Application Name	Confluence Assay
Version	1.0.0
Documentation Version	1318.09.2023 - 1
Input Image(s)	2D (standard and/or WSI) / Time Series / z-Stack / Multichannel Images; RGB or Grayscale (8 bit)
Input Parameter(s)	Regions of interest (optional)
Keywords	confluence, in-vitro, cell growth, growth rate, regeneration, proliferation, migration, microscopy
Short Description	Detection and measurement of the (artificial) gap, the “scratch,” on a confluent cell monolayer for in-vitro wound healing researchpercentage of the analysed image covered by adherent cells. Cell confluence is important for determining timings for splitting and harvesting cells.
References / Literature	For more information regarding the assay cell confluency check e.g. https://pubmeden.ncbi.nlm.nih.gov/15576902/wikipedia.org/wiki/Confluency or https://www.naturebitesizebio.com/63887/articles/nprot.2007.30 ;cell-confluency/

IKOSA Prisma Confluence Assay

You can use this image analysis application or any of our other applications in your account on the IKOSA platform. If it is not on in the list of available applications, please contact your organization's administrator or our team at support@kmlvision.com.

Application description

This application automatically measures the (artificial) gap, the so-called “scratch”, in images showing a cell monolayerpercentage of the analysed image covered by a monolayer of adherent cells. In addition to the scratch widthpercentage area covered with cells, areas that are ( not ) covered with cells are quantified (including/excluding single cells or cell-free areas). A ratio of each area (covered and uncovered) compared to the overall image size is also calculated. The application supports images from phase contrast and brightfield microscopy and arbitrary cell types. This analysis can also be performed on time lapse recordings (time series) to study directional cell migration in vitro, z-stacks, or multichannel images.

In the following sections, we provide the necessary input data requirements that are necessary to obtain accurate image analysis results and a description of the output files.

Input data requirements

Input image(s)

Input for this application is the following image data:

Image type

Color channels

Color depth (per channel)

Size (px)

Resolution (μm/px)

2D (standard and/or WSI),

Time Series,

Multichannel,

Z-Stack*

Check image formats

File formats

3 (RGB)

or

1 (Gray)

8 Bit

Standard images and WSI formats:

Max: 6144 x 6144

typically: 0.3-1.16

Image content

Microscopy (phase contrast or brightfield) image of a cell monolayer showing an (artificial) gap with no adherent cells, typically taken with 10x magnification.

Additional requirements

The measurement of scratch width is only supported for images showing a single (not multiple) scratch.

Info
*Please note: Z-stack images cannot be uploaded into IKOSA but still can be analyzed via IKOSA Prisma API.

Note

Important:

For all images, the following requirements apply:

The illumination must be constant throughout the image(s).
The sample must be in focus, i.e. no blurry regions in image(s).

Input parameter(s)

No additional input parameters are required for this application.

As an optional parameter, a single or multiple regions of interest (ROIs) can be defined in which the analysis should be performed (‘inclusion ROIs’).

Description of output files and their content

Files

File format

Description

1

csv

results.csv:

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

2

jpg

results_vis/vis.jpg (2D image, no ROI), or

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

results_vis/<roi-id>.jpg (2D image, ROI <roi-id>), or

results_vis/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.

Description for visualizations:

Areas and contours
- no color: scratch area cell free (the largest uncovered) area)
- green: adherent cells and cell-covered area
- red: bubbles (=cell-free areas outside the scratch)
- cyan: free/floating cells and cell aggregates inside of scratch
Lines
- magenta: linear fit to scratch “frontlines”, only calculated when the cell-covered area borders are not yet touching anywhere and if the borders are not too curvy

Image Removed

Image Removed Confluence Assay App output visualisation

Image Added Confluence Assay App input file visualisation

Image Added

Reference: Zaritsky A; Natan S; Kaplan D; Ben-Jacob E; Tsarfaty I (2015): Supplemental materials for "Live time-lapse dataset of in vitro wound healing experiments." GigaScience Database. http://dx.doi.org/10.5524/100118

Info
Please note: These files are only created if qualitative result visualization was requested when submitting the analysis job.

Info
Please note: the image quality might be reduced for the presentation in the technical docs.

3

json

annotation_results.json:

JSON file containing detected cells (cell covered regions). The position is measured from the left upper corner (1,1) of the image.

4

json

roiMeta.json:

A json file containing all information regarding the ROIs defined for the analysis job to ensure reproducibility. The file is empty if no ROIs were defined for analysis.

5

jpg

rois_visualization.jpg, or

t<time-step>_z<z-layer>_c<channel>_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, or channel <channel> of a multichannel image.

Info
Please note: This file is only created if inclusion ROIs were defined for analysis.

6

json

jobResultBundleMeta.json:

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

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Please note: This file is only included if bundled or merged analysis jobs are downloaded.

Info

Please note:

The linear fit to the scratch borders (“frontlines”) may not be calculated for quite curvy scratch borders. In this case, frontline-related quantitative values will be zero.In the case of inclusion ROIs that are partially outside of the image, the ROIs are cropped to the areas that lie inside the image.
In the case of inclusion ROIs that are completely outside of the image, no analysis is performed. However, they are still listed in corresponding results files.
A <roi-id> is generated automatically by the application corresponding to the creation date of a ROI. The location of a ROI within an image with its specific <roi-id> can be seen in the file “rois_visualization.jpg.” ROIs that are completely outside of the image are not shown in this file.
All visualizations are downscaled to 25 megapixels (MP), if the original image or inclusion ROI is larger than 25 MP.

Content

results.csv

Single csv-file

If one or more time steps (of a time series), z-layers (of a z-stack), or channels (of a multichannel image) were specified, the results in a specific row refer to the time step/z-layer/channel specified in the corresponding column.

If one or more ROIs were specified, the results in a specific row refer to the ROI specified in the corresponding columns, otherwise (empty ROI columns) the results refer to the whole image.

Column NO.	Column name	Examples	Value range	Description
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	c	2	1 -	Channel, i.e. the position of the channel in the multichannel image.
4	roi_id	ROI-03	ROI-01 -	<roi-id> starting from “ROI-01”. Empty, if no inclusion ROI is specified and the whole image was analyzed.
5	roi_name	“central”	text	Custom text to identify the ROI. Empty, if no inclusion ROI is specified and the whole image was analyzed.
6	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.
7	totalcells_covered_area _without_scratch [Px^2]	1011878	0 - #of pixels in image	Total area without scratch with adherent cells in Pixel^2, includes cell-covered area , and free/floating cells and bubbles (= cell-free areas outside the scratch).
8	totalcells_covered_area_withoutratio_scratch_ratio [%]	68.6	0 - 100	Ratio of total area without scratch with adherent cells to the total area of the image.
9	scratchuncovered_area [Px^2]	36698	0 - #of pixels in image	Total scratch area ( cell-free area in center of image) in Pixel^2. Free/Floating cells inside of scratch are not considered part of the scratch.
10	scratchuncovered_area_ratio [%]	31.4	0 - 100	Ratio of scratch area to total area of image. Free/Floating cells inside of scratch are not considered part of the scratch.	11	scratch_area_incl_free_cells [Px^2]	37580	0 - #of pixels in image	Total scratch area (cell-free area in center of image) in Pixel^2. Free/Floating cells inside of scratch are considered part of the scratch.	12	scratch_area_incl_free_cells_ratio [%]	31.64	0 - 100	Ratio of scratch area to total area of image. Free/Floating cells inside of scratch are considered part of the scratch..
13	scratch_width_area [Px]	400.5	>=0	Scratch width in Pixel, calculated by dividing the scratch_area by the “total length” of the scratch, i.e. the maximum distance between two points on the scratch border. Maximum value depends on the scratch orientation.
14	scratch_width_frontlines_mean [Px]	435	>=0	Scratch width in Pixel, estimated by mean distance between the two linearly fitted “frontlines” (scratch borders).Value is 0, if no frontlines have been fitted. Maximum value depends on the scratch orientation.
15	scratch_width_frontlines_min [Px]	430	>=0	Scratch width in Pixel, estimated by minimum distance between the two linear fitted “frontlines” (scratch borders).Value is 0, if no frontlines have been fitted. Maximum value depends on the scratch orientation.
16	scratch_width_frontlines_max [Px]	440	>=0	Scratch width in Pixel, estimated by maximum distance between the two linear fitted “frontlines” (scratch borders). Value 0, if no frontlines have been fitted. Maximum value depends on the scratch orientation.
17	is_scratch_closed	0	0 or 1	Boolean indicator if scratch is closed (1) or not (0).
18	cells_covered_area [Px^2]	22143	0 - #of pixels in image	Total area of cell-covered region in Pixel^2. Bubbles (= cell-free areas outside the scratch) are not included in this area.
19	cells_covered_area_ratio [%]	68.6	0 - 100	Ratio of cell-covered area to total area of image. Bubbles (= cell-free areas outside the scratch) are not included in this area .
20	cells_covered_area_incl_bubbles [Px^2]	23053	0 - #of pixels in image	Total area of cell-covered region in Pixel^2. Bubbles (= cell-free areas outside the scratch) are included in this area.
21	cells_covered_area_incl_bubbles_ratio [%]	68.9	0 - 100	Ratio of cell-covered area to total area of image. Bubbles (= cell-free areas outside the scratch) are included in this area.

Info
Please note: The parameters marked with an asterisk (*) are calculated using https://scikit-image.org/.

Error information

More information about errors can be found in the Application Error Documentation.

Contact

If you have any questions about this app, as well as suggestions or ideas for new ones, email us at support@kmlvision.com.

Feel free to book a 30-minute meeting to speak with us about IKOSA and the apps!

https://calendly.com/kmlvision/book-the-ikosa-platform-demo

Versions Compared

Old Version 2

New Version 3

Key

Table of contents

IKOSA Prisma Confluence Assay

Application description

Input data requirements

Input image(s)

Input parameter(s)

Description of output files and their content

Files

Content

results.csv

Error information

Contact

Page Comparison

Versions Compared

Old Version 2

New Version 3

Key

Table of contents

IKOSA Prisma Confluence Assay

Application description

Input data requirements

Input image(s)

Input parameter(s)

Description of output files and their content

Files

Content

results.csv

Error information

Contact