Application Name

IKOSA AI Multichannel Instance Segmentation App

Version

2.2.2

Documentation Version

30.01.2024 - 1

Input Image(s)

Multichannel Images (standard and/or WSI; 8/16 bit);

same channel number as in training images (max. 10)

Input Parameter(s)

• Max. outgrowth: For each class (trained label) a maximum outgrowth in the range of 0 - 99 pixels.

• Channel properties

  • channel name

  • channel color

• Regions of interest (optional)

Keywords

fluorescence, multichannel, immunofluorescence, multiplex, phenotype, microscopy

Short Description

Instance Segmentation of objects that resemble ones annotated and labeled in the training data of this application. Extraction of morphometric parameters and intensity/distance/density measures of objects and corresponding outgrowth areas.

References / Literature

-

Multichannel (standard and/or WSI)

Check image format

File formats

same as training images (max. 10)

8 Bit or 16 Bit

WSI (CZI) formats: arbitrary

Standard images: max. 18,000 x 18,000

arbitrary

Image content

Arbitrary

Additional requirements

None

csv

results.csv

A csv file containing the overall analysis results for the input image or all inclusion ROIs.

csv

results_<xx>_<class-name>.csv

A csv file containing the analysis results for all detected objects of class number <xx> with class name <class-name> (in training data) in the input image or inclusion ROIs.

jpg

results_vis/<xx>_<class-name>_vis.jpg (2D image, no ROI), or

results_vis/<xx>_<class-name>_t<time-step>_z<z-layer>_c<channel>.jpg (for time series, z-stack, or multichannel image, no ROI), or

results_vis/<xx>_<class-name>_<roi-id>.jpg (2D image, ROI <roi-id>), or results_vis/<xx>_<class-name>_t<time-step>_z<z-layer>_c<channel>_<roi-id>.jpg for time series, z-stack, or multichannel image, ROI <roi-id>):

A visualization of the analysis result for a specific time step (of a time series), z-layer (of a z-stack), or channel (of a multichannel image) for either the whole image (if no inclusion ROIs selected for analysis) or each individual inclusion ROI, for each class number <xx> with class name <class-name> (in training data).

Each visualization includes two parts:

• Input

  • input image/ROI, channels blended with set channel colors to RGB image

• Objects

  • blended RGB image (same as first part)

  • object instances are shown as an overlay in random color.

  • object instance index number corresponds to the object_id as listed in the results_<xx>_<class-name>.csv file.

• Objects and outgrowth

  • blended RGB image (same as first part)

  • instance contours of detected objects and outgrowth contours from instance are marked with black/white solid lines

json

annotation_results.json

A json file that includes geometries of the objects detected in the input image or inclusion ROIs.

json

roiMeta.json

A json file containing all information regarding the ROIs defined for the analysis job to ensure reproducibility. Also, information regarding image and analysis dimensions is provided.

jpg

rois_visualization.jpg or

t<time-step>_z<z-layer>_rois_visualization.jpg

An overview visualization to show locations of all analyzed ROIs for the 2D image or time step <time-step> of a time series, z-layer <z-layer> of a z-stack.

json

jobResultBundleMeta.json

A json file containing all information regarding the analysis job (application name and version, project, etc.) to ensure reproducibility.

1

t

3

1 - 

Time step, i.e. the position of the image in the time series.

2

z

5

1 - 

z-layer, i.e. the position of the layer in the z-stack.

3

roi_id

ROI-03

ROI-01 - 

<roi-id> starting from “ROI1”. Empty, if no inclusion ROI is specified and the whole image was analyzed.

4

roi_name

“central”

text

Custom text to identify the ROI. Empty, if no inclusion ROI is specified and the whole image was analyzed.

5

roi_size [Px^2]

1212212

1 -

Size of the ROI that was analyzed in pixels^2. The size of the whole image is given if no inclusion ROI is specified and the whole image was analyzed.

6

bit depth [Bit]

8

8, 16

Bit/color depth of each channel of the image

7

class

“cell nuclei”

text

Name of the trained label (<class-name>).

8

object_max_outgrowth [Px]

2

0 - 99

Maximum outgrowth in pixels.

9

total_num_objects

788

0 -

Total number of objects which are detected in the image/ROI.

10

on_border_num_objects

12

0 - total_num_objects

Number of objects which are detected in the image/ROI and touch the border of the image/ROI.

11

holes_num_objects

3

0 - total_num_objects

Number of objects which are detected in the image/ROI and have holes (>=1 pixel).

