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The Cell Analysis recipe detects cells, nuclei, and vesicles in 2D fluorescence microscopy images. The recipe establishes relationships between detected objects so that nucleus and vesicle measurements can easily be done on a per-cell basis and so that measurements between cellular components can be made.

On this page:

Table of Contents

Parameters and Presets

Parameters

Recipe parameters for Cell Analysis and their descriptions are summarized in the table below.

Preset Group

Parameter Name

Min Value

Max Value

Description

Whole Cells Detection

Image Smoothing Filter Size

(without Skip Smooth Image only)

1

100

Specifies the diameter of the filter that is used to smooth the Cells Input before further processing

The available smoothing types are the following:

  • Morphological Smoothing uses a morphological filter consisting of an average filter and morphological opening/closing operations based on the value of the Image Smoothing Filter Size.

  • Average Filter Smoothing uses an average filter that outputs the average intensity value for each pixel across a circular region with a diameter specified by the value of the Image Smoothing Filter Size.

  • Gaussian Filter Smoothing uses a Gaussian filter with a filter width specified by the value of the Image Smoothing Filter Size.

  • Binomial Filter Smoothing uses a binomial filter with a filter width specified by the value of the Image Smoothing Filter Size.

Typical Cell Diameter

(Remove Background only)

0

10,000 (px or µm)

Specifies the diameter of a typical cell in the image; the diameter is used for object enhancement and background removal, with a lower value preserving smaller objects

Contrast Threshold

(Remove Background only)

0

255 (8-bit)

65,535 (16-bit)

Specifies the lower intensity threshold for cell detection on the enhanced image, which has the background removed; a lower value leads to the detection of more and bigger cells

Intensity Threshold

(Skip Remove Background only)

0

255 (8-bit)

65,535 (16-bit)

Specifies the lower intensity threshold for cell detection on the smoothed (if smoothing is opted for) Cells Input

Fill Holes Size

0

1,000,000 (px2 or µm2)

Specifies the maximum size of gaps in detected cells that are filled; a lower value leads to the preservation of more holes in the detected cells

Smoothing Factor

0

100

Specifies the amount of smoothing applied to the detected cell boundaries; a higher value leads to more smoothing

Cells Diameter

0

10,000 (px or µm)

Specifies the range of detected cells to keep based on their diameters

Min Edge-to-Center Distance

(Apply Cells Partition only)

0

10,000 (px or µm)

Specifies the minimum distance from the center of a cell to the edge that is touching its closest neighboring cell; a lower value leads to more aggressive partitioning, which results in smaller, more uniform cell objects

Nuclei Detection

(optional)

Image Smoothing Filter Size

(without Skip Smooth Image only)

1

100

Specifies the diameter of the filter that is used to smooth the Nuclei Input before further processing

The available smoothing types are the following:

  • Morphological Smoothing uses a morphological filter consisting of an average filter and morphological opening/closing operations based on the value of the Image Smoothing Filter Size.

  • Average Filter Smoothing uses an average filter that outputs the average intensity value for each pixel across a circular region with a diameter specified by the value of the Image Smoothing Filter Size.

  • Gaussian Filter Smoothing uses a Gaussian filter with a filter width specified by the value of the Image Smoothing Filter Size.

  • Binomial Filter Smoothing uses a binomial filter with a filter width specified by the value of the Image Smoothing Filter Size.

Typical Nucleus Diameter

(Remove Background only)

0

10,000 (px or µm)

Specifies the diameter of a typical nucleus in the image; the diameter is used for object enhancement and background removal, with a lower value preserving smaller objects

Contrast Threshold

(Remove Background only)

0

255 (8-bit)

65,535 (16-bit)

Specifies the lower intensity threshold for nucleus detection on the enhanced image, which has the background removed; a lower value leads to the detection of more and bigger nuclei

Intensity Threshold

(Skip Remove Background only)

0

255 (8-bit)

65,535 (16-bit)

Specifies the lower intensity threshold for nucleus detection on the smoothed (if smoothing is opted for) Nuclei Input

Fill Holes Size

0

50,000 (px2 or µm2)

Specifies the maximum size of gaps in detected nuclei that are filled; a lower value leads to the preservation of more holes in the detected nuclei

Smoothing Factor

0

100

Specifies the amount of smoothing applied to the detected nucleus boundaries; a higher value leads to more smoothing

