Pairing detailed phenotypic characterization of cells with downstream molecular analysis represents a significantchallenge,especiallyatthelevelofsinglecells.CellMicrosystemshasdevelopedandcommercialized theCellRaft?System,whichiscurrentlytheonlycommerciallyavailablemeansofimagingcellsusingbrightfield and/ormulti-??channelfluorescencepriortosinglecellisolationanddownstreammolecularanalysis.Investigators interestedinpairingphenotypicdatawithgenomicanalysisatthesinglecelllevelhaveemployedtheCellRaft Systemforarangeofinnovativestudiesincludingstemcelldifferentiation,tumorcellphysiologyandscreening ofneurons.However,thematerialsusedinthecurrentlycommerciallyavailableCellRaftArraysarenotoptimized forimagingqualityandwereselectedforotherpropertiessuchasthere-??sealingbehavioroftheelastomerandcell culturecompatibilityoftheplastic.DuringthePhaseIprogram,alternativematerialsfortheCellRaftArraywere exploredforimprovedopticalpropertiessuchasrefractiveindexagreementandreducedautofluorescence.One particular alternative materials combination exhibited favorable performance in both criteria leading to dramatically improved cell imaging performance. Here, we refer to this version of the product as the High Resolution, or HR-??CellRaft. The focus of our Phase II proposal is to commercialize the HR-??CellRaft for users interested in pairing detailed phenotypic imaging not possible on the current CellRaft Array (e.g. subcellular protein localization, organelle physiology, and semi-??quantitative fluorescent signals), as well as automate the CellRaft System workflow. In a parallel program, we are developing a highly automated instrument, the Automated Isolation and Retrieval (AIR?) System for single cell imaging and isolation employing the current CellRaftArray.DuringPhaseIIwewillimplementseveraldesignrevisionstotheopticalcapabilitiesoftheAIR System as well as its software-??based image analysis algorithms to better leverage the HR-??CellRaft?s improved optical properties. The new proposed instrument is referred to here as the CLEAR AIR? System. Additional fluorescencedetectionchannels(sixinsteadofthreeintheAIR?System),highermagnificationobjectives(10X, 20Xand40Xasopposedtothesingleobjective-??4Xor10X-??foundontheAIR?System)andahigherresolution camera will enhance the imaging capabilities of the system. For external validation of HR-??CellRaft Array and CLEAR AIR System performance, we have identified two investigators from our Early Adopter Program with researchneedsuniquelysatisfiedbytheHR-??CellRaftandCLEARAIRSystem.Onewillisolatesinglecellsbased onsubcellularlocalizationofareporterproteinandthesecondwillisolatecellswithvaryinglevelsoftransgene expression, with both groups coupling these phenotypic observations to downstream single cell molecular analysis. The HR-??CellRaft and the improved optical subsystem in the CLEAR AIR System meets an urgently unmetneedforpairinghigh-??resolutionphenotypicimagingwithdownstreammolecularanalysisofsinglecells.
Cell Microsystems, Inc. has developed the CellRaft? System, a cost-??effective, user friendly means of imaging,isolatingandretrievingsinglelivecellsinanunperturbedstate.Here,wepresentourworkindeveloping anewversionoftheCellRaft?Array,thehigh-??resolution,orHR-??CellRaft,whichdramaticallyimprovesimaging quality,permittinghighcontentimagingofsinglecellspriortomolecularanalysis.Anewinstrument,theCLEAR AIR System, will also be developed enabling dramatic improvements in imaging quality and automation. Two academic investigators will receive CLEAR AIR prototypes for validation prior to full commercialization of the automatedsystem.
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