Quail Egg Image Analysis

We are working with the Childrens Cancer Therapy Development Center to help automate testing with quail eggs. This project aims to automate the process of checking eggs for current and future viability during treatment.

=Problem Definition=

Background
Preclinical research goes from in vitro studies that are inexpensive and not necessarily indicative of the disease to mouse studies that are better models but prohibitively expensive at scale. Chorioallantoic membrane (CAM) assays utilizing Japanese quail (Coturnix Japonica) are a cost-effective screening method to precede murine xenografts. To increase the throughput of CAM assays, an optimized platform has been built for processing 100-200 quail a week to validate one or more in vitro hit per week. Concordance and divergence from in vitro data and concordance with murine in vivo studies is demonstrated, suggesting a role for ex ovo studies in the validation of new anticancer agents.

Automatic detection of shell-free quail embryos that will not be viable is needed before tumor cell engraftment and drug testing are done. The Egg Buddy MK2 digital heart monitor provides proof of concept that EKG is possible.

Deliverables
Our goal is to create an automated system that monitors viability parameters of quail embryos in six-well plate for the use in cancer drug therapy tests.

This device must be able to determine heart rate, oxygen saturation, pH monitoring, and CAM blood vessel area and branch point counting, while also using minimal to non-invasive, using aseptic techniques. This also needs to be able to communicate with a desktop application for easier and faster use.

Specifications
The device should determine the following metrics by a combination of touch-free or minimally-invasive, aseptic electrodes.
 * Heart rate
 * Oximetry (oxygen saturation)
 * Angiogenesis Quantification (Vessel Branch Counting)

These results need to be viewed and parameters modified from an easy to use interface.

Imaging can be taken from above or below using visible or near-infrared light. Nothing should touch the internal components of the plate if possible. Components should have minimal luminescence or light-scattering.

The device should keep its environment within the following parameters to support normal incubation of Coturnix Japonica:

=Design Considerations= All processes must be conducted as non-invasively as possible to avoid affecting the eggs development. The measurements should be taken with as little human involvement as possible. In an ideal scenario, this design will be fully automated, only requiring intervention based on its output.

We have looked at a number of different sensors and ways to apply them. We are using using NASA’s Vessel Generation Analysis (VESGEN) Software for vessel branch counting. Acoustocardiography and Ballistocardiography for heart rate, and image processing for a pH sensor.

Andiogenesis Quantification
Measuring the growth and formation of new blood vessels in Chorioallantoic Membrane (CAM). The following parameters will need to be measured: These metrics are taken at specific sites and used to help determine viability. VESGEN will automatically compile these parameters as described in the Software section.
 * Dv Vessel Diameter
 * Nv Vessel Number Density
 * Bry Vessel Branch Point Density
 * Lv Vessel Length Density
 * Av Vessel Area Density

The program ImageJ handles the binary conversion of the source image. The VESGEN plugin for this software further processes this input and provides us with a wide range of potentially useful metrics, such as vessel count and the length diameter and tortuosity of individual vessels.

The current challenge is automating VESGEN itself. The current version does not support macros or calls, and the VESGEN plugin must be reloaded after each generation. We have communicated with the VESGEN devs, and they confirmed the lack of macro support, but said that it is a desired feature in a future update. We can automate the image preprocessing, binarization and defining a region of interest within the image, as well as opening images with ImageJ. Converting the results is also automatable, but the ImageJ functions are not. We are looking for potential workarounds to fully or mostly automate this process.

We can convert the dialogue output to a text file. Because the output has a consistent format, we use a python script that converts this text file into a Comma-Separated format. This file can contain any set of values we need and can be easily utilized in other programs.

Once we are receiving images from our final design prototype, the binarization will need to be adjusted based on lighting and positioning. A Calibration Factor will also need to be determined. VESGEN provides its' output based upon pixels. Once we have a consistent scale, we can convert this value to a meaningful measurement.

Heart Rate Monitoring
Heart rate is a useful metric for determining viability. The method needs to be non invasive, but still fairly accurate. The following methods are under consideration.


 * Ballistocardiography: Ballistocardiograph is a non-invasive method used to measure heart rate based on the size of the motion generated by the ejection of the blood at each cardiac cycle. A Piezoelectric Sensor produces a charge created across certain materials when a mechanical stress is applied to it. Which is then read and recorded as a voltage spike.

To test this technique, we are using an Arduino UNO and a DFROBOT SEN0209 piezoelectric sensor. When force is applied to this sensor, depending on its severity, it creates a voltage of +/- 90V, giving a wide range. It has a built-in sensitivity adjustment to increase/decrease threshold potential, and has a dynamic range of 0.001HZ-1000MHz, which is definitely precise enough to read Quail Heart beats.


 * Ultrasound: Using sub-20kHz waves a positive doppler shift can be detected. Measuring this shift can give a useful metric for heartrate.


 * Photoplethysmography: Using a standard SpO2 sensor underneath the eggs to measure O2 saturation and heart rate. This method will need to filter out regular embryo movement.

pH Monitoring
We want to determine minimally invasive and sterile way to monitor pH without hampering the quail embryo. The idea behind observing the pH is to observe the effect of change in pH on the viability of the eggs. We are using three different methodology which are pH probe, pH strips and pH dye.

To test the pH, we are using Atlas EZO ORP circuit and Atlas Ph to build the pH circuit. We have tested the pH using the pH probe, strips and dye and result for all three are accurate. To increase the precision of the result from the dye we are using image processing tool in MATLAB.

Hardware
The enclosure should contain a camera for imaging and additional sensors for tracking metrics such as heart rate, as well as environmental variables such as humidity and temperature. A system on a chip (SOC) should be mounted externally and connected to all sensors. A driver program connecting to this SOC should be able to run on various user devices. This connection can be made either by physical connection or a wireless protocol, such as Bluetooth.

Software
Vessel area and branch point count will be handled by NASA licensed software: Vessel Genration Analysis, (VESGEN). Receiving input for heart rate and oxygen saturation may require additional software, running on the SOC or driver program on a separate system. A driver program will run on the separate system, handling the processing and UI. This program should be multi-platform, running on Windows, OSX, and potentially Linux. Inputs should all be received by the SOC and then sent to the driver for processing and display.

This software should display all pertinent gathered or processed information. It should provide the status of each egg's viability in an intuitive and user-friendly form. It may also provide projections for future viability.

=Project Learning=

=Final Design=

=Validation=

=Team Members=

=Additional Documentation=

Project Schedule

Meeting Minutes

Presentations


 * Meet The Team
 * Snapshot I