ContentslistsavailableatScienceDirect
Annals
of
Anatomy
j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a a n a t
INVITED
REVIEW
Advances
in
cryo-electron
tomography
for
biology
and
medicine
Roman
I.
Koning
a,b,∗,
Abraham
J.
Koster
a,b,
Thomas
H.
Sharp
a,∗aDepartmentofCellandChemicalBiology,LeidenUniversityMedicalCenter,2300RCLeiden,TheNetherlands bNeCEN,InstituteBiologyLeiden,LeidenUniversity,2300RALeiden,TheNetherlands
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received4September2017
Receivedinrevisedform2February2018 Accepted5February2018 Keywords: Electronmicroscopy Cryo-electrontomography Energyfilter Phaseplate
Directelectrondetector
a
b
s
t
r
a
c
t
Cryo-electrontomography(CET)utilizesacombinationofspecimencryo-fixationandmulti-angle elec-tronmicroscopyimagingtoproducethree-dimensional(3D)volumereconstructionsofnative-state macromolecularandsubcellularbiologicalstructureswithnanometer-scaleresolution.Inrecentyears, cryo-electronmicroscopy(cryoEM)hasexperiencedadramaticincreaseintheattainableresolutionof 3Dreconstructions,resultingfromtechnicalimprovementsofelectronmicroscopes,improved detec-torsensitivity,theimplementationofphaseplates,automateddataacquisitionschemes,andimproved imagereconstructionsoftwareandhardware.Thesedevelopmentsalsogreatlyincreasedtheusability andapplicabilityofCETasadiagnosticandresearchtool,whichisnowenablingstructuralbiologiststo determinethestructureofproteinsintheirnativecellularenvironmenttosub-nanometerresolution. Theserecenttechnicaldevelopmentshavestimulatedustoupdateonourpreviousreview(Koning,R.I., Koster,A.J.,2009.Cryo-electrontomographyinbiologyandmedicine.AnnAnat191,427–445)inwhich wedescribedthefundamentalsofCET.Inthisfollow-up,weextendthisbasicdescriptioninorderto explaintheaforementionedrecentadvances,anddescriberelated3Dtechniquesthatcanbeappliedto theanatomyofbiologicalsystemsthatarerelevantformedicine.
©2018ElsevierGmbH.Allrightsreserved.
Contents
1. Introduction...83
2. Samplepreparation...83
2.1. Vitrificationofthinsamples...83
2.2. Highpressurefreezingforthicksamplesandwholecells ... 85
2.3. Cryo-sectioningthicksamples...85
2.4. Stabilityofvitreoussamplesintheelectronmicroscope ... 86
3. Datacollection...86
3.1. Tiltseriesacquisition...86
3.2. Phaseplatesimprovetomograminterpretability...86
3.3. Energy-filteringreducesnoise...88
3.4. Directelectrondetectors...89
3.5. Automateddatacollectionandstorage...89
4. Imageprocessing...90
4.1. Tomogramreconstructionautomation...90
4.2. Themissingwedgelimitsinterpretationoftomographicvolumes...91
Abbreviations:3D,three-dimensional;cryoEM,cryo-electronmicroscopy;CCD,charged-coupleddevice;CEMOVIS,cryo-electronmicroscopyofvitreoussections;CET, cryo-electrontomography;CLEM,correlativelightelectronmicroscopy;CMOS,complementarymetaloxidesemiconductor;CT,computedtomography;CTF,contrasttransfer function;DED,directelectrondetector;DQE,detectorquantumefficiency;FIB,focusedionbeam;fLM,fluorescencelightmicroscopy;GFP,greenfluorescentprotein;HPF, high-pressurefreezing;MRI,magneticresonanceimaging;NMR,nuclearmagneticresonance;PET,positronemissiontomography;PSF,point-spreadfunction;SBF,solidblock face;SEM,scanningelectronmicroscopy;SIRT,simultaneousiterativereconstructiontechnique;SPA,singleparticleanalysis;SXT,softX-raytomography;TEM,transmission electronmicroscope;VPP,voltaphaseplate;WBP,weightedback-projection.
