Our integrated automated molecular cytogenetic system has been designed to assist the cytogeneticist in rapidly selecting the optimal metaphase spreads for final data capture and optimization. The resulting expert system will
Ø Substantially increase the throughput of these procedures.
Ø Reduce errors due to eye strain, and in particular mitigate fluorescent bleaching due to prolonged exposure of the specimen to microscope’s xenon lamp.
Ø More comprehensively identify chromosomal abnormalities.
Ø Efficiently manage image data from large research and clinical projects.
A brief overview of the project is given below.
Visual basic is used as the interface between the user and all other software. Image processing is done using MatLab Image Processing tool box. Scope Tool (Active X) is used for the automation of the microscope’s stage. IC-PCI frame grabber is used for the configuration of camera.
Image Processing Steps
1) Autofocusing: All the images taken by the microscope should be in good focus for the subsequent image processing algorithms to give good results. The optimal focus position (Z) is obtained by moving the stage in the direction of maximum contrast.
2) Detection of Cover slip: The portion of the slide with dense population of metaphases is covered with thin plastic cover slip and sealed. Image processing algorithms have been developed to detect the location of the cover slip based on the opacity of the sealant.
3) Low power scan of the slide: The area under the cover slip is first scanned at low magnification (10X). Image processing algorithms have been developed to detect the metaphases and store their location. It takes around 360-400 images to cover the entire cover slip.
4) High magnification scan of the metaphases: Locations of the metaphases detected during low power scan are revisited and images of only metaphases are taken at high magnification (100X). The following steps are followed to get the final High magnification image.
a. Centering on the metaphase:
b. Capturing the metaphase with different filter sets.
5) Capture of Probe signals: (In Progress)
a. Detection of scFISH probe signals on the chromosomes.
b. Merging the images from different filter sets and pseudo coloring the probe signals.
6) Ranking: Ranking the images based upon a predefined criterion: The metaphase images are ranked according to their suitability for analysis. The ranking algorithm is benchmarked upon human quality measures of metaphase data and ranks the high magnification images according to those measures.
7) OME: Uploading the images into OME (Open Microscopy Environment) database: Open Microscopy Environment (www.openmicroscopy.org ) is used to handle image data from different projects. During image collection a configuration file is developed describing detailed information about the image. The image along with its configuration data is uploaded to the OME. This would facilitate efficient storage and retrieval of images.