Aivia Software

Nuclei Tracking

The Nuclei Tracking recipe in Aivia detects nuclei and tracks their motility over time in fluorescence time-lapse microscopy images.

This recipe measures morphological, intensity, and motion attributes of the detected nuclei for comprehensive characterization of nuclei migrations. In addition, the recipe tracks nuclei divisions and generates lineages for all detected nuclei.

Parameters and Presets

Parameters

Recipe parameters for Nuclei Tracking and their descriptions are summarized in the table below.

Preset Group

Parameter Name

Min Value

Max Value

Description

Preset Group

Parameter Name

Min Value

Max Value

Description

Detection

Background Removal Factor

(Remove Background only)

0

100

Adjusts the sensitivity of the background removal operation; a lower value will preserve larger objects and more background variations

Contrast Threshold 

(Remove Background)

Intensity Threshold

(Skip Remove Background)

0

255 (8-bit)

65,535 (16-bit)

Adjusts the detection sensitivity on the segmentation image:

  • When Remove Background is selected, the threshold will be applied to the background-removed image

  • When Skip Remove Background is selected, the threshold will be applied to the input image

A lower value will detect bigger and more objects

Fill Holes Size

0

50,000 px2 or µm2

Adjusts the maximum size threshold for filling in gaps inside a detected object; a lower value will preserve more holes in the detection

Smoothing Factor

0

100

Adjusts the amount of smoothing applied to the outlines of the detected objects; a lower value will preserve more of the objects' morphological features

Tracking

Object Size

0

50,000 px2 or µm2

Specifies the range of objects to be included in the analysis results based on the areas of the detected objects

Separation Factor

0

100

Adjusts the sensitivity of the object separation operation; a lower value will preserve larger objects with multiple intensity peaks

Max Search Range

0

50,000 px or µm

Specifies the maximum distance for track-point matchmaking between successive time frames; a higher value will expand the search distance for fast-moving cells

Min Track Length

0

50,000

Specifies the minimum number of time frames before a detected object is considered a valid track; a lower value will generate more, and often shorter, tracks

Matchmaking Option

Not applicable

Toggles the tracking algorithm used for track-point matchmaking between time points; the available options are the Greedy Algorithm and the Hungarian Algorithm

Track Lineage Option

Not applicable

Specifies whether to track divisions for quantifying lineages

 

Presets

There are two preset groups in the recipe: Detection and Tracking. Each group has three pre-configured parameter groupings to help you get started on the analysis. The default preset values are as follows:

 

Detection

Parameter Name

Low

Medium

High

Background Removal Factor

75

75

75

Contrast Threshold

Intensity Threshold

23 (8-bit)

8 (8-bit)

1 (8-bit)

5,898 (16-bit)

1,966 (16-bit)

262 (16-bit)

Fill Holes Size

50 px2 or µm2

50 px2 or µm2

50 px2 or µm2

Smoothing Factor

4

4

4

 

Tracking

Parameter Name

Low

Medium

High

Object Size

10 - 500 px2 or µm2

50 - 3,000 px2 or µm2

100 - 6,000 px2 or µm2

Separation Factor

75

75

75

Max Search Range

20 px or µm

20 px or µm

20 px or µm

Min Track Length

4

4

4



 

Tutorial

Before beginning the tutorial, please download the Nuclei Tracking Demo image. For information on how to select presets or modify parameter values, please refer to the tutorial on how to use the Recipe Console.

  1. Unzip the demo file and load the demo image, “NucleiTrackDemo.tif,” into Aivia.

  2. In the Recipe Console, click on the Recipe selection dropdown menu and select the Nuclei Tracking recipe.

  3. Select the Low presets for both the Detection and Tracking preset groups.

  4. Click on the caret to the left of the Tracking preset group to show the preset parameters.

  5. Modify the parameter value as follows, leaving the other parameters intact:

    • Separation Factor: 92

  6. Click the Start button or press the F4 key on your keyboard to begin applying the recipe to the image.

The detected object outlines and tracks will be overlaid on the image.

 

Results

 

Nuclei Tracking tutorial results

 

Measurements

The Nuclei Tracking recipe generates morphological, intensity, position, and count measurements for each detected cell. You can add additional measurements to the analysis results with the Measurement Tool in Aivia and view measurement definitions on the Measurement Definitions page. The measurements generated by the recipe are given in the table below.

Morphology

Intensity

Position

Count

Morphology

Intensity

Position

Count

  • Area

  • Circularity

  • Pixel-based Mean Intensity

  • Pixel-based Standard Deviation Intensity

  • Pixel-based Total Intensity

  • Total Time

  • First Frame

  • Last Frame

  • Acceleration Magnitude

  • Velocity Magnitude

  • Centroid X

  • Centroid Y

  • Current Lineage Track Count

  • Min Track Generation

  • Max Track Generation

  • Mean Track Generation

 

 

Image Credits

Harvard Stem Cell Institute, Harvard University, Boston MA

 

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