12

recombined_num_objects

4

0 - total_num_objects

Number of objects which are detected at tile borders of image (tile size: 2048x2048 pixels) and are recombined.

13

density_num_objects [1/Px^2]

0.0007514953

0 - 1

Total number of objects divided by area of image/ROI.

14

total_area_objects [Px^2]

101863

8 - #pixels

Total area of objects which are detected in the image/ROI.

15

density_area_objects

0.0971441268

0 - 1

Total area of objects divided by area of image/ROI.

16

total_area_outgrowth [Px^2]

125326

0 - #pixels

Total area of object’s outgrowth which are detected in the image/ROI.

17

density_area_outgrowth

0.11952018737

0 - 1

Total area of outgrowth divided by area of image/ROI.

18

total_area_objects_incl_outgrowth [Px^2]

227189

8 - #pixels

Total area of objects including outgrowth which are detected in the image/ROI.

19

density_area_objects_incl_outgrowth

0.2166643142

0 - 1

Total area of objects including outgrowth divided by area of image/ROI

20

mean_area_objects [Px^2]

14.324523

8 - #pixels

Mean area of detected objects in Pixels^2.

21

stddev_area_objects [Px^2]

1.213133

0 - #pixels

Standard deviation of area of detected objects in Pixels^2.

22

median_area_objects [Px^2]

13.983252

8 - # pixels

Median area of detected objects in Pixels^2.

23

mean_area_outgrowth [Px^2]

21.363824

0 - #pixels

Mean area of outgrowth in Pixels^2.

24

stddev_area_outgrowth [Px^2]

2.573912

0 - #pixels

Standard deviation of areas of outgrowth in Pixels^2.

25

median_area_outgrowth [Px^2]

20.874623

0 - #pixels

Median area of outgrowth in Pixels^2.

26

mean_area_objects_incl_outgrowth [Px^2]

35.836274

8 - #pixels

Mean area of detected objects including outgrowth in Pixels^2.

27

stddev_area_objects_incl_outgrowth [Px^2]

2.309003

0 - #pixels

Standard deviation of area of detected objects including outgrowth in Pixels^2.

28

median_area_objects_incl_outgrowth [Px^2]

35.473222

8 - #pixels

Median area of detected objects including outgrowth in Pixels^2.

29

mean_distance [Px]

13.5016475693

0 -

Mean distance (center-to-center) between detected objects in Pixels. The distance is calculated by the nearest-neighbor distance to other objects of the same class.

30

stddev_distance [Px]

5.5485792042

0 -

Standard deviation of distances between detected objects in Pixels. The distance is calculated by the nearest-neighbor distance to other objects of the same class.

31

median_distance [Px]

12.529964086

0 -

Median distance between detected objects in Pixels. The distance is calculated by the nearest-neighbor distance to other objects of the same class.

32

mean_bbox_area [Px^2]

202.03426395

8 -

Mean area of bounding box of detected objects in Pixels^2.

33

mean_area_ratio [%]

0.0124492385

0 - 100

Mean area of detected object as percentage of overall  image area or ROI area inside the image.

34

mean_perimeter [Px]

40.434010152

8 -

Mean perimeter of detected objects in Pixels.

35

mean_circularity

0.92114213197

0 - ~1

Mean circularity factor of detected objects.

Circularity = 4*pi*area/(perimeter^2).

The circularity of a circle is 1.

Note: Detected objects with a circularity of infinity are ignored when calculating mean_circularity.

36

mean_circularity_ISO

0.85114213197

0 - ~1

Mean circularity factor of detected objects calculated by ISO defintion.

37

mean_solidity

0.9310659898

0 - 1

Mean solidity value of all detected objects.

Solidity is calculated out of the ratio of an object’s area to the area of its convex hull.

38

mean_eccentricity

0.7109644670

0 - 1

Mean eccentricity of all detected objects.

Eccentricity of the ellipse that has the same second-moments as the object area. The eccentricity is the ratio of the focal distance (distance between focal points) over the major axis length. When it is 0, the ellipse becomes a circle.

39

mean_equivalent_diameter [Px]

11.737309644

1 -

Mean equivalent diameter of all detected objects.

The equivalent diameter is the diameter of a circle having the same area as the detected object.

40

mean_extent

0.709022842

0 - 1

Mean extent of all detected objects.

The extent value is the ratio of the object area to the total bounding box area.

41

mean_minor_axis_length [Px]

9.4835025380

1 -

Mean minor axis length of all detected objects.

