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Proximal Correlation Fluorescence Sensing for HTS Microarray Imaging
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The motivation behind this project is that normally to run microarray you need work with 2 different types of instruments. The first being an being an instrument that is used for microarray fabrication. The second being an scanner that generates a high resolution image of the slide. These are normally classified as being capital equipment purchases (ie., items greater then $20K per unit). Added to the cost of array substrate surfaces, as explained in the application note describing how to develop a recycleable bioassay surface, it is clear that despite the potential of conserving high cost biological material and for increasing the throughput using the microarray format, the materials are still too high for many research labs.
So this was project was developed to see if is possible to combine the scanning process with the fabrication process thereby using one instrument to do both tasks. In this example, demonstrated is a proximal correlation fluorescence sensing technique that involves measuring fluorescence microarray spots that are positioned at a sufficiently defined distance from the laser beam diameter. This allows for the spots having fluorescence dye to be consistently excited from the dissipated light from the laser beam diameter. Using this trick, there then generating one high resolution image that involves doing computational intensive image processing and filling data storage, this involves generating many very small pictures that cropped for image processing process where the spots are located as the gantry head containing microscope and light source moves.
It is a very straightforward imaging technique that is fast, quantitative, accurate and scaleable for high throughput. It is also portable and can be configured on standard microarray fabrication instrumentation and gantries like repstraps. This data compares how you can set up a laser and low light sensitive camera to a standard photomultiplier tube confocal microarray scanner.
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Video uScope
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PMT Laser Scanner
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Dye Conc |
Video uScope 100mW 532nm |
PMT Scanner %80L %55PMT |
PMT Scanner %80L %65PMT |
PMT Scanner %80L %75PMT |
3.3uM |
Sat: % 91.338 CV: % 1.113
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Sat: % 96.480 CV: % 2.519
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Sat: % 97.083 CV: % 0.875
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Sat: % 96.410 CV: % 0.907
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820nM |
Sat: % 91.999 CV: % 2.712
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Sat: % 78.905 CV: % 12.237
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Sat: % 92.263 CV: % 5.910
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Sat: % 97.274 CV: % 1.063
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200nM |
Sat: % 70.504 CV: % 10.892
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Sat: % 25.021 CV: % 24.004
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Sat: % 69.649 CV: % 17.767
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Sat: % 85.148 CV: % 4.104
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50nM |
Sat: % 33.240 CV: % 19.797
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Sat: % 5.517 CV: % 24.906
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Sat: % 16.839 CV: % 25.133
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Sat: % 43.450 CV: % 24.606
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13nM |
Sat: % 18.136 CV: % 19.958
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Sat: % 1.639 CV: % 28.856
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Sat: % 5.426 CV: % 27.995
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Sat: % 13.986 CV: % 28.827
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