Posted on Sep 4, 2018

Assay Option: COLORIZE-TRACKING-IMAGE

ACAS by default does not colorize the loops. You can enable this feature by adding the following text to the experiment-set description:

COLORIZE-TRACKING-IMAGE

Here is a sample of colorized closed loops:

colorized angiogenesis tube formation
 

Setting Assay Options

Assay Option: COLORIZE-TRACKING-IMAGE

ACAS by default does not colorize the loops. You can enable this feature by adding the following text to the experiment-set description:

COLORIZE-TRACKING-IMAGE

Here is a sample of colorized closed loops:

colorized angiogenesis tube formation
 

Setting Assay Options

Posted on Sep 4, 2018

Assay Option: FAINT-EDGE-DETECTION

As the experiment progresses, the tubes become thinner until the point when the tubes visually disappear. During this transition the human reader can find faint tubes that are beyond the capability of automated analysis. In the image shown below, the red boxes indicate areas where the tubes are in various stages of disappearing from the image.

angiogenesis tube formation analysis faint edges

The automated system must also face a large variety of input images of varying resolution and quality.  An aggressive linear edge detection can introduce un-wanted artifacts in noisy images.  So we have chosen to be less aggressive on faint edge detection, by default, to provide the best possible result for the maximum number of researchers.  However, we have provided an option to increase the aggressiveness of the faint edge detection.

In this example, a manual analysis indicates the presence of 21 closed loops.

angiogenesis tube formation analysis faint edges manual analysis

In this example, the default analysis mode (less aggressive faint linear edge detection) will produce the following output, finding a total of 15 closed loops:

angiogenesis tube formation analysis faint edges no enhancements

To increase the aggressiveness of the faint linear edge detection, add this text to the experiment-set description field:

FAINT-EDGE-DETECTION

With this option enabled, the following output is produced, finding a total of 22 closed loops.

angiogenesis tube formation analysis faint edges with enhancements

While the total loops in this analysis is one more than the manual analysis, there are artifacts which missed one loop and created false loops.

  1. loop 10 from the manual analysis was lost (-1)
  2. number 20 from the manual analysis was split into two by the image margin (+1)
  3. number 12 from the manual was lost (-1),
  4. number 16 from the manual was split into three due to the margin and bubble (+2)
  5. number 10 from the manual was lost (-1)

for a net gain of 1.

Related Posts

 

Setting Assay Options

data azat height

Assay Option: FAINT-EDGE-DETECTION

As the experiment progresses, the tubes become thinner until the point when the tubes visually disappear. During this transition the human reader can find faint tubes that are beyond the capability of automated analysis. In the image shown below, the red boxes indicate areas where the tubes are in various stages of disappearing from the image.

angiogenesis tube formation analysis faint edges

The automated system must also face a large variety of input images of varying resolution and quality.  An aggressive linear edge detection can introduce un-wanted artifacts in noisy images.  So we have chosen to be less aggressive on faint edge detection, by default, to provide the best possible result for the maximum number of researchers.  However, we have provided an option to increase the aggressiveness of the faint edge detection.

In this example, a manual analysis indicates the presence of 21 closed loops.

angiogenesis tube formation analysis faint edges manual analysis

In this example, the default analysis mode (less aggressive faint linear edge detection) will produce the following output, finding a total of 15 closed loops:

angiogenesis tube formation analysis faint edges no enhancements

To increase the aggressiveness of the faint linear edge detection, add this text to the experiment-set description field:

FAINT-EDGE-DETECTION

With this option enabled, the following output is produced, finding a total of 22 closed loops.

angiogenesis tube formation analysis faint edges with enhancements

While the total loops in this analysis is one more than the manual analysis, there are artifacts which missed one loop and created false loops.

  1. loop 10 from the manual analysis was lost (-1)
  2. number 20 from the manual analysis was split into two by the image margin (+1)
  3. number 12 from the manual was lost (-1),
  4. number 16 from the manual was split into three due to the margin and bubble (+2)
  5. number 10 from the manual was lost (-1)

for a net gain of 1.

