Simultaneous whole-animal 3D-imaging of neuronal activity using

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).

Download Report