extraction process

  • Version 1.1.0.0 • Released on: 2011-05-27 15:57:33
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    Description:

    - New: added a method to output the connected components found in the sequence
    - New: the total number and ID of the components are displayed in the console and the output sequence.

  • Version 1.0.0.1 • Released on: 2011-05-19 17:33:49
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    Description:

    - switched to non-beta state
    - adjusted default parameters

  • Version 1.0.0.0b • Released on: 2011-05-19 17:23:54
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    Description:

    initial submission