Research And Development Of Two-Wavelength Confocal Fluorescent Microarray Analyzer

The microarray technology is a new branch of biology and life science, which emerges at a tremendous pace. It is based on the glass surface, silicon wafer, nylon substrate etc, on which large quantities of biomaterials are immobilized in high density. The microarray technology allows an accurate, rapid and parallel analysis of thousands of genes, proteins, cells and other biomolecules in a single experiment. It is becoming a common tool in many areas of biology and life research, including drug discovery, clinical diagnosis, genome structure and function research etc. Microarray experiments depend on the hybridization reaction. The test samples are tagged with the fluorescent dyes and hybridized with the microarrays. A fluorescent analyzer scans the microarrays and interprets the fluorescent signals, so it is a necessary instrument to obtain the microarray experimental results and a key one which boosts the microarray technology to be used widely.How to design and implement a laser scanning confocal microarray analyzer was introduced in the dissertation, mainly including four aspects: the design scheme, performance analysis, experimental testing and result discussion. The analyzer would be reasonable cost, good performance and own intellectual property. From the view of industrialization, we did some meaningful work and made an experimental prototype. In this prototype, the Cy3 and Cy5 fluorophores were excited by a 532nm green laser and a 635nm red laser respectively. The emitted fluorescent signal was detected using a photomultiplier tube (PMT) sequentially. One dimension scanning of the microarray slide was performed by a telecentric f-theta objective with a moving coil optical scanner; the other dimension was scanned through a stepping motor driving the precision guidance. The features and specifications of the analyzer, such as the resolution, signal-to-noise ratio, detection limit and dynamic range, were analyzed based on the actual parameters. The distortion of the f-theta objective and the frequency response error of the optical scanner were analyzed too, they would affect the field uniformity of the instrument. The reproducibility and cross-talk reduction were discussed and an auto gain control system for the PMT was proposed. When scanning by green laser, the resolution of the analyzer would be 5uim and the detection limit would be 1 fluor/um2 for Cy3. Its dynamic range of detection would be linear over 3 orders of magnitude. When scanning the microarray slide us' ig two lasers and at 5 microns resolution, it cost 400 seconds, faster than most of the confocal microarray analyzer under the same condition. The prototype was integrated with the embedded operating system, monitor and printer. The data were processed and analyzed by a DSP. The fluorescent image and the experimental results could be displayed on the monitor or printed out by the printer. It was designed according the requirement of the hospital. All the procedures of scanning and analyzing were automated and straightforward.The basic image processing technique was also included in the dissertation. An algorithm for image enhancing was designed. It not only reduced the impulsive noise, but also corrected the background variation in the image. The microarray gridding was performed according image projections. To identify the spots from the background accurately, both the adaptive circle and adaptive shape segmentation algorithm were discussed in detail.