Note: The Colony Analyzer recipes has been replaced with the Colony Analysis recipe in Aivia 7. For information about the new Colony Analysis recipe, please go here.


Recipe Description


The Colony Analyzer recipes in Aivia detects colonies in phase contrast or fluorescence microscopy images. The recipes provide a count of the number of colonies and measure the morphology and of the detected cells.

Three (3) Colony Analyzer recipes are provided in Aivia: MEF, Matrigel, and Fluorescence. The MEF and Matrigel recipes are for the detection of colonies plated on a substrate layer of mouse embryonic fibroblasts (MEF) and Matrigel respectively in phase contrast microscopy images; the fluorescence recipe is for the detection of fluorescently-labeled colonies.

Parameters and Presets


Parameters

MEF / Matrigel

Parameters and their descriptions for the Colony Analyzer (MEF) and Colony Analyzer (Matrigel) recipes are summarized in the table below.

Preset Group
Parameter Name
Min Value
Max Value
Description
Colony Detection
Colony Detection Sensitivity
0
255
Adjusts the sensitivity of the detection; a lower value will detect bigger and more objects
Colony Refinement
Minimum Colony Area
0
2,000,000
Specifies the minimum size for objects to be included in the analysis results based on the area of the detected objects
Colony Refinement
Fill Holes Size
0
500,000
Adjusts the maximum size threshold for filling in gaps inside a detected object; a lower value will preserve more holes in the detection
Colony Refinement
Smoothing Factor / Kernel
0
100
Adjusts the amount of smoothing applied to the outline of the detected objects; a lower value will preserve more of the object's morphological features

Fluorescence

Parameters and their descriptions for the Colony Analyzer (Fluorescence) recipe are summarized in the table below.

Preset Group
Parameter Name
Min Value
Max Value
Description
Detection
Background Removal Factor
1
100
Adjusts the sensitivity of the background removal operation; a lower value will preserve larger objects and more background variations
Detection
Contrast Threshold
0
255 (8-bit)
65,535 (16-bit)
Adjusts the detection sensitivity on the background removed image; a lower value will detect bigger and more objects
Detection
Fill Holes Size
0
250,000
Adjusts the maximum size threshold for filling in gaps inside a detected object; a lower value will preserve more holes in the detection
Detection
Smoothing Factor
0
100
Adjusts the amount of smoothing applied to the outline of the detected objects; a lower value will preserve more of the object's morphological features
Subset Filtering
Object Size
0
10,000,000
Specifies the range of objects to be included in the analysis results based on the area of the detected objects
Subset Filtering
Separation Factor
1
100
Adjusts the sensitivity of the object separation operation, a lower value will preserve larger objects with multiple intensity peaks

Presets


MEF / Matrigel

There are two preset groups in the recipe: Colony Detection and Colony Refinement; each group has three pre-configured parameter grouping to help you get started on the analysis. The default preset values are as follows:

Colony Detection

Parameter Name
Low
Medium
High
Colony Detection Sensitivity
20
10
5

Colony Refinement (MEF)

Parameter Name
Small
Medium
Large
Minimum Colony Area
50,000
200,000
400,000
Fill Holes Area
1,000
50,000
200,000
Smoothing Kernel
15
25
35

Colony Refinement (Matrigel)

Parameter Name
Small
Medium
Large
Minimum Colony Area
10,000
50,000
200,000
Fill Holes Area
1,000
5,000
10,000
Smoothing Factor
1
4
15

Fluorescence

There are two preset groups in the recipe: Detection and Subset Filtering; each group has three pre-configured parameter grouping to help you get started on the analysis. The default preset values are as follows:

Detection

Parameter Name
Low
Medium
High
Background Removal Factor
20
49
67
Contrast Threshold
51 (8-bit)
13,107 (16-bit)
41 (8-bit)
10,486 (16-bit)
10 (8-bit)
2,621 (16-bit)
Fill Holes Size
5,000
20,000
50,000
Smoothing Factor
18
18
18

Subset Filtering

Parameter Name
Low
Medium
High
Object Size
2,000 - 100,000
4,000 - 250,000
12,500 - 500,000
Separation Factor
10
65
90

Measurements


The Colony Analyzer recipes generates morphological and intensity measurements for each detected colony. Normalized morphological measurements are included to account for the size difference between colonies. You can add additional measurements to the analysis results by using the Measurement Tool in Aivia. The measurements generated by the recipes are as follows.

MEF / Matrigel

  • Morphological
    • Area
    • Circularity
    • Normalized Area
    • Normalized Confidence
  • Intensity (measured on the input channel)
    • Mean
  • Summary
    • Colony Area Mean
    • Colony Area Maximum
    • Colony Area Minimum
    • Colony Circularity Mean
    • Colony Circularity Maximum
    • Colony Circularity Minimum

Fluorescence

  • Morphological
    • Area
    • Circularity
    • Component Count
  • Intensity (measured on the input channel)
    • Mean
    • Total
    • Standard Deviation
  • Summary
    • Total Component Count
    • Average Mean Intensity


Tutorial


Before beginning the tutorial, please download the Colony Analyzer Demo Image (ColonyDemo.zip). For information on how to select presets or modify parameter values, please refer to the tutorial on how to use the Recipe Console. You can download the image directly here.
  1. Unzip the demo file and load the demo image, ColonyDemo.tif, into Aivia
  2. In the Recipe Console, click on the Recipe selection dropdown menu and select the Colony Analyzer (MEF) recipe
  3. Select the Medium preset for the Colony Detection group and the Medium preset for the Colony Refinement group
  4. Click on the caret (>) to the left of the Colony Refinement preset group to show the preset parameters
  5. Modify the parameter values as follows: leaving the other parameters in tact
    • Fill Holes Area: 10,000
    • Minimum Colony Area: 25,000
  6. Click the From beginning button or press the F4 key on your keyboard to begin applying the recipe to the image

The detected objects outline and tracks will be overlaid on the image.

Results

ColonyDemo_output.png
Colony Analyzer (MEF) results