Eliminating Interference from Autofluorescence in Flow Cytometry

SUMMARY Flow cytometry is a powerful cell analysis toolproviding information on cell countshapesizeDNA contentredox statemembrane permeabilityand surface receptorsamong other featuresIt can be used on both live and fixed cellsand can be used to non destructively sort cells based on these characteristicsFlow cytometry has utility in research and clinical fields as diverse and important as tumor biology researchcancer and AIDS diagnosticsstem cell therapyimmunophenotypingand cancer immunotherapyFlow cytometry relies extensively on fluorescent labeling cellswith the emission light collected from the interrogation region divided spectrally by filters and beam splittersand directed to detectors that measure intensityOne common problem derives from the fact that the exogenously added fluorophores are not the only source of fluorescence in the sampleSo called cellular autofluorescence can bein some circumstancesa major contributor to the signal present in each of the detection channelsA number of solutions have been proposedbut they often involve complex experimental protocolsor complex processing of dataThis leaves the field wanting for a solution to the nagging problem of interference from cellular autofluorescenceHere we propose to use fluorescence lifetime as a second discriminating parameter in order to distinguish autofluorescence interference from the desired signalWe propose to build a simple system using a single pulsed laserwhich will be sufficient to demonstrate feasibility of our methodThe system will include a highspeed pulsednm laser capable ofns pulses and the required fast electronics andemission channelswhich span the range of the broad cellular autofluorescenceWe will leverage the deep expertise of Kinetic River in designing and building complexinnovative cytometry instrumentsimplementing novel hardware solutions to enable lifetime measurements in flow cytometryand innovativeproprietaryand patent protected techniques for rapid analysis of complex lifetime decay curvesWe will be assisted by consultants and collaborators with deep experience in fluorescence lifetime measurementautofluorescence signalscell based assays and algorithm developmentTo demonstrate the feasibility of our approachwe are pursuing the following strategybuild prototype instrument and optimize system hardwarecharacterize instrument performanceandvalidate using relevant biological samplesSuccessful completion of this work will pave the way for further development in Phase IIwhich will see the addition of other laser lines with lifetime capabilities to create a general purpose instrumentThis instrument will be similar in cost to similarly equipped cytometersbut will have the advantage of automated elimination of cellular autofluorescenceSuch an instrument will have wide ranging utilityincreased ease of useand will allow for clearer interpretation of experimental results NARRATIVE Cellular autofluorescence is ever present in both live and fixed cellsIn flow cytometrythis can interfere with the desired signal and cause loss of sensitivity or erroneous resultsWe are developing a methodbased on fluorescence lifetimethat can automatically eliminate autofluorescence from standard flow cytometry assays