Nuclei Diameter

0

10,000 (px or µm)

Specifies the range of detected nuclei to keep based on their diameters

Min Edge-to-Center Distance

(Apply Nuclei Partition only)

0

10,000

Specifies the minimum distance from the center of a nucleus to the edge that is touching its closest neighboring nucleus; a lower value leads to more aggressive partitioning, which results in smaller, more uniform nucleus objects

Nuclei Correction

Not applicable

Specifies how to deal with detected nuclei that lie partially outside of detected cells

  • When No Nuclei Correction is selected, no post-processing is done to detected nuclei and cells, even when nuclei are partially outside of cells

  • When Extend Cell To Nucleus is selected, the detected cells are expanded so that they fully encapsulate their detected nuclei

  • When Crop Nucleus To Cell is selected, detected nuclei are cut off at the edges of their detected cells so that all cells fully encapsulate their nuclei

Vesicles [#] Detection

Up to five (5) vesicle sets can be detected.

(optional)

Image Smoothing Filter Size

(without Skip Smooth Image only)

1

100

Specifies the diameter of the filter that is used to smooth the Vesicles [#] Input before further processing

The available smoothing types are the following:

  • Morphological Smoothing uses a morphological filter consisting of an average filter and morphological opening/closing operations based on the value of the Image Smoothing Filter Size.

  • Average Filter Smoothing uses an average filter that outputs the average intensity value for each pixel across a circular region with a diameter specified by the value of the Image Smoothing Filter Size.

  • Gaussian Filter Smoothing uses a Gaussian filter with a filter width specified by the value of the Image Smoothing Filter Size.

  • Binomial Filter Smoothing uses a binomial filter with a filter width specified by the value of the Image Smoothing Filter Size.

Typical Vesicles Diameter

(Remove Background and Enhance Bright Spots only)

0

10,000 (px or µm)

Specifies the diameter of a typical vesicle in the image; the diameter is used for object enhancement and background removal, with a lower value enhancing/preserving smaller objects

Contrast Threshold

(Remove Background and Enhance Bright Spots only)

0

255 (8-bit, Remove Background)

65,535 (16-bit, Remove Background)

255 (Enhance Bright Spots)

Specifies the lower intensity threshold for vesicle detection on the enhanced image, which has the background removed or bright spots enhanced

Intensity Threshold

(Skip Remove Background only)

0

255 (8-bit)

65,535 (16-bit)

Specifies the lower intensity threshold for vesicle detection on the smoothed (if smoothing is opted for) Vesicles [#] Input

Fill Holes Size

(Remove Background and Skip Remove Background only)

0

1,000,000 (px2 or µm2)

Specifies the maximum size of gaps in detected vesicles that are filled; a lower value leads to the preservation of more holes in the detected vesicles

Smoothing Factor

(Remove Background and Skip Remove Background only)

0

100

Specifies the amount of smoothing applied to the detected vesicle boundaries; a higher value leads to more smoothing

Vesicles Diameter

0

10,000 (px or µm)

Specifies the range of detected vesicles to keep based on their diameters

Min Edge-to-Center Distance

(Apply Vesicles Partition only)

0

10,000 (px or µm)

Specifies the minimum distance from the center of a vesicle to the edge that is touching its closest neighboring vesicle; a lower value leads to more aggressive partitioning, which results in smaller, more uniform vesicle objects

Presets

There are three (3) preset groups in the recipe: Whole Cells Detection, Nuclei Detection, and Vesicles Detection. Each group has three (3) pre-configured parameter groupings to help you get started on the analysis. In the Input and Output section, you may opt to not detect nuclei and/or to not detect vesicles, in which case that/those parameter group(s) will not appear. Additionally, you may choose to detect up to five (5) vesicle sets at once. Each vesicle set has its own Vesicles Detection section, and all Vesicles Detection sections have the same default preset values. The default preset values are given in the sections to follow.

