Autofluorescence background, the natural fluorescence from biological materials, reduces sensitivity of detection of localized fluorescence in multicellular organisms. For example in the case of a transgenic C. elegans that expresses GFP in two cells, that fluorescence can be masked by the autofluorescence of the remaining 1000 somatic cells. We have solved this problem with an add-on to our COPAS analysis and sorter technology that allows the instrument to measure and store the pattern of fluorescence along the length of the animal. That information was used to restrict fluorescence analysis to the region of the animal that contained the fluorescent cells thus improving the signal to autofluorescence ratio more than 20-fold. We used the Profiler system to perform a genetic screen in which we isolated mutants of PY1089 (kindly provided by Piali Sengupta), a transgenic C. elegans that expresses GFP in the AWB sensory neurons under control of the
str-1 promoter. Proper expression of the odorant receptor, STR-1, in the AWB sensory neurons is required for appropriate chemotactic response to the repulsive odorant 2-nonanone. Without the Profiler technology the COPAS system was unable to distinguish PY1089 from N2 wild type animals, and it could not be used in a genetic screen for mutants with reduced GFP expression. With the addition of Profiler we were able to clearly distinguish PY1089 from N2 and were further able to sort animals that had either reduced or increased GFP expression in the sensory neurons. F2 progeny of mutagenized PY1089 were analyzed and sorted using the COPAS BIOSORT with prototype Profiler hardware. First, 100 animals were run and their peak fluorescence intensities were determined. That information was used to set a range of normal peak heights in the Profiler. The Profiler was instructed to dispense those animals whose peak heights were outside of the normal range, either higher or lower, one per well into 96-well plates. Mutants were confirmed by regrowing the sorted animals and running them through the Profiler a second time. Approximately 50,000 animals from 8 separate pools were screened and 1,000 were dispensed to wells in the first pass. We have retested 109 lines from the sorted animals to date. We have isolated 5 independent mutant lines with decreased GFP expression, 5 independent lines with increased GFP expression and 2 lines with a mix of increased, wild-type, and decreased GFP expression. So far, we have tested 5 mutant lines for their chemotactic response to 2-nonanone. Of these one line has demonstrated defective chemotaxis