Related Posts

 

Setting Assay Options

data azat height

Posted on Sep 4, 2018

Some researchers prefer to include loops which are detected partially, others prefer to exclude loops which are only partially in the captured image.  The ACAS default setting is to exclude partial loops. This can be overridden by adding this text in the experiment-set description:

DONT-EXCLUDE-EDGE-LOOPS

In this example, with manual analysis, we found 9 closed loops that are fully enclosed in the captured image.

angiogenesis tube formation faint edges manual analysis exclude edge loops

In automated analysis, seven closed loops are detected that are completely enclosed in the image, less the margin.  A key distinction between the auto and manual analysis is the image margin. The algorithms are not able to accurately detect structures to the very edge of the image. The greater the resolution (increased number of pixels), the smaller the margin will be (that is, the algorithms will do a better job up to the edge. In this low-resolution image (0.65 pix/µm), the margin is relatively large.

angiogenesis tube formation faint edges auto analysis exclude edge loops

Some researchers prefer to include loops which are detected partially, others prefer to exclude loops which are only partially in the captured image.  The ACAS default setting is to exclude partial loops. This can be overridden by adding this text in the experiment-set description:

DONT-EXCLUDE-EDGE-LOOPS

In this example, with manual analysis, we found 9 closed loops that are fully enclosed in the captured image.

angiogenesis tube formation faint edges manual analysis exclude edge loops

In automated analysis, seven closed loops are detected that are completely enclosed in the image, less the margin.  A key distinction between the auto and manual analysis is the image margin. The algorithms are not able to accurately detect structures to the very edge of the image. The greater the resolution (increased number of pixels), the smaller the margin will be (that is, the algorithms will do a better job up to the edge. In this low-resolution image (0.65 pix/µm), the margin is relatively large.

angiogenesis tube formation faint edges auto analysis exclude edge loops

Posted on Sep 3, 2018

For a three-dimensional collagen lattice with a final volume of 150 µl, the following protocol is recommended. The collagen lattice is prepared in two parts: 100 µl collagen preparation and 50 µl cell suspension

Part A. Collagen Preparation

5 µl bicarbonate (e.g. Sigma-Aldrich S8761-100ML)

10 µl 10x concentrated minimum essential Eagle’s medium (e.g. Sigma-Aldrich M0275-100ML)

85 µl PureCol (e.g. Inamed, Fremont, USA, catalog #5005-100ML)

Mix components at room temperature under sterile conditions.

Links to recommended reagents:

https://www.sigmaaldrich.com/catalog/product/sigma/s8761?lang=en&region=US

https://www.sigmaaldrich.com/life-science/cell-culture/classical-media-salts/mem-media.html

https://www.advancedbiomatrix.com/collagen-type-i/purecol-bovine-collagen-product-3-mgml/

Part B. Cell Suspension

Use regular cell culture medium without serum that is recommended for the cell line or use PBS.

Adjust amount of cells to 1.5x105 for tumor cells (final concentration 5x104 tumor cells) or 4.5x105 for leukocytes (final concentration 1.5x105 leukocytes) in 50 µl. When using mixed cell cultures, lower number of each cell type by half. Suspend each cell type in 25 µl and mix then. Further adjustments of cells numbers might be necessary due the size of the cells.

When a pharmacological substance is tested, suspend cells in 35 µl and add 15 µl of solubilized drug at 10fold of the desired final concentration (cells generally tolerate a final concentration of 0.1% ethanol or 0,05% DMSO, if the substance is not soluble in PBS-buffer).

Mix 50 µl cell suspension and 100 µl collagen preparation, pipette into chamber. Let it form a collagen lattice for 30 min in an incubator at 37° C in a humidified atmosphere with 5% CO2.

Fill remaining compartments of the chamber as per recommendation of the supplier. Seal chamber before recording.

Recommended Chambers:

For a three-dimensional collagen lattice with a final volume of 150 µl, the following protocol is recommended. The collagen lattice is prepared in two parts: 100 µl collagen preparation and 50 µl cell suspension

Part A. Collagen Preparation

5 µl bicarbonate (e.g. Sigma-Aldrich S8761-100ML)

10 µl 10x concentrated minimum essential Eagle’s medium (e.g. Sigma-Aldrich M0275-100ML)

85 µl PureCol (e.g. Inamed, Fremont, USA, catalog #5005-100ML)

Mix components at room temperature under sterile conditions.

