Details

Name
Sox2 Vessel Proximity Analysis
IQbot ID
IQBOT-0017
Version
1.0.0
Research Area(s)
Angiogenesis, Cancer Research, Genetics, Neurology
Processing Time Expectation
Less than 2 Minutes per Image
Acquisition Hardware
Fluorescence Optical Microscopy (Confocal or Wide-Field); Optimized for Single Field Images (5-20x), but Compatible with Large Field-of-View Images
Techniques, Dyes, Stains, & Proteins
Samples Must be Fluorescently Stained and Label Sox2 Using Dyes of Antibodies that have a Nuclear Component of Staining, a Marker that Stains the Vessel Borders (i.e., Vessel or Endothelial Cell Markers), and a Nucler Dye not Dependent upon the Presence of the Protein of Interest*
Sample Context
In Tissue

 

Name
Sox2 Vessel Proximity Analysis
IQbot ID
IQBOT-0017
Version
1.0.0
Research Area(s)
Angiogenesis, Cancer Research, Genetics, Neurology
Processing Time Expectation
Less than 2 Minutes per Image
Acquisition Hardware
Fluorescence Optical Miscroscopy (Confocal or Wide-Field); Optimized for Single Field Images (5-20x), but Compatible with Large Field-of-View Images
Techniques, Dyes, Stains, & Proteins
Samples Must be Fluorescently Stained and Label Sox2 Using Dyes of Antibodies that have a Nuclear Component of Staining, a Marker that Stains the Vessel Borders (i.e., Vessel or Endothelial Cell Markers), and a Nucler Dye not Dependent upon the Presence of the Protein of Interest*
Sample Context
In Tissue

Output Parameters

Total Cells
The total number of cells in an image (defined by nuclear marker, i.e., DAPI).
Sox2-Positive Cells
The total number of Sox2-positive cells in an image (defined by antibody staining, mean nuclear intensity >= 40).
Sox2-Positive Cell-Vessel Distances (pixels)
The distance of given Sox2-positive nucleus (centroid) from the closest vessel border (labeled using a secondary marker, i.e., von Villebrand Factor, smooth muscle cell actin, etc.).
Sox2-Positive Cell-Vessel Intensity
The mean nuclear intensity of the Sox2-positive Cell (DAPI-segmented nuclear mask applied to Sox2 channel) whose vessel distance is defined above.
Sox2-Negative Cell-Vessel Distances (pixels)
The distance of given Sox2-negative nucleus (centroid) from the closest vessel border.
Sox2-Negative Cell-Vessel Intensity
The mean nuclear intensity of the Sox2 Negative Cell (DAPI-segmented nuclear mask applied to Sox2 channel) whose vessel distance is defined above.

 

This section will identify any output parameters that are only output by the IQbot in its advanced configuration.

 

Total Cells
The total number of cells in an image (defined by nuclear marker, i.e., DAPI).
Sox2-Positive Cells
The total number of Sox2-positive cells in an image (defined by antibody staining, mean nuclear intensity >= 40).
Sox2-Positive Cell-Vessel Distances (pixels)
The distance of given Sox2-positive nucleus (centroid) from the closest vessel border (labeled using a secondary marker, i.e., von Villebrand Factor, smooth muscle cell actin, etc.).
Sox2-Positive Cell-Vessel Intensity
The mean nuclear intensity of the Sox2-positive Cell (DAPI-segmented nuclear mask applied to Sox2 channel) whose vessel distance is defined above.
Sox2-Negative Cell-Vessel Distances (pixels)
The distance of given Sox2-negative nucleus (centroid) from the closest vessel border.
Sox2-Negative Cell-Vessel Intensity
The mean nuclear intensity of the Sox2 Negative Cell (DAPI-segmented nuclear mask applied to Sox2 channel) whose vessel distance is defined above.

Image Acquisition


Tissue cross-section samples must be fluorescently stained and include the protein or marker of interest (i.e., Sox2) labeled using dyes or antibodies that have a nuclear component of staining, a marker (dye or antibody) that stains the vessel borders (i.e., vessel or endothelial cell markers), and a nuclear dye not dependent upon the presence of the protein of interest (i.e., DAPI). Images may be acquired using a fluorescence optical microscope (confocal or wide-field) with the appropriate filters. This IQbot has been optimized for single field images (5-20x) but may be applied to large field-of-images.

 
 

The Sox2 Vessel Proximity Analysis IQbot has been built to accept .tif or .tiff files which must be 24-bit overlays of all channels.

As we reference for IQbots in general here, the Sox2 Vessel Proximity Analysis 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.

 

     (216) 678-9258           help@imagequantify.com

 

Validation Information


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 High Magnification Cell Division 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., with varying Sox2 and vessel characteristics). 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.

 

In fluorescently stained tissue cross-sections, the Sox2 Vessel Proximity Analysis IQbot calculates the shortest distance of each Sox2-positive (mean nuclear threshold >= 40) and Sox2-negative cell from a vessel border labeled with either antibodies for smooth muscle cell actin or von Willebrand Factor. The distances are measured from the centroid of each cell's nucleus labeled with DAPI (4',6-diamidino-2-phenylindole) to the closed segmented vessel edge pixel. Relative distances or proximity of cells negative and positive for Sox2, the SRY (sex determining region Y)-box 2 transcription factor (a stem cell marker), to vessels can provide a method for defining metastatic potential for a given tumor. Outputs of the IQbot include distance of each and every cell (positive or negative) within a given image to the closest vessel along with the mean intensity of Sox2 labeling within that particular cell's nucleus (since each cell will have corresponding intensity reported in the output data generated by the IQbot, it is possible for end user to redefine the threshold post-analysis if the 40 gray level cutoff is not appropriate). Additionally the IQbot will generate pseudo-colored overlays including 1) an image of the DAPI channel with an green outline around Sox2-positive nuclei, blue outline around nuclei negative for Sox2, and red outline around vessel borders (*_pseudo.tif), 2) an image of the DAPI channel where the centroid of each positive nucleus contains a dot that is pseudo-colored using a blue-red look-up table - red represents the nucleus furthest away from a vessel border and blue is closest to a vessel border, red outlines delineate vessel borders (*_pseudo_pos_map.tif), and 3) an image of the DAPI channel where the centroid of each negative nucleus contains a dot that is pseudo-colored using a blue-red look-up table - red represents the nucleus furthest away from a vessel border and blue is closest to a vessel border, red outlines delineate vessel borders (*_pseudo_neg_map.tif).

DEMO IMAGE
IQBOT APPLIED IMAGE

 

SAMPLE DATA
Image Total Cells Sox2-Positive Cells Sox2-Positive Cell-Vessel Distances
(pixels)
Sox2-Positive Cell-Vessel Intensity Sox2-Negative Cell-Vessel Distances
(pixels)
Sox2-Negative Cell-Vessel Intensity
Sample 120 17 44.16 47.62 212.81 25.48
16 Additional Rows 16 Additional Rows 102 Additional Rows 102 Additional Rows