The minor axis length is the length of the minor axis of the ellipse that has the same normalized second central moments as the object.

42

mean_major_axis_length [Px]

15.843908629

1 -

Mean major axis length of all detected objects.

The major axis length is the length of the major axis of the ellipse that has the same normalized second central moments as the object.

For the different channels [CC] in the multichannel image:

43/ 55/ …

channel_#[CC]_index

1

1, 2, 3

Index of channel

44/ 56/ …

channel_#[CC]_name

“DAPI”

text

Name of channel

45/ 57/ …

channel_#[CC]_total_intensity

170486667

0 - 65535 x #pixels

Total intensity (sum) of this channel CC in the image/ROI.

46/ 58/ …

channel_#[CC]_objects_mean_intensity

272.93074030

0 - 65535

Mean intensity of this channel CC over the areas of all detected objects.

47/ 59/ …

channel_#[CC]_objects_stddev_intensity

130.84258715

0 - 65535

Standard deviation of intensity of this channel CC over the areas of all detected objects.

48/ 60/ …

channel_#[CC]_objects_intensity_dominant

265

0 - total_num_objects

Number of objects for which mean intensity is highest (=dominant) in this channel CC, compared to all other channels.

49/ 61/ …

channel_#[CC]_outgrowth_mean_intensity

273.26551765

0 - 65535, or

”” (empty)

Mean intensity of this channel CC over the areas of the outgrowths of all detected objects.

Is empty ““ if no outgrowth was set (max. outgrowth was set to 0 pixels).

50/ 62/ …

channel_#[CC]_outgrowth_stddev_intensity

203.15327249

0 - 65535, or

”” (empty)

Standard deviation of intensity of this channel CC over the areas of the outgrowths of all detected objects.

Is empty ““ if no outgrowth was set (max. outgrowth was set to 0 pixels).

51/ 63/ …

channel_#[CC]_outgrowth_intensity_dominant

724

0 - total_num_objects

Number of objects for which mean intensity of outgrowth areas is highest (=dominant) in this channel CC, compared to all other channels.

52/ 64/ …

channel_#[CC]_objects_incl_outgrowth_mean_intensity

273.115521066

0 - 65535

Mean intensity of this channel CC over the areas of the objects including outgrowths of all detected objects.

53/ 65/ …

channel_#[CC]_objects_incl_outgrowth_stddev_intensity

174.47822028

0 - 65535

Standard deviation of intensity of this channel CC over the areas of the objects including outgrowths of all detected objects.

54/ 66/ …

channel_#[CC]_objects_incl_outgrowth_intensity_dominant

530

0 - total_num_objects

Number of objects for which mean intensity of areas of detected objects incl. outgrowth is highest (=dominant) in this channel CC, compared to all other channels.

1

t

3

1 - 

Time step, i.e. the position of the image in the time series.

2

z

5

1 - 

z-layer, i.e. the position of the layer in the z-stack.

3

roi_id

ROI-03

ROI-01 - 

<roi-id> starting from “ROI1”. Empty, if no inclusion ROI is specified and the whole image was analyzed.

4

roi_name

“central”

text

Custom text to identify the ROI. Empty if no inclusion ROI is specified and the whole image was analyzed.

5

roi_size [Px^2]

1212212

1 -

Size of the ROI that was analyzed in pixels^2. The size of the whole image is given if no inclusion ROI is specified and the whole image was analyzed.

6

bit depth [Bit]

8

8, 16

Bit/color depth of each channel of the image

7

max_outgrowth [Px]

2

0 - 99

Maximum outgrowth in pixels.

8

object_id

5

1 -

ID of detected object. More information regarding numbering can be found in Numbering (IDs) of objects in analysis results.

9

object_center_x

741

1 - image/ROI width

x-coordinate of object center (centroid, starting from upper left corner, along image/ROI width).

10

object_center_y

26

1 - image/ROI height

y-coordinate of object center (centroid, starting from upper left corner, along image/ROI height).

11

is_on_border

“True”

“True” or “False”

Boolean indicator to show if object is touching the border of the image/ROI.

12

has_holes

“False”

“True” or “False”

Boolean indicator to show if object has holes (>=1 pixel).

13

is_recombined

“False”

“True” or “False”

Boolean indicator to show if object was recombined because it was detected at tile borders of image (tile size: 2048x2048 pixels).

14

object_area [Px^2]

174

8 -

Area of object in pixels^2.