∗ Correspondingauthorsat:DepartmentofCellandChemicalBiology,LeidenUniversityMedicalCenter,2300RCLeiden,TheNetherlands. E-mailaddresses:R.I.Koning@lumc.nl(R.I.Koning),T.Sharp@lumc.nl(T.H.Sharp).
Anatomyinvolvesimagingatdifferentlengthscales,ranging fromcompleteorganisms,metersinsize,downtosinglecellswith dimensionsontheorderofmicrometers,andevennanometer-scale sub-cellularstructures.Consequently,manydifferenttechniques arerequiredtovisualizeanatomicalprocessesoverthiswiderange (Fig.1).Tomographygeneratesthree-dimensionalreconstructions andisusedindiverseareasofscienceandmedicine,formingthe basisofcomputedtomography(CT),magneticresonanceimaging (MRI),andpositronemissiontomography(PET).WhilstCT,MRI, andPETcanimagewholeorganismsandachievesub-millimeter resolution,cryo-electrontomography(CET,alsoknownascryo-ET orelectroncryo-tomography;ECT)canimagethemicrometerto sub-nanometerlengthscales(Fig.1).Infact,CETistypicallyusedto visualizeindividualorganellesandbiomoleculeswithincells, yield-inginsightsintothemolecularandcellularpathologyofdiseases (Bauerleinetal.,2017)andpathogens(Wanetal.,2017).
ThemethodologyofCETisfundamentallysimilartoother tomo-graphictechniques; a volumeof material (molecule,virus, cell, organism,etc.)isimaged frommultipleangles(different orien-tations),andisthenreconstructedcomputationallytoforma3D volume(Fig.2).WhereasinMRI,CTandPETtheimagingsystem isrotatedfullyaroundthespecimen(i.e.thepatient),withCET thespecimenisrotateduptoalimitedangularrangewithinthe electronmicroscope(Fig.2A).Theresultingseriesoftilt-images (Fig. 2B) iscomputationally back-projected toreconstructa 3D volume(Fig.2C).
Inalltomographicimagingtechniques,themaximumallowable samplesizeandtheattainableresolutionareimportantpractical parameterstoconsider,asthesedeterminethelimitsofwhatcanbe observed.InCET,thefieldofviewistypicallyintheorderofmicrons. Thesamplethicknessshouldbelessthan500nminordertoallow electronstotransmitthroughthesamplewithlimited specimen-damaging effects due to the coulomb-force interactions of the imagingelectronbeamwiththeatomsofthesample.Therefore,in practice,wholecellsandtissuesmustbeslicedintosufficientlythin sectionsbeforeimaging, whilesmall biologicalspecimens, such aspurifiedproteins,virusesandbacteria,canbeimagedwithout sectioning.
TheresolutionofCETisdeterminedbyacombinationofthe electronradiationdosethatcanbeusedtoimagethesample with-outdetectabledamage,detectorsensitivity,samplethickness,and thequalityoftheelectromagneticlenses.Firstly,aselectron radi-ationstrongly interactswithatoms(Henderson,1995), andcan thereforebehighlydamagingforcryo-fixedspecimens,the imag-ingdosemustbekeptaslowaspossible.Moreover,inCETthis allowableelectrondosehastobedistributedovermanyangular viewsfrom which the3Dtomogram isreconstructed, resulting inindividualimageswithalowsignal-to-noiseratio.Therefore, detectorsthatefficientlydetectelectronswithlowintrinsicnoise levels, are paramountto obtain thehighest possible single-to-noiseratioandresolutioninthetomogram.Withincreasingsample thickness,electronsaremorelikelytoundergomultiplescattering
thatareusedforimaging areimperfect,which resultsinimage aberrationsandlossofresolution.Theselensimperfectionsare,to someextent,wellknownandcharacterizedfortransmission elec-tronmicroscopy,andcanthereforebecomputationallycorrected toachievethemaximumpossibleresolution(Haideretal.,2009).
Recentadvances incryo-electron microscopy (cryoEM)have givenrisetoa“resolutionrevolution”(Kuhlbrandt,2014),which hasincreasedthenumberofnear-atomicresolutionstructuresof proteinsandproteinassembliesthatemergedusingsingle parti-clecryoEM(Fernandez-LeiroandScheres,2016).Theadvancesthat enabledthisincludetechnicalimprovementsoftheelectron micro-scope,improveddetectorsensitivity,theimplementationofphase plates,automateddataacquisitionschemes,andimprovedimage reconstructionsoftwareandhardware,which,takenalltogether greatlyincreasetheusabilityandpracticalapplicabilityofCETasa diagnosticandresearchtool.
