Zeiss Elyra User manual

ZEISS ELYRA

Sample Preparation for Superresolution Microscopy –

a Quick Guide

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General Guidelines for

Superresolution Structured Illumination Microscopy (SR-SIM)

Sample support

In general, we recommend using high-precision coverslips (always no. 1.5 thickness) to mount the samples.

High-precision cover glasses feature an exceptionally accurate thickness of 170 ± 5 µm (= 0,170 ± 0,005 mm).

Also mind thickness issues when ordering glass-bottom petri dishes or multi-well plates.

For cover glasses see:

• ZEISSHighprecisionCoverGlasses18x18mm;

ZEISSorderNumber:474030-9010-000

• MarienfeldSuperior:www.marienfeld-superior.com

 •18x18mm(Cat.No.0107032).

 •22x22mm(Cat.No.0107052).

 •roundwith18mmdiameter(Cat.No.0117580).

• CellPathLtdUKHighperformancecoverslips

no1.5H18x18mm(Cat.NoSAN-5018-03A)

For glass-bottom petri dishes and multi-well plates see:

• Willco(http://www.willcowells.com/)

• Lab-tek(offeredthroughThermo)

• MatTek(http://glass-bottom-dishes.com)

ZEISS ELYRA

Sample Preparation for Superresolution Microscopy –

a Quick Guide

Authors: Dr. Sylvia Münter

  Dr.YilmazNiyaz

  CarlZeissMicroscopyGmbH,Germany

Date:  November2013

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Please make sure that the coverslip is centered on the glass slide in order to ﬁt into the sample holder.

SeebelowpicturesoftheZEISSleveladjustablepiezostageinsert.

Fix your cells according to your standard protocol. For SR-SIM imaging, a thoroughly clean glass surface plays a crucial

role.Thereforeitisbenecialtosealorattachthecoverslipinawaythatfacilitatescleaningwithethanol,without

moving the coverslip.

Fluorescent Labels for SR-SIM

Allcommontypesoforganicdyesusuallyconjugatedtoantibodiesoruorescentproteinsaresuitabletobeusedfor

SR-SIM. Make sure to have a highly speciﬁc labeling with low background to obtain a good signal to noise ratio.

Formulticolorsamplestheuorophoresshouldbeselectedforminimalspectraloverlaptoavoidcrosstalk.Available

ltersetsforELYRAPS.1areareoptimizedfortheavailablelaserlines:405,488,561and642nm.

Note

Cytosolicorothernon-specicuorescentproteinexpression(e.g.GFP)willresultinstainingofextendedareas.

Since well-deﬁned structures are missing to interfere with the grid pattern, modulation contrast will be low and the

ﬁnal image will lack high resolution information.

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Figure 1 ZEISS level adjustable piezo stage insert

Embedding

Ideally, the sample should be embedded in a medium that matches the refractive index (RI) of the immersion oil

(n=1,518).Thefollowingmediaperformwell,despitehavingalowerrefractiveindex.

• Fluoromout-G(SouthernBiotech)withRIof1,40.

• 86%Glycerolwith2.5g/lDABCO(1,4-deazabicyclo[2.2.2]octane,SigmaCat.D2522)inTris-HCl.1MpH8.0.

 Forfurtherreading:http://www.nanoimaging.de/homepages Sample requirements

• Non-hardeningVECTASHIELDMountingMediumwithRIof1,44.

• ProLongGold(LifeTechnologies).Beawarethisembeddingmediumneeds2daytoharden(inordertoreacha

constant RI). In addition your sample may shrink during this process. Refractive index (RI) of the cured product:

1,46.

• SlowFadeGold(LifeTechnologies)withRIof1,42.

• 2,2’-thiodiethanol(TDE)–aqueous.RIcanbevariedrangingfrombeingthatofwater(1.33)tothatofimmersion

oil(1.52)byappropriatelydilutingwithwater.

Note

In order to have stable imaging conditions, especially concerning the RI of the mounting medium we would urge

you to prepare the slides at least one week before use. Please note, that during curing the RI will rise.

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General Guidelines for Photoactivated Localization Microscopy (PALM)

Sample support

SamplesforPALManddSTORMareideallypreparedon

NuncLab-Tekchambersorglassbottomdisheswithcover

glassthicknessNo1.5.

• NuncLab-TekIIChamberedCoverglassno1.5

(orderno.:155409)

• GreinerBio-One(orderno.:672860CELLview)

• NuncLab-Tekchambers(orderno.:

 P35G-0.17-10-CorP35G-0.17-14-C)

Alltheseformatswilltintotheadjustablepiezostage

insert.

Pleasebeaware,inordertoobtainidealTIRFillumination

you need a sufﬁcient mismatch in refractive indices.

In addition you may want to change imaging buffer

concentration therefore we recommend no embedding

afterxationofPALManddSTORMsamples.

Note

NuncLab-Tekchambersarewellsuitedtotransportsamplesbetweenlabs.Theytperfectlyintoa50mlFalcontube,

thatcanbelledwithPBSandsealedwithparalm.

