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MicroscopyZinZThickZSamples
LydiaZZajiczekbFZKevinZO2HolleranAZandZMichaelZShawb
6

NationalxPhysicalxLaboratoryYx.nalyticalxSciencex'ivisionYxEamptonxRoadYxTeddingtonYxMiddlesexYxTW66xLWYxUX
4
"ambridgex.dvancedx/magingx"entreYxUniversityxofx"ambridgeYx.natomyx7uildingYx"ambridgeYx"74x8'YYxUX

lydia(zajiczek@npl(co(uk

CurrentZIssuesZwithZStructuredZIlluminationZMicroscopyZWSIMR
S/Mxhasxemergedxasxonexofxthexmostximportantxsuper-resolutionxmicroscopyxtechniquesxinxrecentxyearsOx/tsxprimaryxdrawbacksxarexthexnumberxofximagesxrequiredxtoxreconstructxaxsuper-resolutionximagexandxthex
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WeightedZLinearZReconstruction

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SpatialZ
LightZ
ModulatorZ
WSLMR:

L8
L

PatternsQ
:[

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orientationsxofx
illuminationxgivesxx
near-isotropicx
resolutionx
enhancement

:64

-Structuredxilluminationxallowsxforxextractionxofxhigh-spatialxfrequencyxinformationxfromxaxsamplexthatx
isxoutsidexthexopticalxtransferxfunctionxfOTJBxsupportxofxaxwidefieldxmicroscopexduextoxfrequencyx
mixingxbetweenxthexilluminationxpatternxandxnormallyxunobservablexfeaturesxinxthexsamplex[4]x
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locationxinxJourierxspaceYxout-of-focusxlightxgeneratesximagexartefactsxnotxrelatedxtoxrealxstructure
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multiplyingxfrequencyxpassbandsxwithxweightingxfunctionsxfseexbelowB
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frequencyxofxilluminationxpatternxandxreducingxabsoluteximprovementxinxspatialxresolution

kz

Quarterwavex
PlatexfQWPB
Pockelsx"ell

Laserxenginex
coupledxintox
PMxfiberx

Microscopex7ody

-LocatedxatxJourierxplanexofxSLM
-7locksxzerothxorderxandxhigherxharmonicsxofxJTx
ofxSLMxpatternsYxallowsx6stxordersYx8xorientations
-Resultingxinterferencexinxfocalxplanexofxmicroscopexisxsinusoidal
-"anxbexmodifiedxtoxmatchxpolarizationxandxintensityxofxzerothxandx
firstxordersxforx8xbeamxS/M

-.zimuthalxpolarizationxmaximizesxcontrastxofxilluminationxpatternx[6]
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PockelsxcellxandxQWPYxalsoxaccountsxforxrotationxfromxSLM
:
:[
:64

kr

SR-S/M

PassbandZ
Weighting

Figure:xOTJxsupportxftopBxforx4'xS/Mxwithxspatialxfrequencyxofxilluminationxpatternxequalxtoxhalfxthex
incoherentxcutoffxfrequencyxofxthexsystemxandxweightingxfunctionsxfbottomBxappliedxtoxpassbandsxtox
attenuatexout-of-focusxlightOxRedxcolorxisxzeroth-orderxpassbandYxbluexisxfirst-orderxpassbandOx

x:x5wwxnm
x:xz[6xnm
x:x[8wxnm

PolarizationZ
Rotation:

OS-S/M

WLR-S/M

8N.=

OTFZSupport

SpatialZ
Filter:

"ollimator

Widefield

4N.=

widefield

SR-S/M

WLR-S/M
WLR-S/M

8'-S/M

AZm

Above:ZTailxregionxof C. elegans animalximagedxviax

diffraction-limitedxwidefieldxmicroscopeYxconventionalx4'x
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weightedxlinearxreconstructionxfWLRBxS/M
Opposite:Zcross-sectionxofxaboveximagesxshowingx
reductionxofxout-of-focusxlightxpresentxinxSR-xandxOS-S/M Widefield
Bottom:xreconstructedximagesxofxclusterxofx'ilxlabelledx
OS-S/M
structuresxinsidexpharynxxofxC. elegans animal
WLR-S/M

Widefield

OS-S/M

AZm
AZm

Left:Z"utxthroughxofxwidefieldxfupperxrightBxandxWLR-S/MxflowerxleftBximagesxofxalphaxtubulinxfredBxandx
G-cadherinxfgreenBxinxepidermisxofxDrosophiliaxembryoOx"ontrastxenhancedxOz2xforxclarityO
Right:ZSinglexplanexS/Mximagesxofxalpha-tubulinxfdashedxboxxinxlowerxleftximageBxwithxdifferentx
reconstructionxtechniquesOx"ontrastxenhancedx62xforxclarityO
.rtefactsxpresentxinxthexSR-S/Mxreconstructedximagexduextoxout-of-focusxlightxhavexbeenxremovedxinx
thexWLR-S/MximageOxThex8'-S/MximagexwasxobtainedxwithxaxcommercialxsystemxforxcomparisonOx8'S/Mxshouldxhavexthexhighestxsignal-to-noisexratioxofxallxtechniquesxpresentedxherexbutxthisxrequiresx
imagingxthexentirexvolumexofxthexsamplexwithoutxanyxmovementOxThisxparticularxcommericalx
reconstructionxalgorithmxwasxnotxasxeffectivexinxsuppressingxout-of-focusxlightOx
WLR-S/M

Summary
-MainxproblemsxwithxS/MQxdifficultyximagingxdynamicxprocessesxandxartefactsxfromxout-of-focusxlightx
-Eigh-speedxS/Mxsystemxpresentedxherexcanxexceedximagingxspeedsxofx65xEzxforxsuitablexsamples
-WLRxreconstructionxmethodxattenuatesxout-of-focusxlightxpresentxinxopticallyxthickxsamplesxfupxtox
z-]xmxinxdiameterBxwithoutxneedxtoximagexmultiplexplanes
-4'xWLR-S/Mxreconstructionxrequiresxoverlapxbetweenx0missingxcones0xofx6stxorderxpassbandxandx
widefieldxpassbandxfusuallyxmeansxsacrificingximprovementxinxspatialxresolutionB
-"urrentxsystemxandxreconstructionxmethodxcanxpotentiallyxbexmodifiedxtoximplementx8-beamxS/Mxforx
improvementxinxaxialxresolutionxasxwell

Acknowledgements
ThexauthorsxgratefullyxacknowledgexMunaxGlmixfromxUniversityx"ollegexLondonxforxpreparationxofx"Oxelegansx
samplesYx%uanxManuelx9omezxfromxthex7rownxLabxatxthex9urdonx/nstituteYxUniversityxofx"ambridgexforxpreparationx
andximagingxofx'rosophiliaxembryosxandxNicolaxLawrencexfromxthex9urdonx/nstituteYxUniversityxofx"ambridgexforx
helpfulxdiscussionsxaboutxthexOMXxmicroscopexsystemOxXevinxOIEolleranxisxsupportedxbyxthex/saacxNewtonxTrustx
andxthexMR"xNextx9enerationxOpticalxMicroscopyx/nitiativeOxThisxworkxwasxpartiallyxfundedxbyxthexUXIsxNationalx
MeasurementxSystemO

References
[6]xXOxOIEolleranYxMOxShawYxOptOxLettOYx8]xf464Bx5[8-5[zO
[4]xMO9OLOx9ustafssonxetOxalOYx7iophysOx%OYxk5xf4wBx5kz]-5k]O
[8]xXOxOIEolleranYxMOxShawYx7iomedOxOptOxGxpressYxzxf465Bx4zw-4zk

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