Thisreviewfollowsuponourearlierpublication(Koningand Koster,2009)andfocusesondescribingthetechnicaladvancesthat haveevolvedandemergedsince.Afterrecapitulatingthegeneral detailsofcryoEM,therecentimprovementsonCETaredescribed anddiscussedintheframeworkofthepracticalcryoETworkflow.
2. Samplepreparation
ForimagingsamplesusingCET,thefirststepistocryogenically fixthespecimenbyamethodreferredtoasvitrification. Depend-ingonsamplethicknesstherearetwovitrificationmethods(Fig.3). Samplesthatarethinnerthan10m,suchasisolatedprotein com-plexes,viruses,bacteriaorthincells,maybevitrifieddirectlyby plunge freezingintoacryogen(Dubochetetal.,1988).Samples uptoathicknessof200m,suchascellpelletsortissuebiopsies, requirefreezingathighpressure(so-calledhigh-pressurefreezing; HPF)for vitrification(Vanheckeetal.,2008).Afterthe vitrifica-tionstep,samplesthickerthan500nmrequirethinningpriorto imaging.Thinningcanbeperformedbycryo-ultramicrotomy( Al-Amoudietal.,2004b),usingadiamondknifetoslicethevitreous iceintosections,orafocusedionbeam(FIB)ofaheavymetal(e.g. gallium)toablatethematerialtoaspecifiedthickness.
2.1. Vitrificationofthinsamples
Fig.1.Scalesandresolutionofmedicalimagingtechniques.
Specimensofdifferentlengthscalesrequirediverseimagingtechniqueswithparticularlimitsinimagingdepthsandresolutions.
Fig.2.Cryo-electrontomographymethodology.
(A)Inthetransmissionelectronmicroscope,aseriesofimagesisacquiredonadetectorfromasampleofinterest,asthesampleisrotatedtospecifiedangles.Thesampleis embeddedinvitreousiceandcanbeverydiverse,e.g.wholecells,cellularextracts,proteinsorotherbiomolecules.(B)Theresultingprojectionimagesofthespecimenfrom differentangularviewsyieldsaso-calledtilt-series.(C)Thistilt-seriesiscomputationallyback-projectedtoforma3Dvolume,thetomogram.
Themoststraightforwardwaytovitrifyistouse instrumen-tationdevelopedforplunge-freezing.Thesedevicesallowcontrol overcryogentemperature,aswellasparameterssuchasblottime, humidityandtemperatureinthevicinityofthegridbeforeand dur-ingplunging,whichimprovesreproducibilityandenablesfreezing oflivecellsculturedongrids.Thinsamplesarevitrifiedbydirectly plungingthemintoacryogenwithhighheatcapacityandthermal conductivity,suchasliquidethaneatitsmeltingtemperatureof ∼90K.Practically,forpurifiedproteinsamples,typically2–5lis appliedonthegridand,inordertomakethesamplesufficiently thinforvitrification,mostfluidis removedbyblottingwith fil-terpapertoleaveathinlayerofliquid(typically30–100nm)over
Fig.3. Cryoelectronmicroscopysamplepreparationflowchart.
SamplepreparationflowchartforcryoEMinwhichchoices(grey)forseveralpreparationandvisualizationtechniques(blue)dependon:(i)thesamplethickness,and(ii) thedesiredstructurestobevisualized.Samples(green)thinnerthan10m(e.g.proteins,viruses,bacteria)canbevitrifiedbyplunge-freezing,whilethickersamples(e.g. cellpelletsortissueblocks/biopsies)canbecryo-fixedbyHPF.Samplesthickerthan∼500nm(e.g.cellsandtissue)mustbethinnedpriortoimaginginordertoensure electrontransparency.Thinningcanbeperformedeitherbycryo-ultramicrotomyorcryo-FIB-milling.Finally,cryo-preservedsamplesthinnerthan500nmcanbeimagedby 2DcryoEM(projectionimagingorsingleparticleanalysis)or3DCET(tiltseriesacquisitionfortomographyorsub-tomogramaveraging).(Forinterpretationofthereferences tocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
liposomes.ForcryoEMofcellularsamplesthatarecultureddirectly onthegrid,goldgridsareusedasasubstratetoavoidthe cytotox-icityofcopper.