Links to recommended reagents:

https://www.sigmaaldrich.com/catalog/product/sigma/s8761?lang=en&region=US

https://www.sigmaaldrich.com/life-science/cell-culture/classical-media-salts/mem-media.html

https://www.advancedbiomatrix.com/collagen-type-i/purecol-bovine-collagen-product-3-mgml/

Part B. Cell Suspension

Use regular cell culture medium without serum that is recommended for the cell line or use PBS.

Adjust amount of cells to 1.5x105 for tumor cells (final concentration 5x104 tumor cells) or 4.5x105 for leukocytes (final concentration 1.5x105 leukocytes) in 50 µl. When using mixed cell cultures, lower number of each cell type by half. Suspend each cell type in 25 µl and mix then. Further adjustments of cells numbers might be necessary due the size of the cells.

When a pharmacological substance is tested, suspend cells in 35 µl and add 15 µl of solubilized drug at 10fold of the desired final concentration (cells generally tolerate a final concentration of 0.1% ethanol or 0,05% DMSO, if the substance is not soluble in PBS-buffer).

Mix 50 µl cell suspension and 100 µl collagen preparation, pipette into chamber. Let it form a collagen lattice for 30 min in an incubator at 37° C in a humidified atmosphere with 5% CO2.

Fill remaining compartments of the chamber as per recommendation of the supplier. Seal chamber before recording.

Recommended Chambers:

Posted on Sep 3, 2018

For fluorescence, phase-contrast, bright field, and dark-field imaging, we recommend the following ibidi chambers. These chambers have high-resolution bottoms and chamber walls that will not interfere with the side-illumination used in dark-field.

In all live culture experiments, meniscus formation is a concern. A meniscus forms if the culture is allowed to dry out during the course of the experiment. The meniscus forms something of a lens effect and distorts the image around the edges. To eliminate the formation of a meniscus, it's important to have a properly sealed chamber. Also, a larger chamber is better because it keeps the edge meniscus outside of the center field of view.

The following ibidi chambers are recommended:

µ-Slide VI 0.4

µ-Slide 4 Well Ph+

The larger 35 mm dishes will also work since the have a quite large well where the meniscus is less prominent:

µ-Dish 35 mm, high

ibidi has a free sample program:  https://ibidi.com/module/ibidifreesample/request

For fluorescence, phase-contrast, bright field, and dark-field imaging, we recommend the following ibidi chambers. These chambers have high-resolution bottoms and chamber walls that will not interfere with the side-illumination used in dark-field.

In all live culture experiments, meniscus formation is a concern. A meniscus forms if the culture is allowed to dry out during the course of the experiment. The meniscus forms something of a lens effect and distorts the image around the edges. To eliminate the formation of a meniscus, it's important to have a properly sealed chamber. Also, a larger chamber is better because it keeps the edge meniscus outside of the center field of view.

The following ibidi chambers are recommended:

µ-Slide VI 0.4

µ-Slide 4 Well Ph+

The larger 35 mm dishes will also work since the have a quite large well where the meniscus is less prominent:

µ-Dish 35 mm, high

ibidi has a free sample program:  https://ibidi.com/module/ibidifreesample/request

Posted on Sep 3, 2018

About Plate Selection and Reports

Because the final report will compare results from all wells in the plate, the plate selection is important for creating the correct number of wells. For example, if you choose a 24 well plate, 24 wells will be created and the report will compare the results for 24 wells.  The reports will not be automatically generated until all 24 wells have been populated with input images and those images have been processed.

If you only upload say 20 wells in a 24 well plate, you will not get a report. If you don't intent to upload all 24 wells, you can delete the remaining open wells, then click the report generation icon: report icon

Note: If you choose a plate with multiple wells but do not upload images for all of the wells, a report will not be generated. The report tool waits for results from all wells before generating a final report.

The Custom Plate

The Custom plate is the most flexible. The Custom plate is very flexible because the number of wells is determined when you upload your files. Any number starting from 1 is allowed.

Your well names will be assigned based on the name of the files uploaded or the folders containing the files. These well names will be propagated to your final report.

Manually Generating A Report

You can always edit the well names and regenerate a report by clicking on the report icon shown here: report icon

Well Naming

If you use the wizard to upload files, the wells will be renamed using the file name (or folder name if you have one folder per well).  But if you upload one image file for each well manually, then you will have the option to keep the default well name or use the file name.

