Details

Name
Eotaxin Analysis
IQbot ID
IQBOT-0018
Version
1.0.0
Research Area(s)
Neurology, Pulmonology
Processing Time Expectation
Less than 2 Minutes per Image
Acquisition Hardware
Wide-Field, Optical Microscope in Fluorescence Mode Using a Monochrome CCD with the Appropriate Fluorescence Filters
Techniques, Dyes, Stains, & Proteins
DAPI, Eotaxin Antibodies
Sample Context
In Tissue
Name
Eotaxin Analysis
IQbot ID
IQBOT-0018
Version
1.0.0
Research Area(s)
Neurology, Pulmonology
Processing Time Expectation
Less than 2 Minutes per Image
Acquisition Hardware
Wide-Field, Optical Microscope in Fluorescence Mode Using a Monochrome CCD with the Appropriate Fluorescence Filters
Techniques, Dyes, Stains, & Proteins
DAPI, Eotaxin Antibodies
Sample Context
In Tissue

Output Parameters

Number of Cells
The number of nuclei or cells in the image.
IOD
The integrated optical density (the sum of pixel intensities) of eotaxin around a given nucleus (within an ROI ring around each nucleus formed by a 40-pixel extrusion of the nuclear boundary).
Intensity
The mean density of eotaxin around a given nucleus (within an ROI ring around each nucleus formed by a 40-pixel extrusion of the nuclear boundary).
Area
The area (in pixels) of the ROI around each nucleus that extends 40 pixels from the surface of the nucleus.
Background Intensity
The intensity of the background pixels in the eotaxin channel.

 

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

 

Number of Cells
The number of nuclei or cells in the image.
IOD
The integrated optical density (the sum of pixel intensities) of eotaxin around a given nucleus (within an ROI ring around each nucleus formed by a 40-pixel extrusion of the nuclear boundary).
Intensity
The mean density of eotaxin around a given nucleus (within an ROI ring around each nucleus formed by a 40-pixel extrusion of the nuclear boundary).
Area
The area (in pixels) of the ROI around each nucleus that extends 40 pixels from the surface of the nucleus.
Background Intensity
The intensity of the background pixels in the eotaxin channel.

Image Acquisition


This IQbot was developed to analyze images captured with a wide-field, optical microscope in fluorescence mode using a monochrome CCD with the appropriate fluorescence filters. Input images must be overlays of a DAPI (blue) and eotaxin (green or red) channels.

 
 

The Eotaxin Analysis IQbot has been built to accept .tif/.tiff files or 24-bit RGBs only.

As we reference for IQbots in general here, the Eotaxin 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.

 

     (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 purposes of the Eotaxin 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 to high eotaxin intensity and nuclear presence). 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 Eotaxin Analysis IQbot analyzes cytoplasmic eotaxin (-1, -2, or -3) fluorescence in cytospun (non-adherent) cells. Eotaxin is a chemokine involved in eosinophil chemotaxis activation during inflammation. In images of non-adherent cells (eosinophils) that have been cytospun onto a slide, fixed, and fluorescently stained with a nuclear marker (i.e., DAPI) and eotaxin antibodies, this IQbot segments and counts the total number of nuclei (Number of Cells). Subsequently, the Eotaxin Analysis IQbot extrudes the boundary of each nucleus a fixed distance into the cytoplasm, forming a ring-shaped region of interest (ROI) for analysis of eotaxin mean intensity (Intensity) and integrated optical density (IOD, the sum of pixel intensities) for each cell. These values, in addition to the area of the ROI (Area), are exported to Excel for each cell in a given image. Additionally, the IQbot also generates a pseudo-colored overlay image that contains outlines of the ring-shaped ROIs around each cell utilized for eotaxin intensity analysis (*_overlay1.tif).

DEMO IMAGE
IQBOT APPLIED IMAGE

 

SAMPLE DATA
Image Number of Cells IOD Intensity Area Background Intensity
Sample 37 67834 7.365 9210 55.01
36 Additional Rows 36 Additional Rows 36 Additional Rows