Simultaneouswhole‐animal3D‐imagingofneuronalactivity usinglight‐fieldmicroscopy RobertPrevedel*,Young‐GyuYoon*,MaximilianHoffmann,NikitaPak,Gordon Wetzstein,SaulKato,TinaSchrödel,RameshRaskar,ManuelZimmer,EdwardS. BoydenandAlipashaVaziri *Theseauthorscontributedequallytothiswork. CorrespondenceshouldbeaddressedtoE.S.B.([email protected])orA.V. ([email protected]). TutorialforBuildingLightFieldMicroscope(Hardware) Overview AnLFMsystemappendedtoanepifluorescencemicroscopecanbeimplementedas follows. Any of these parts can be replaced by a proper alternative, so we briefly discusstheconsiderations. Thekeycomponentsaremicrolensarray,amicrolensstage,a1:1relaylensanda camera. o Microlensarray:MicrolensarrayisthemostimportantcomponentforaLFM system.UsermayrefertoRef.1,2toproperlydeterminethefocallengthandthe pitch. o Microlensstage:Thevolumereconstructionqualitydependsalotonthe alignmentofthesystem,souserneedtomakesurethatthemicrolensarrayis wellalignedwithrespecttotheimageplaneandthecamera.Inordertoachieve goodalignment,amicrolensstagethatallowsbothsolidfixationandangle adjustmentisneeded. o 1:1relaylens:Whereasthecameracanbeplacedrightbehindthemicrolens arrayintheory,re‐imagingusinga1:1relaylensprovidesalotofmechanical flexibilityinpractice.Imagecirclesize,distortionandaberrationshouldbe consideredwhendeterminingtherelaylens. o Camera:Asthevolumetricimagingspeedissolelydeterminedbytheframerate ofthecamera(assumingthatthespeedisnotlimitedbythenumberofphotons), sCMOScameraisgenerallyareasonablechoiceasitcanprovidelargefieldof view,largenumberofpixelsandhighframerate.Otherspecificationofcameras thataregenerallyimportantforscientificimagingincludingreadnoise,dark currentanddynamicrangeshouldalsobeconsidered. PartList Vendor Zeiss Zeiss Andor Nikon OkoTech Thorlabs Thorlabs Thorlabs Thorlabs Thorlabs Thorlabs PartNumber Axiovert200 Description Microscopewithimagingport MicroscopeObjective (20x0.5dry;40x0.95dry) Zyla ZylasCMOScamera10tap Nikon AF‐S 105mm Macro‐lens(usedin1:1relaysetting) 2.8GVRIF‐EDMicro OKO Tech Microlenses with lens' f# matched to objective (e.g. 150um pitch,f=3mm) APO-Q-P150-R1.37 K6XS 6‐AxisLockingKinematicOpticMountand CM1‐4ER/M CompactClamping4‐PortPrism/Mirror30mmCageCubeM4 Tap KB75/M Complete 75mm x 75mm KinematicBase Top and Bottom PlatesMetric MT1/M 13mmTranslationStage PT1/M 25mmTranslationStagewithStandardMicrometerM6Taps various(min.3x)posts,postholderandclampsdependenton beamheight tomountcameraandmicrolensarraytotranslationstage Alignment Sinceadjacentpixelsinlight‐fieldimagesmaycontainvastlydifferentimplicationonthe volume,aligningthemicroscopewelliscrucialforgettinghighquality3Dvolume. sCMOS Green Collimated Light Source <Setupforaligning&takingcalibrationimage> Dichroic Filter Green Fluorescent Slide Blue Excitation Light Source Thisfigure(notdrawntoscale,relaylensisomitted)illustratesthesetupforaligning LFMaswellastakingcalibrationimagesforLFM.Thelightfromcollimatedlightsource willappearasanarrayofspotsontheimageandthelightfromgreenfluorescentslide willappearasanarrayofcircles.Iftheintensityoftwolightsourcesisadjustedsuch thatonedonotdominateanother,boththearrayofspotsandthearrayofcircleswillbe visibleasshowninthefollowingfigure. <CalibrationImagefromwell‐alignedLFM> Asonecaninferfromthisfigure,thekeyfeaturesofthecalibrationimageofwell‐aligned LFMareasfollows. ‐ Thesizeofthespotswillbecomessmall(in‐focus) ‐ Thearraysofspotsandcirclesappearuniformoverwholefieldofview ‐ Thecentersofspotsandcirclescoincide ‐ Thearrayofcirclesisperfectlyalignedwithrespecttothepixelarrayintermsof rotation Oncethemicroscopeisaligned,thiscalibrationimagecanbeusedforfinding rectificationparameters,whicharealsocrucialforobtaininghighqualityreconstruction results. References 1. M.Levoy,R.Ng,A.Adamsetal.,ACMTrans.Graph.25(3),924(2006). 2. M.Broxton,L.Grosenick,S.Yangetal.,OpticsExpress21(21),25418(2013).
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