2.2. Highpressurefreezingforthicksamplesandwholecells High-pressurefreezing(HPF) canbe usedto vitrifysamples upto200minthickness(DahlandStaehelin,1989;McDonald and Auer, 2006; Moor and Riehle, 1968), and is essential for cryo-fixationofadherentcells, cellpellets,tissuesandbiopsies. WithHPF,highpressureisappliedtoa sampleduringfreezing, theexpansionofwaterduringcrystallizationiscounteracted(le Chatalier’s principle),inhibiting theformation of hexagonal ice crystals(thecommonformoficefoundinfreezers)andthereby promotingtheformationofvitrifiedice.
DedicatedHPFapparatuses(Studeretal.,2001)freezesamples within∼20ms at200MPausinga jetofliquid nitrogenat77K (−196◦C)inacontrolledandreproducibleway.Withtheaddition ofarapidtransfersystem(Verkade,2008),correlativelightelectron microscopyimaging(CLEM,seebelow)isfacilitatedbymakingit practicallypossibletovitrifythesamplewithin∼4safterimaging with(fluorescent)lightmicroscopy.
Arecentlyproposed,relativelystraightforwardmethodforHPF utilizesself-pressurization,inwhichthesampleiscontainedwithin aclosedcoppercapillarytube(LeunissenandYi,2009).Upon cool-ingthetubeinliquidnitrogen,theclosedtubefreezesfromthe outside,whichincreasesthepressureinthecenter.Thismethod excludestheneedforspecialistHPFequipment,althoughtheuse
ofcryo-protectantswithinthesamplesappearstobenecessary tomaximizethesuccess-rateofhigh-qualitysamplepreservation (Hanetal.,2012).
2.3. Cryo-sectioningthicksamples
Specimens thicker than 500nm, prepared by either plunge-freezingorHPF,requirethinningpriortoimagingwithCET.This thinningmustbeperformedbelow∼140K(∼−130◦C),the tem-peratureabovewhichvitreousicebeginstorecrystallize(known asdevitrification). Thinning canbeperformedusing adiamond knifetoslicethesampleintomanythinsections(e.g.50–150nm) usingultramicrotomyatcryogenictemperature,oftenreferredto as cryo-sectioningor cryo-electron microscopy of vitreous sec-tions(CEMOVIS) (Al-Amoudi et al.,2004a).CEMOVIScombines cryo-preservationwiththepossibilitytoimageslicesfromthick samples,andenableshighresolutionimagingofe.g.vitrifiedcells thatcouldnotbevisualizedotherwise(Al-Amoudietal.,2004b). Cryo-sectioningofvitreoussamplesistechnicallydemandingand canintroduce artifactswhich distort thesample, suchasblade marksandcompressionfractures(Richter,1994).Manyofthese cryo-sectionscanbetransferred ontoan EMgrid,and multiple sectionscanbeimagedbyCET.
1999). Recently, dedicated scanning electron microscopes have beendevelopedtofacilitateFIBmillingofvitrifiedbiological mate-rialpreparedbybothHPF(Haylesetal.,2010)andplunge-freezing (Markoet al.,2007)sampleswhich cannotbethinnedby cryo-sectioning(Fig.3).WhileFIBmillingresultsinasinglecellularslice, cryo-sectioningcanbeusedtoimagemultiple,sequentialsections. Galliumionsareexpectedtodepositonmilledsurfacesandinthe sections,andirregularitiesofthemilledsurfaceoftenoccur,which inextremecasescanleadtoproblemsduringimaging(Rigortetal., 2012a;RigortandPlitzko,2015).
