Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images 英文参考文献.docVIP

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Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images 英文参考文献.doc

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Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images 英文参考文献

BrightFieldMicroscopyasanAlternativetoWholeCell FluorescenceinAutomatedAnalysisofMacrophage Images JyrkiSelinummi1,2,PekkaRuusuvuori1,2,IrinaPodolsky1,AdrianOzinsky1,ElizabethGold1,OlliYli- Harja2,AlanAderem1,IlyaShmulevich1,2,3,4* 1InstituteforSystemsBiology,Seattle,Washington,UnitedStatesofAmerica,2DepartmentofSignalProcessing,TampereUniversityofTechnology,Tampere,Finland, 3Department of Bioengineering, University of Washington, Seattle, Washington, United States of America, 4Department of Electrical Engineering, University of Washington,Seattle,Washington,UnitedStatesofAmerica Abstract Background: Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes,overlappingexcitationandemissionspectraofthestains,andphototoxicity. Methodology:Weherepresentandvalidateamethodtoautomaticallydetectcellpopulationoutlinesdirectlyfrombright fieldimages.Byimagingsampleswithseveralfocuslevelsformingabrightfieldz-stack,andbymeasuringtheintensity variationsofthisstackoverthez-dimension,weconstructanewtwodimensionalprojectionimageofincreasedcontrast. Withadditionalinformationforlocationsofeachcell,suchasstainednuclei,thisbrightfieldprojectionimagecanbeused insteadofwholecellfluorescencetolocatebordersofindividualcells,separatingtouchingcells,andenablingsinglecell analysis.UsingthepopularCellProfilerfreewarecellimageanalysissoftwaremainlytargetedforfluorescencemicroscopy, wevalidateourmethodbyautomaticallysegmentinglowcontrastandrathercomplexshapedmurinemacrophagecells. Significance: Theproposed approach frees upafluorescence channel, which canbe used for subcellular studies. It also facilitates cell shape measurement in experiments where whole cell fluorescent staining is either not available, or is dependent on a particular experimental condition. We show that whole cell area detection results using our projected bright