|Cell Invasion Analysis|
|Angiogenesis & Common Tools|
|Processing Time Expectation|
|Less than 2 Minutes per Image|
|Wide-Field, Optical Microscope in Bright-Field (Transmission) Mode Using a Color Mosaic CCD or Monochrome CCD with RGB Filter|
|Techniques, Dyes, Stains, & Proteins|
|Processing Time Expectation|
|Less than 4 Minutes per Image|
|Techniques, Dyes, Stains, & Proteins|
|The area of collagen plug, given in pixels.|
|Mean Plug Diameter|
|The mean diameter of the collagen plug, given in pixels. This is measured as the average of all diameters passing through the plug centroid in two-degree increments around the plug.|
|Mean Cell Invasion Distance|
|The mean distance of the furthest cell from the perimeter of the plug, given in pixels. This is measured as the average of distances determined by using a caliper that originates from the centroid and rotates around the plug in one-degree increments to generate 360 distance measurements.|
|Standard Deviation of Invasion Distance|
|The standard deviation of the 360 measurements of the distance between the plug perimeter and furthest cell, given in pixels.|
|Maximum Cell Invasion Distance|
|The maximum value of the 360 measurements of the distance between the plug perimeter and furthest cell, given in pixels.|
|Minimum Cell Invasion Distance|
|The minimum value of the 360 measurements of the distance between the plug perimeter and furthest cell, given in pixels.|
|The area outside of the edge of the collagen plug within which cell invasion is contained, given in pixels. This is measured by connecting the furthest cells from the plug to define the outer invasion border, filling this region, and subtracting the plug area.|
|Cell Area in Invasion Area|
|The sum of pixels representing segmented cells within the Invasion Area.|
This section will identify any output parameters that are only output by the IQbot in its advanced configuration.
This IQbot was developed to analyze images captured using a wide-field, optical microscope in bright-field (transmission) mode using a color mosaic CCD or monochrome CCD with RGB filter. Images for this IQbot must be in color to properly segment the plug. Images submitted for analysis should depict the entire collagen-cell plug and surrounding cells.
The Cell Invasion Analysis IQbot has been built to accommodate the following imaging types only.
As we reference for IQbots in general here, the Cell Invasion Analysis IQbot is only ultimately effective if you upload high quality images for processing. Please ensure that any images you upload follow the below guidelines specific to this IQbot.
High-Resolution: The quality of the output data you receive is largely dependent on the input image resolution. Please provide high-resolution images to ensure that the IQbot can most effectively differentiate between features.
In-Focus: Please produce the most in-focus images possible for analysis. Images with high background contrast will result in more accurate and useful data and overlays.
Properly Saturated: If your submitted images are significantly undersaturated or oversaturated, it is possible that the IQbot will be unable to detect structures, or may group structures together. Please ensure that the images you upload are saturated appropriately.
Clear of Artifacts: Please take care to avoid creating bubbles, debris, or otherwise inducing artifacts into your image. Such additional features may corrupt the resulting data.
Clear of Accessary Information: Please do not submit images retaining any annotations, legends, timestamps, or other markings. Every part of the images that you upload will be analyzed and included in the output data.
If you are uncertain whether or not your images meet the quality standards for which this IQbot was validated, or have additional questions relating to image quality, please view our FAQs or contact the ImageQuantify.com support team.
This IQbot has been successfully validated for use, and a copy of its validation certificate accompanies the results of each order it analyzes. In accordance with general ImageQuantify.com validation procedures, this IQbot has been found to provide quantitative information equivalent to that of a manual observer, but with no variability and increased breadth of information.
The first step of the validation process was to establish the ground truth against which the IQbot’s efficacy would be compared. For the purpose of the Cell Invasion Analysis IQbot, ground truth was defined on a manual observer basis. Trained observers were guided through a standardized, semi-automated process to produce output data from a specially selected validation image set. This image set represented the entire range of expected output values (e.g., low and high cell presence and density). Images were presented for analysis in a randomized order and deidentified to protect against observer biases. The observer mean for each parameter measured was accepted as ground truth.
By analyzing the inter- and intraobserver variability present in the manual observation process, acceptable criteria were established so as to compare the IQbot’s output to that of a trained replacement observer. Ultimately, the IQbot’s performance proved to offer significant upgrades over manual or semi-automated analysis techniques, and validation was considered successful. Further considerations ensured that the IQbot rejected and flagged concerning image-specific outputs and performed within operation time constraints.
The Cell Invasion Analysis IQbot quantifies the density, distance, and aggregate area of cells that have migrated away from a collagen plug into a surrounding matrix. For each bright-field, color image of a collagen plug surrounded by cells, the IQbot segments the plug and outputs its area and mean diameter. Cells migrating away from the plug’s edge are subsequently segmented and a caliper originating from the plug centroid is rotated around the plug periphery (in one-degree increments) to determine the location and distance of the furthest segmented cell from the plug edge. These distances are used to calculate the mean, maximum, and minimum distance of cells from the plug edge. Additionally, the furthest cells from the edge of the plug are connected to form a contiguous cell invasion boundary. The invasion area is then calculated by filling the region formed by this boundary and subtracting the collagen plug area. Lastly, summing the segmented cell pixels within the invasion boundary, cell area in the invasion region can be determined and subsequently used in the calculation of cell invasion density. To enable verification of IQbot cell segmentation and invasion area delineation, two pseudo-colored output images are also generated for each image analyzed. The first output image consists of segmented cells, the invasion boundary, and the collagen plug edge pseudo-colored green, red, and blue, respectively, and superimposed upon the original image (*_overlay.tif). Similarly, a second image is generated in which cell distances from the collagen plug to the outer invasion boundary are pseudo-colored with a blue-red heat map superimposed upon the original image where blue represents a distance of 0 and red is a maximum distance of 125 pixels (*_pseudo.tif).
IQBOT APPLIED IMAGE