2.4. Stabilityofvitreoussamplesintheelectronmicroscope
Aftercryo-fixation,duringstorage,transport,thinning,transfer intotheTEMandimageacquisition,thetemperatureofthesample mustbemaintainedbelow∼140K(∼−130◦C)toavoid devitrifica-tion.Inaddition,samplesmustbekeptfreefromcontamination, suchastheadhesionofsmallice-crystalsthatmayfloatinliquid nitrogen,atalltimes.Therefore,cryoEMgridsaretypicallykeptand transferredinclosedstorageboxesundernitrogenliquidorgasat ∼77K(−196◦C)beforebeinginsertedintotheTEM.
Mostelectronmicroscopesareusedforbothconventional(room temperature)EMandcryoEM.Theyusearemovablecryo-holder whenperformingcryoEM,which isinsertedintothesideofthe TEMcolumn(aso-calledside-entrysystem).Thecryo-holder com-prisesaDewarfilledwithliquidnitrogentomaintainthegridat 95K(−178◦C).TheDewarmustberefilledmanuallywithliquid nitrogeneveryfewhours,whichlimitstheabilitytoautomatedata collectionforaprolongedperiodoftime.Themechanicalstability ofthesampleisalsolimitedbythetemperatureinstabilityofthe cryo-holdercausedbydiminishingliquidnitrogenlevelsandheat flowbetweentheholderandthemicroscope.Thiscausesthe sam-pletodriftovertime,furtherlimitingtheabilitytoautomatedata collectionoverthecourseofhours.
Whenworkingwithaside-entrysystem,EMgridsareloaded individually,andthereforeeachnewsamplerequiresre-insertion ofthecryo-holderintotheTEM.Eachinsertiondeterioratesthe low-pressureenvironmentwithintheTEM-columnnearthe sam-ple,andsubsequentlyincreasesthechanceofcontaminationofthe EMgrid.Moreover,overtime thesamplethicknesscanincrease duetoicegrowthonthesamplewithintheTEMcolumn,limiting themeasuringtimethatcanbeusedtocollectatiltseries.
Duringthelastfiveyears,manyoftheselimitationshavebeen removedwiththedevelopmentandintroductionofnoveltypesof electronmicroscopesdedicatedtoperformcryoEMforaprolonged time-period.ThesetypesofTEMhaveanautomatedspecimen load-ingsystemthatholdsmultiplegridsandautomaticallytransfers themintoacontinuouslycryogenically-cooledandstablespecimen stagewithoutdisruptingthehighvacuumenvironment. Addition-ally,thequalityofthevacuumsystemofthesetypeof TEMsis significantlyenhanced,whichresultsinnegligibleicegrowthon thesampleoverthecourseofdays,allowingextendedautomated datacollection.
3. Datacollection
3.1. Tiltseriesacquisition
Electronsdamagebiologicalspecimens(Glaeser,1971)andas suchtheelectrondoseexposedtothesamplemustbeminimized. Thiscanbeachievedusingasearch,focusandimagescheme(a so-calledlow-dosescheme)duringdatacollection,wherebythe desiredimaginglocationsareidentifiedatlowmagnification(and hencelow electrondose)whilefocusingisperformedata loca-tionadjacenttotheareaofinterestpriortoimageacquisition.This
minimizestheamountofdose,andhencedamagethatthearea ofinterestisexposedtoandmaximizestheachievableresolution. Thetotaldoseusedforimagingmuststillbeverylow,typicallyless than100e−/Å2,toachievenanometerresolution(Fig.3,toprow). Becausetomographyisbasedonimagingthesameregionfrom multipleangles,thisdosehastobedistributed(fractionated)over thenumberofimagesthatisusedtorecordatiltimage.Tiltseries aretypicallycollectedfrom±60◦,withaseparationof2◦between images;eachtiltseriestherefore imagesthesamelocation∼61 times,resultinginadoseoflessthan2e−/Å2 foreachtiltimage. Duringtilting,theapparentthicknessofthesampleincreasesas 1/cos(),whereisthetiltangle.Samplesappear2×thickerat 60◦tilt,hencethedistancethatelectronsmusttransmitthrough, andthelikelihoodof multiplescatteringeventswiththeatoms withinthesample,alsoincreases.Consequently,thefractionated electrondoseofthetiltseriesresultsinlowcontrastandhigh lev-elsofnoiseintheindividualimages(Fig.3,bottomrow,leftimage), whichmakesaccuratealignmentofatiltserieschallenging.To facil-itateandimprovetheaccuracyofalignment,goldfiducialmarkers between5–25nmindiameteraregenerallyaddedtothesample(or onthespecimensupport)immediatelypriortovitrification.These fiducialmarkershavehighcontrastinthelow-doseimages,and canbecomputationallytrackedaspoint-likeobjectstoalignthe imagespriorto3Dreconstruction(Lutheretal.,1988;Walzetal., 1997).
Overall,theresultinglowdosethathastobeusedforimaging tominimizeradiation-inducedspecimendamageresultsinnoisy imagesinthetiltseries(Fig.4),whichhampersaccuratealignment ofatiltseries.Asaconsequence,cryo-electrontomogramswill typ-icallyexhibithighnoiselevels,lowcontrastandlimitedresolution. Technicalimprovementsthatincreasethesignal-to-noiseratioin theindividualimagesofatiltserieshaveadirecteffecton improv-ingthequalityoftheresultingcryo-tomograms.Duringthelast threeyears,thequalityofdatacollectedforcryo-tomographyhas improvedsignificantly,primarilybythreemajor developments: phaseplates,energyfiltersanddirectelectrondetectors.
3.2. Phaseplatesimprovetomograminterpretability
As cryoEMsamples are very thin (less than a few hundred nanometers)andarecomposedofrelativelylightelements (typi-callycarbon,hydrogen,oxygenandnitrogen),thecontrastforming mechanism canbevery welldescribed by (weak−) phase con-trastimaging.Phasecontrastisgeneratedbyphasedifferencesthat occurbetweenanunscatteredelectronwave(themajorityofthe electronsthatdonotinteractwiththesample)andtheelectron wavesthatinteractwithatomswithinthesample.Totranspose thisphaseshiftintoameasurablecontrast,imagesaretypically takenoutoffocus,whichincreaseslow-resolutionimagecontrast butnegativelyaffectsthehighresolutionintheimages.Theway thatthephase(andamplitude)oftheelectronwavesareinfluenced bytheobjective lensofthemicroscope(e.g.defocusing)canbe mathematicallydescribedbythecontrasttransferfunction(CTF) (Marabinietal.,2015).TheCTFdescribesaninverserelationship betweencontrastandresolution, andadditionallypredicts con-trast reversalsat certainfrequencies,resulting infringes in EM images.Fortunately,imagescanbe(partly)correctedfortheCTF, whichreliesonaccuratedefocusdetermination.CTFcorrectionin CETisnotalwaysatrivialtasksinceimagesaretiltedandfocus varieswithinindividualimages.Nonetheless,CTFcorrectioncan alsoincreasetheresolutionoftomograms(Fernandezetal.,2006; Turonovaetal.,2017;Xiongetal.,2009).
Fig.4. Radiationdamageandelectrondose.
Electronradiationdamageofcryo-samplesof(gammaproteo)bacteriaoccursdirectlyuponimagingandincreaseswithdose.High-resolutionatomicfeaturesarelostwithin afewelectronsperÅ2(e/Å2)whilevisibledamageoccurslater(toprow,totalelectrondose).Becauseofradiationdamagetheelectrondoseforimagingislimited,which
greatlyaffectsthesignal-to-noiseratiointypicalcryoEMimages(bottomrow,electrondoseperimage).
Fig.5. TheVoltaphaseplateincreasesthecontrastofelectrontomograms.
(A)Incomingelectronwaves(blue)haveacertainphaseandamplitude.Transmitted,non-diffracted,electrons(blue)donotinteractwiththespecimenandtheirphaseis unaffected.Incontrast,someoftheelectronwaveisdiffractedbythespecimen(red),whichretardsthephaseofthediffractedwavebyquarterofawavelength.Thissmall phaseshiftcausesonlyminimalcontrastofthemagnifiedspecimen(B).InthepresenceoftheVPPthediffractedwaveisretardedfurther,ideallybyanotherquarterofa wavelength(green),placingit180◦outofphasewiththenon-diffractedtransmittedwave(blue),resultinginmaximalnegativephasecontrast(C).Micrographsacquiredat
focusofvitrifiedliposomesandlacey-carbonfilmintheabsence(B)andpresence(C)oftheVPP,demonstratingthecontrastimprovement.
correction,comparedtoimagesacquiredusingdefocuscontrast. Electronsarediffractedbyinteractionwiththinbiological speci-mens,whichbehaveasweakphaseobjectsandretardthephaseof thediffractedelectronwavewithrespecttothetransmitted (non-diffracted)electronwavebyaquarterofawavelength(Fig.5A),but nottheamplitude,sothatverylittlecontrastisvisibleatlow fre-quency(Fig.5B).Unfortunately,thephaseinformationislostand onlyamplitudedataiscollectedbythedetectorintheformof inten-sity.So-calledZernike-typephaseplatesfurthershiftthediffracted electronwaves,generatinganapproximatehalf-wavelengthphase shiftwithrespecttothenon-diffractedelectrons,turningthisphase informationintomeasurableamplitudecontrastwithouttheneed todefocus(Fig.5C)(DanevandNagayama,2001).Thisresultsin
destructiveinterferenceandmaximalcontrastatlowresolution (Fig.5C),whichmakesprojectionimagesandreconstructed tomo-gramsmuchmorereadilyinterpretable(Fukudaetal.,2015;Sharp etal.,2016).
Fig.6.Energyfiltering.
Electronswithacertainenergy(here200keV±1eV)areemittedfromanelectronsource(FEG).Elasticinteractionswiththespecimendonotresultinenergyloss(black wave)whileinelasticinteractionsleadtolowerenergyelectronsthathavealongerwavelength(redwave).Aprismlenstransfersthisdifferenceintoashiftoftheelectrons thathavelostenergy,whichareremovedbyaslit.Insetshowsthatanenergyfilteredimageislessbrightbuthasmorecontrastthananunfilteredimage.(Forinterpretation ofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
phaseplate.ThetransmittedelectronspassthroughthislocalVolta potentialwithouttheirphasebeingaffected,whilstthediffracted electronsinteractwiththerestofthephaseplateandareretarded byafurtherquarter-wavelengthbyinteractionwiththecarbonof specificthickness,therebygeneratingahalf-wavephaseshiftwith respecttothetransmittedelectrons(Fig.5).Becausethe transmit-tedelectronbeamcreatesthelocalphaseshift,theVPPrequires minimalphysicalalignment,andtheVoltapotentialcanbe gen-eratedonthecarbonfilm whenand where desired, facilitating automateddatacollection.However,sincethephaseshiftis gen-eratedbyirradiationwithelectrons(knownasconditioning)the amountisdependentontheelectrondose,whichresultsin unsta-blephaseshifts(Danevetal.,2014;Sharpetal.,2017).Nevertheless, thevariablephaseshiftsstillincrease thecontrastofthe tomo-gram,andtheVPPhasalreadyshownpotentialforinsitustructure determinationofcomplementactivation(Sharpetal.,2017)and membraneattackcomplexporeformation(Sharpetal.,2016)on liposomes,aswellasthecellulardistributionofproteasomes, ribo-somesandnuclearporecomplexes(Asanoetal.,2015;Mahamid etal.,2016).
3.3. Energy-filteringreducesnoise
Whenelectronsareincidentonasampletheycaninteractwith thesampleineitheranelasticevent,wherenoenergyistransferred betweenelectronandspecimen,aninelasticevent,whereenergy islosttothespecimen,ortheelectronsdonotinteractwiththe sampleandaretransmittedunaffected(Egerton,2011). Inelasti-callyscatteredelectronscontributetothenoisewithintheimage anddamagethespecimenbybreakingmolecularbonds,localized heating,andevolutionofhydrogengas,amongothereffects(Baker
andRubinstein,2010).Theratioofscatteredversusunscattered electronscanbepredictedbasedonthemeanfreepathofthese electrons,theaveragedistancetravelledbetweentwosuccessive scatteringevents.Thismeanfreepathfor200–300keVelectrons isontheorderof200–300nm(FejaandAebi,1999;Grimmetal., 1996)andisdifferentforelasticandinelasticevents.Thelikelihood oftheseeventsthereforeshiftsdependingonsamplethickness.In cryoEMsamplesthatarelessthan100nmthick,mostelectrons passthroughthesampleunscattered,whiletherestmainlyscatter onlyonce,eitherelasticallyorinelastically.However,thelikelihood thatelectronsscattermorethanonceincreasesquicklywith speci-menthickness(Hanetal.,1995).Sincemultiplescatteredelectrons resultinnoiseintheimage,samplesarepreferablynotthickerthan themeanfreepath.Withsamplesthat havea thicknessofone totwotimesthemeanfreepath,suchasbacterialcellsandthin adherentcells,removaloftheinelasticallyscatteredelectronscan significantlyincreaseimagecontrast.Thiscanbeachievedbyusing anenergyfiltertoblockelectronsthathavelostorgainedenergy.
pri-Fig.7. Electrondetectors.