15

outgrowth_area [Px^2]

310

0 -

or ““ (empty)

Area of outgrowth in pixels^2.

Is empty ““ if no outgrowth was set (max. outgrowth was set to 0 pixels).

16

object_incl_outgrowth_area [Px^2]

484

8 -

Sum of areas of object and outgrowth in pixels^2.

17

distance_nearest_neighbor [Px]

22.090722034

0 -

Distance (center-to-center)  to the nearest other object of the same class in pixels.

18

bbox_area [Px^2]

304

8 -

Area of bounding box of detected object in pixels^2.

19

area_ratio [%]

0.02

0 - 100

Area of detected object as percentage of overall image area or ROI area inside the image.

20

perimeter [Px]

53

8 -

Perimeter of detected object in Pixels.

21

circularity

0.78

0 - ~1

Circularity of detected object.

Circularity = 4*pi*area/(perimeter^2).

The circularity of a circle is 1.

22

circularity_ISO

0.65

0 - ~1

Circularity of detected object calculated by ISO definition.

23

solidity

0.97

0 - 1

Solidity of detected object.

Solidity is calculated out of the ratio of an object’s area to the area of its convex hull.

24

eccentricity

0.92

0 - 1

Eccentricity of detected object.

Eccentricity of the ellipse that has the same second-moments as the object area. The eccentricity is the ratio of the focal distance (distance between focal points) over the major axis length. When it is 0, the ellipse becomes a circle.

25

equivalent_diameter [Px]

14

1 -

Equivalent diameter of detected object.

The equivalent diameter is the diameter of a circle having the same area as the detected object.

26

extent

0.57

0 - 1

Extent of detected object.

The extent value is the ratio of the object area to the total bounding box area.

27

minor_axis_length [Px]

9

1 -

Minor axis length of detected object.

The minor axis length is the length of the minor axis of the ellipse that has the same normalized second central moments as the object.

28

major_axis_length [Px]

23

1 -

Major axis length of detected object.

The major axis length is the length of the major axis of the ellipse that has the same normalized second central moments as the object.

For the different channels [CC] in the multichannel image:

29/ 40/ …

channel_#[CC]_index

1

1, 2, 3

Index of channel

30/ 41/ …

channel_#[CC]_name

“DAPI”

text

Name of channel

31/ 42/ …

channel_#[CC]_object_mean_intensity

272.93074030

0 - 65535

Mean intensity of this channel CC over the area of the detected object.

32/ 43/ …

channel_#[CC]_object_stddev_intensity

130.84258715

0 - 65535

Standard deviation of intensity of this channel CC over the area of the detected object.

33/ 44/ …

channel_#[CC]_object_intensity_dominant

“True”

“True”, or

”False”, or

”nan”

Boolean indicator to show if the mean intensity of this channel CC over the area of the object is the highest (=dominant), compared to all other channels).

34/ 45/ …

channel_#[CC]_outgrowth_mean_intensity

273.26551765

0 - 65535, or

”” (empty)

Mean intensity of this channel CC over the area of the outgrowth for the detected object.

Is empty ““ if no outgrowth was set (max. outgrowth was set to 0 pixels).

35/ 46/ …

channel_#[CC]_outgrowth_stddev_intensity

203.15327249

0 - 65535, or

”” (empty)

Standard deviation of the intensity of this channel CC over the area of the outgrowth for the detected object.

Is empty ““ if no outgrowth was set (max. outgrowth was set to 0 pixels).

36/ 47/ …

channel_#[CC]_outgrowth_intensity_dominant

“True”

“True”, or

”False”, or

”nan”

Boolean indicator to show if the mean intensity of this channel CC over the area of the outgrowth for the detected object is the highest (=dominant), compared to all other channels.

Is “False“ if no outgrowth is set (max. outgrowth is 0 pixels).

37/ 48/ …

channel_#[CC]_object_incl_outgrowth_mean_intensity

273.115521066

0 - 65535

Mean intensity of this channel CC over the combined area of the detected object and outgrowth.

38/ 49/ …

channel_#[CC]_object_incl_outgrowth_stddev_intensity

174.47822028

0 - 65535

Standard deviation of intensity of this channel CC over the combined area of the detected object and outgrowth

39/ 50/ …

channel_#[CC]_object_incl_outgrowth_intensity_dominant

“True”

“True”, or

”False”, or

”nan”

Boolean indicator to show if the mean intensity of this channel CC over the combined area of the detected object and outgrowth is the highest (=dominant), compared to all other channels.