Click here to view the plate guide:

selecting plates

About Plate Selection and Reports

Because the final report will compare results from all wells in the plate, the plate selection is important for creating the correct number of wells. For example, if you choose a 24 well plate, 24 wells will be created and the report will compare the results for 24 wells.  The reports will not be automatically generated until all 24 wells have been populated with input images and those images have been processed.

If you only upload say 20 wells in a 24 well plate, you will not get a report. If you don't intent to upload all 24 wells, you can delete the remaining open wells, then click the report generation icon: report icon

Note: If you choose a plate with multiple wells but do not upload images for all of the wells, a report will not be generated. The report tool waits for results from all wells before generating a final report.

The Custom Plate

The Custom plate is the most flexible. The Custom plate is very flexible because the number of wells is determined when you upload your files. Any number starting from 1 is allowed.

Your well names will be assigned based on the name of the files uploaded or the folders containing the files. These well names will be propagated to your final report.

Manually Generating A Report

You can always edit the well names and regenerate a report by clicking on the report icon shown here: report icon

Well Naming

If you use the wizard to upload files, the wells will be renamed using the file name (or folder name if you have one folder per well).  But if you upload one image file for each well manually, then you will have the option to keep the default well name or use the file name.

Click here to view the plate guide:

selecting plates

Posted on Sep 3, 2018

We find that two-channel fluorescence imaging with a zoom factor of 5X to 10X and a high-resolution camera produce the best results. To achieve optimal tracking we suggest you should attempt to duplicate the sample images below in terms of contrast and resolution.

Imaging Mode fluorescence on inverted microscope
Camera Resolution 2048 pixels wide or greater
Field-of-view approximately 1 mm x 1 mm
Microscopy Resolution optimal 3.0 pix/µm, minimum: 0.5 pix/µm (calculating microscopy resolution tech note)
Glass Slide See 'Recommended Chambers' section below.
Time-lapse Recording mp4 movie file containing H.264 encoded images
Time-between-frames One frame recorded each 30 seconds

Sample Fluorescence-Based Imaging

FIBROBLASTS IN 2D COLLAGEN

Imaging Mode Dark-field, inverted microscope
Camera Resolution 1280 x 1024
Field-of-view 1,200 µm to 1,612µm (cropped from original)
Microscopy Resolution 0.623 pix/µm
Glass Slide high-quality flat bottom glass ibidi µ-Dish 35 with high resolution glass bottom

darkfield fibroblast microscopy image

BREAST CANCER CELLS IN 3D COLLAGEN

Imaging Mode Dark-field, inverted microscope
Camera Resolution 2080 x 1584
Field-of-view approximately 1 mm x 1 mm
Microscopy Resolution 2.06 pix/µm
Glass Slide standard glass slide
darkfield breastcancer microscopy cells

Sample mp4/H.264 encoded movie from the microscopy setup described above:

We find that two-channel fluorescence imaging with a zoom factor of 5X to 10X and a high-resolution camera produce the best results. To achieve optimal tracking we suggest you should attempt to duplicate the sample images below in terms of contrast and resolution.

Imaging Mode fluorescence on inverted microscope
Camera Resolution 2048 pixels wide or greater
Field-of-view approximately 1 mm x 1 mm
Microscopy Resolution optimal 3.0 pix/µm, minimum: 0.5 pix/µm (calculating microscopy resolution tech note)
Glass Slide See 'Recommended Chambers' section below.
Time-lapse Recording mp4 movie file containing H.264 encoded images
Time-between-frames One frame recorded each 30 seconds

Sample Fluorescence-Based Imaging

FIBROBLASTS IN 2D COLLAGEN

Imaging Mode Dark-field, inverted microscope
Camera Resolution 1280 x 1024
Field-of-view 1,200 µm to 1,612µm (cropped from original)
Microscopy Resolution 0.623 pix/µm
Glass Slide high-quality flat bottom glass ibidi µ-Dish 35 with high resolution glass bottom

darkfield fibroblast microscopy image

BREAST CANCER CELLS IN 3D COLLAGEN

Imaging Mode Dark-field, inverted microscope
Camera Resolution 2080 x 1584
Field-of-view approximately 1 mm x 1 mm
Microscopy Resolution 2.06 pix/µm
Glass Slide standard glass slide
darkfield breastcancer microscopy cells

Sample mp4/H.264 encoded movie from the microscopy setup described above: