3.1.0 CAM Assay Application Documentation

 

Application Name

CAM Assay

Version

3.1.0

Documentation Version

13.12.2022 - 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

CAM, chorioallantoic membrane, in vivo, angiogenesis, chick, vessel, growth, microscopy, assay

Short Description

Quantification of blood vessels on chick chorioallantoic membrane (CAM) assay used in ex-vivo angiogenesis research.

References / Literature

For more information regarding the assay check e.g. https://www.ncbi.nlm.nih.gov/pubmed/19007659 

Table of contents

IKOSA Prisma CAM 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 the list of available applications, please contact your organization's administrator or our team at support@kmlvision.com.

Application description

This application automatically segments blood vessels in microscopy images that are taken from chick chorioallantoic membrane (CAM) Assay samples and measures their total area, length, mean thickness and the number of branching points. Vessels that are in the background (lower layers) that appear blurry in the images are not detected. This analysis can also be performed on time-lapse recordings (Time Series), z-stacks, or multichannel images, uploaded as multipage TIFF files.

In the following, the requirements for an accurate analysis are given and the output of the application is described.

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)

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

https://kmlvision.atlassian.net/wiki/spaces/KB/pages/3406725124

3 (RGB)

or

1 (Gray)

8 Bit

WSI formats: arbitrary**

Standard images: max. 25,000 x 25,000**

typically: 1-7

Image content

Microscopy image of CAM Assay region.

Additional requirements

  • Vessel thickness has to be between 3 and 220 pixels.

  • Vessels that should be detected have to be in focus.

  • Avoid reflections, e.g. by using  a polarization filter for imaging.

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

**Please note: For images having a dimension larger than 5,000 pixels, skeletonization artifacts occur that may affect the obtained values for length, mean thickness and the number of branching points.

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

File format

Description

1

csv

results.csv

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

2

jpeg

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.

A visualization of the detected vessels contains:

  • Areas of detected vessels are shown in blue overlay.

  • Vessel paths are shown as green lines.

  • Vessel branching points are indicated by red dots.

Results visualization
Input image

Reference: Dr. Nassim Ghaffari Tabrizi-Wizsy, Otto Loewi Research Center, Medical University of Graz.

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

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

3

json

annotation_results.json:

JSON file containing detected vessel structures. 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.

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.

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.

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

Please note:

  • 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

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

Vessels_total_area [Px^2]

3668

0 - #of pixels in image

Total area covered by  detected vessels in Pixel^2.

8

Vessels_total_length [Px]

12092

>=0

Total length of all detected vessels in pixels.

9

Vessels_mean_thickness [Px]

6.98

>=0

Mean thickness of detected vessels in pixels.

10

vessels_num_branching_points

776

>=0

Number of detected branching points.

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

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