Whole Cells Detection

Parameter Name

Low

Medium

High

Image Smoothing Filter Size

3

9

13

Typical Cell Diameter

5 px or µm

20 px or µm

50 px or µm

Contrast Threshold

23 (8-bit)

8 (8-bit)

1 (8-bit)

5898 (16-bit)

1966 (16-bit)

262 (16-bit)

Intensity Threshold

23 (8-bit)

8 (8-bit)

1 (8-bit)

5898 (16-bit)

1966 (16-bit)

262 (16-bit)

Fill Holes Size

10 px2 or µm2

25 px2 or µm2

50 px2 or µm2

Smoothing Factor

4

4

4

Cells Diameter

1-30 px or µm

2-100 px or µm

20-200 px or µm

Min Edge-to-Center Distance

4 px or µm

8 px or µm

25 px or µm

Nuclei Detection

Parameter Name

Low

Medium

High

Image Smoothing Filter Size

1

3

5

Typical Nucleus Diameter

3 px or µm

8 px or µm

15 px or µm

Contrast Threshold

23 (8-bit)

8 (8-bit)

1 (8-bit)

5898 (16-bit)

1966 (16-bit)

262 (16-bit)

Intensity Threshold

23 (8-bit)

8 (8-bit)

1 (8-bit)

5898 (16-bit)

1966 (16-bit)

262 (16-bit)

Fill Holes Size

0 px2 or µm2 (Remove Background)

0 px2 or µm2 (Remove Background)

0 px2 or µm2 (Remove Background)

50 px2 or µm2 (Skip Remove Background)

50 px2 or µm2 (Skip Remove Background)

50 px2 or µm2 (Skip Remove Background)

Smoothing Factor

2 (Remove Background)

2 (Remove Background)

2 (Remove Background)

4 (Skip Remove Background)

4 (Skip Remove Background)

4 (Skip Remove Background)

Nuclei Diameter

1-10 px or µm

2-20 px or µm

5-50 px or µm

Min Edge-to-Center Distance

3

6

10

Nuclei Correction

No Nuclei Correction

Vesicles Detection

Parameter Name

Low

Medium

High

Image Smoothing Filter Size

1

1

1

Typical Vesicles Diameter

0.2 px or µm

0.5 px or µm

1 px or µm

Contrast Threshold

23 (8-bit, Remove Background)

8 (8-bit, Remove Background)

1 (8-bit, Remove Background)

5898 (16-bit, Remove Background)

1966 (16-bit, Remove Background)

262 (16-bit, Remove Background)

125 (Enhance Bright Spots)

65 (Enhance Bright Spots)

15 (Enhance Bright Spots)

Intensity Threshold

23 (8-bit)

8 (8-bit)

1 (8-bit)

5898 (16-bit)

1966 (16-bit)

262 (16-bit)

Fill Holes Size

0 px2 or µm2

0 px2 or µm2

0 px2 or µm2

Smoothing Factor

0

0

0

Vesicles Diameter

0.1-5 px or µm

0.2-10 px or µm

0.5-20 px or µm

Min Edge-to-Center Distance

0.2 px or µm

0.6 px or µm

2 px or µm

Measurements

Cell Analysis generates a few morphological, intensity, position, and count measurements. You can add additional measurements to the analysis results by using the Measurement Tool in Aivia. The measurements generated by the recipe are in the table below.

Object Set

Morphological

Intensity

Position

Count

Cell Membranes

  • Area

  • Perimeter

  • Length

  • Breadth

  • Circularity

  • Mean Intensity - [Cells Input]

  • Centroid X

  • Centroid Y

  • Contained Vesicle Count

Nuclear Membranes

  • Area

  • Perimeter

  • Length

  • Breadth

  • Circularity

  • Mean Intensity - [Nuclei Input]

  • Centroid X

  • Centroid Y

  • Contained Vesicle Count

Cells

  • Cell/Nucleus Area Ratio

None

  • Center to Nucleus Center Distance

  • Cytoplasm to Nucleus Vesicle Ratio

  • Min Edge to Nucleus Edge Distance

  • Centroid X

  • Centroid Y

  • Contained Vesicle Count

Cytoplasm

  • Area

  • Area to Contained Vesicle Area Ratio

None

  • Centroid X

  • Centroid Y

  • Contained Vesicle Count

Nucleus

  • Area to Contained Vesicle Area Ratio

None

  • Min Center of Mass to Cell Edge Distance

  • Centroid X

  • Centroid Y

  • Contained Vesicle Count

Vesicles

  • Area

  • Perimeter

  • Length

  • Breadth

  • Circularity

None

  • Distance to Center of Mass of Cell

  • Distance to Center of Mass of Nucleus

  • Distance to Nearest Edge of Cell

  • Distance to Nearest Edge of Nucleus

  • Centroid X

  • Centroid Y

None

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