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1、OpticalMicroscopyApplication:BrightfieldIlluminationBrightfieldillumination,whichyieldsdarkobjectsonabrightbackground,isthesimplesttechniqueforopticalmicroscopy.Inbrightfieldillumination,thelightsourceispositionedbelowthesample.Lightthenpropagatesthroughthesample,andisobservedbytheobje
2、ctivelensandsensor,whicharepositionedabovethesample.Thedarkerthesample,thedenserthespecimen,asdensersamplesabsorbmorelight.Thesimplicityofbrightfieldilluminationisthemainreasonthistechniqueissopopularinoptical microscopy.IMAGEAPPEARANCEAtypicalbrightfield illumination imagehasadarksamp
3、lewithawhitebackground.Thedarkertheregionsonasample,themoreabsorptionoflightthathasoccurred.Forexample,plantcellswouldappeardarkestatthenucleusandcentralregionwherecellularmatterismostdense,andlighterinthecytoplasmvoidofribosomes,theendoplasmicreticulum,andotherintracellularcomponents.
4、Animalcellsaremoredifficulttoimagewiththistechniquewithoutstainingofthesample,whichultimatelykillslivecells.Figure1: BrightfieldIlluminationImageofTissuePaperTECHNICALDETAILSTherearefourkeycomponentsinthelightpathforbrightfield illumination.1.LightSource: trans-illuminationbelowsamplet
5、hatpropagatesthroughtocondenserandobjectivelens.Typicallyabroadbandsourcesuchasaquartzhalogenbulb.2.CondenserLens: collectstrans-illuminatedlightandfocusestosample.3.ObjectiveLens: collectslightwhichpropagatesthroughsampleandenhancesdetailsbyafactorofmagnification.4.Eyepiece/Camera: vi
6、ewsorrecordstheimage.Therearesomelimitationstothebrightfieldilluminationtechnique,whichincludeverylowcontrastforcellularorbiologicalsamples,lowopticalresolutionduetolimitationsoflight,andtherequirementforstainedsamplespriortoimagingorviewing.However,simplicityofthebrightfieldtechniquei
7、sahugebenefitwhenfirstimaginganunknownsample.Additionalopticalmicroscopyapplicationsinclude darkfieldillumination, phasecontrast, fluorescence,and differentialinterferencecontrast.