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Figure 2 ZEISS level adjustable piezo stage insert

Fluorescent Proteins for PALM

Theadvantageofphotoswitchableuorescentproteins(PS-FPs)liesintheiroutstandingspecicityandtheirsmallsize,

whichisaround2nm.Thelatterfeaturepotentiallyallowsforhighlabelingdensities.Amongphoto-switchableproteins

photoconvertable ones are the easiest to use as they - and the structure they stain – can be visualized at a different spec-

tralrangebeforeconversion.Alsoonecaneasilychecktransfectionefcienciesandexpressionlevels.E.g.tdEOSor

mEOScanbecheckedinthegreenspectralrange,whilethePALMexperimentwillbecarriedoutdetectingphoto-

switchedEOSmoleculesinamoreredshiftedspectralband.ThereforetdEOSoritsmonomericvariantmEOShave

been in extensive use as they also yield reasonable photon numbers.

Figure 3 Examples of ﬂuorescent proteins that can be used for PALM imaging

Mostoftheseproteinsareavailablethroughe.g.Addgene,Cambridge,MA02139,USA

Recommended FP-Pairs for dual-color PALM

Pair1:mEOS2+Dronpa

Pair2:NeonGreen+PA-mCherry1

Pair3:Padron+Dronpa

IftwodifferentlystainedmoleculesinthesamesamplearesubjectedtoPALM,itisadvisabletorstimagethehigher

wavelength dye as this does not cross-talk into the shorter wavelength channel. Hence, cross-excitation of the longer

wavelength dye by the shorter wavelength and cross-emission of the shorter wavelength dye into the longer wave-

lengthchannelareminimized.Underexperimentalconditionsmanymoleculesoftheshorterwavelengthdyeare

equallyactivatedwiththe405nmlaserline,thatisusedforconversion/PALMimagingofthelongerwavelengthdye.

Thereforereversibleswitchableuorophoresarethepreferredchoicefortheshorterwavelengthastheycanberecov-

ered and are not irretrievably lost.

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Recommended Dyes for dSTORM

dSTORMusesorganicdyes,whichcanbeswitchedbyemployingreducingagentsinthebuffer.Dyeswithahighphoton

yield per switching event, low on-off duty cycles, high survival fraction and a large number of switching cycles are

preferential.ManyXanthene,CoumarinandCyaninederivativesfulllthesecriteria.Companiesoftentradetheseunder

specialgroupnameslikeBodipy,AlexaFluor(bothfromInvitrogen),Atto(fromSigmaAldrich),DyLightuor(from

ThermoScientic)andFluoProbes(fromInterchim).Intheliterature,Alexa647hasbeenmostlyusedasithasproved

to be a dye that matches all imaging criteria very well and can be switched easily between the dark and bright state.

TheinuenceoftheselecteduorophorehasbeennicelyshownbyG.T.Dempseyetal.(Evaluationofuorophores

foroptimalperformanceinlocalization-basedsuper-resolutionimaging,NatureMethods2011).Pleaseseebelowan

extract of the data from this publication.

Figure 4 (a-c): Effect of number of photons per on-switching event and the on-off duty cycle on STORM image quality for an example structure

(a ring-like object). (p, t and x): Images of CCPs (clathrin-coated pits) using Alexa Fluor 647 (p), Atto 655 (t) and Cy5.5 (x). Shown are composite

x–y cross-sections for ten CCPs aligned to their respective centers of mass along with the radial density distributions of localizations derived from

the composite x–y cross-sections. Scale bars: 100 nm.

ForamulticolorexperimentanycombinationbetweenAtto488,Cy3B/Alexa561andAlexa647/DyLight654

willwork.SpecicallythecombinationofAtto488withAlexa647hasproventobeuseful(seeG.T.Dempseyetal.:

Evaluationofuorophoresforoptimalperformanceinlocalization-basedsuper-resolutionimaging,NatureMethods

2011)Recommendationsfordoublelabeling:Atto488+Atto565orAlexa647

Aniceoptioncouldalsobe:tdEOS/mEOS+Alexa647

Antibodies for dSTORM

If we consider the size of antibodies it is preferable to do direct antibody labeling without a primary and secondary

AB.Smallerantibodiessuchasnanobodies(cameloidlikeantibodiesfromcamels,llamasandsharks)withasizeinthe

rangeof2nmmaybepreferred.

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Post-ﬁxation Following Antibody Labeling

A post-ﬁxation step can be valuable. Herein, you ﬁx cells a second time after staining in order to improve the stability

ofthelabel.Thiscanpreventthelabelfromdetachingandoatingtheimagingmedium.

Imaging Buffer dSTORM

(pleasesee:G.T.Dempsey,NatureMeth2011andS.vanLinde,NatureMeth2011)Itisrecommendedtofreshly

preparetheimagingbuffereveryday.Theoxygenscavengersystemwillonlylastforafewhoursandismainlyneeded

forCyaninedyeslikeAlexa647andCy5(RhodaminesandOxazinesdonotrequireit.)

Imaging Buffer

100µlPBS10x(Phosphatebufferedsaline;e.g.fromSigma:D1408)

100µlMEA(CysteamineHydrochloride)stockconcentration1M(e.g.fromSigmaM6500-25G)(toxic!!)

Optional:

Oxygenscavengersystem(forcyaninedyes)

500µlGlucose20%(e.g.insolutionfromSigma(49163-100ML))

25µlGlucoseOxidase(e.g.24mg/mlGluOxfromAspergillusniger;SigmaG0543-50KU)

5µlCatalase(e.g.:12.6mg/mlCatalasefromBovineliver;SigmaC3155-50MG)

AddH2Otonalvolumeof1ml

Veryimportant:adjustpHto7.5-8.5with5MNaOHor4.5MKOH

(TotestpHyoucanusepHpaperintheindicatedrange)

Note

1MMEA(SigmaM6500-25G):0,113gin1ml

StoreMEA(solid)at4ºC.Preparefreshas1Mworkingstocksolutioninwater.Thisstockcanbekeptat4ºCand

usedwithin1-2weeksofpreparation.Alternativelyyoucanfreezesmallaliquotsat-20ºCandkeepthemforseveral

months.

TheMEAconcentrationdependshighlyontheuorophoreandtheexperimentalcondition.Thereforethebest

concentration has to be tested by trial and error for each sample (between 10mM and 100mM).

IfyouuseCysteamine(nottheHydrochloride)youhavetouse37%HCltoadjustthepHto7.5-8.5

As a general advice:

• Ifblinkingratesaretoohigh:increaseMEAconcentrationand/orincreasepH

• Ifblinkingratesaretoolow:decreaseMEAconcentrationand/ordecreasepH

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Fiducial Markers for Localization Microscopy

Fiducial Markers (FM) are used to:

1) correct for small drifts (tens of nanometer range) during the course of an experiment

2) toalignchannelsinmulticolorexperiments

3) tocorrectforchromaticaberrationsinmulticolorexperiments

4) serveasareferenceforthesoftwareautofocus

For PALM imagingwerecommenduorescentbeads(e.g.TetraspekbeadsfromInvitrogen)orgoldnanoparticles,

dependantonthelaserpower,asducialmarkers.Choosethebeadsaccordingtotheuorophoresusedinyour

experiment.

For dSTORM imaging we recommend nanoparticles (gold colloids) of which the photoluminescence persists through

the entire measurement and which should be immobilized on the coverslip.

Ideallyonehasabout1-3ducialmarkersintheeldofview.Youcaneither:

I) ﬁx ﬁducial markers on coverslip before seeding of cells:

• sonicateFMsolutioninanultrasoundbathforatleast5min

• addFM(100nm,BBI1:1000diluted)todH2Osolution

• vortexFMsolution

• coatcoverslipswithPoly-L-lysine(PLL)foradhesionofFM

• addFMMixsolutionontoPLLcoatedcoverslips

• checkdensity(1-3FMineldofview)

• preparesampleontheslide

II) add ﬁducial markers to sample before experiment

• sonicateFMsolution

• add2-5µlFMsolutioninto2mldH2Oandvortexseveralminutes

• applyontoxedcells

• wait~1hforFMtosettle

Note

OnlydissolveFMinwater(nosaltsolution),otherwisetheywillaggregateandnotsticktothecoverslip.

Fiducial Marker Order Info

• BBInternational[www.british-biocell.co.uk]&[http://www.bbigold.com]

 GoldColloid80nm(Cat.No.GM.GC80)and100nm(Cat.No.GM.GC100)

• Microspheres-Nanospheres(http://www.microspheres-nanospheres.com/)

 40nm(Cat.No.790122-010)and80nm(Cat.No.790120-010)nanospheresAuparticles

• Nanopartz[www.nanopartz.com]

 Nanorods550(Cat.No.30-25-550)and600(Cat.No.30-25-600)

• TetraspekBeads[www.invitrogen.com]

Fiducial coverslips

• HestzigLLC(http://www.hestzig.com)

• Fiducialcoverslips500±50nmspectralrangeattwodensities(Cat.No.550-30AuF&550-100AuF);

• Fiducialcoverslips600±100nmspectralrangeattwodensities(Cat.No.600-30AuF&600-100AuF)

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Literature References

Reviews on Superresolution Microscopy

1. G.Pattersonetal.:Superresolutionimagingusingsingle-moleculelocalization,AnnuRevPhysChem2010

2. 2. B.Huangetal.Breakingthediffractionbarrier:superresolutionimagingofcells,Cell2010

3. L.Schermellehetal.:Aguidetosuperresolutionuorescencemicroscopy,JCB2010

4. D.ToomreandJBewersdorf:Anewwaveofcellularimaging,Annu.Rev.CellDev.Biol.2010

Superresolution Structured Illumination Microscopy (SR-SIM)

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