(A)TypicallayoutsofCCD(chargecoupleddevice)cameraandCMOS(complementarymetaloxidesemiconductor)directelectrondetector(DED).CCDcamerasdetect electrons(e−)viaaluminescentscintillatorlayerthatconvertselectronsintolight(␥).ThislightistransferredviaafiberopticcouplingintoaCCDchip,whichconvertsthe lightintoavoltageonasquarearrayofpixelsthatisreadoutinafewseconds,resultinginadigitalimage.(B)Incontrast,adirectelectrondetectorCMOSchipdirectly detectsincomingelectronsandconvertsthemintoavoltageoveranarrayofpixelsthatcanbereadoutathighspeed.
maryelectronbeamoperateinaso-calledzero-lossimagingmode, removingthebackgroundnoiseduetoinelasticallyscattered elec-trons.Consequently,thecontrastoftheresultantimageisincreased by∼16%comparedtounfilteredimages(Fukudaetal.,2015).Apart frombeingusefulforthickerspecimens,itisalsousefulin tomogra-phy,where,upontilting,theslab-shapedspecimeneffectivelygets thickerathightiltangles.Zero-lossenergyfilteringcanbeused togetherwithphaseplateimaging,andprovidean∼68%increase incontrastwhenusedincombination(Fukudaetal.,2015).
3.4. Directelectrondetectors
Electronmicrographswereoriginallyacquiredonphotographic film,whichneedstobedevelopedandscannedbeforedata anal-ysisortomogramreconstruction.Digitalcamerasaremuchmore convenientduetotheirnear-instantaneousreadout.Thefirst dig-italdetectorsforEMwerecharged-coupledevice(CCD)cameras (Dierksenetal.,1995;Kosteretal.,1992;KrivanekandMooney, 1993;SpenceandZuo,1988)(Fig.7A).CCDcamerasincorporate aluminescentscintillatorlayerthatconvertsincidentelectronsto photons,andthescintillatorlayerisconnectedtoaCCDchipby fiber-coupledoptics(Fig.7A),which spreadstheelectronsignal overa largeareadependingonthethicknessofthescintillator, resultingin lower resolutionimages. Thisspreading of the sig-nalcanbedescribedbya so-calledpoint-spreadfunction(PSF). Anotherimportanteffectofthisdesignisthatnotallincoming elec-tronsareregisteredasphotonsonthechip.Thelikelihoodthatan incidentelectronisactuallyregisteredisdescribedbythedetector quantumefficiency(DQE).Ideally,allelectronsaredetectedandno signalcomingfromthesampleislost.Furthermore,itisimportant thatdigitalcamerashaveahighdynamicrange,candetectbothlow andhighlevelsofelectrons,andtheirresponseoverthisbrightness rangeislinear.So,overall,gooddetectorscanbedefinedbyhaving asmallPSF,ahighDQEandideallyalinearsensitivityoverahigh dynamicrange.
TheprimaryreasonfortheresolutionrevolutionimpactingEM istheadventofdirectelectrondetectors(DEDs)(Xuongetal.,2007). DEDsdirectlyconvertelectronsintoanelectricalsignalforoutput detection(Fig.6B).Theyveryefficiently detectelectrons,which resultsin a highDQE,much betterthan film(McMullanet al., 2014).Therefore,alsothePSFnowdependsonscatteringof elec-tronsinsidethedetectoritself,whichcanbeminimizedbymaking thedetectorasthinaspossible.AnotheradvantageofDEDsistheir fastreadout,whichcanreachafewhundredframespersecond. Insteadofsingleimages,moviesofthesamplecanberecorded. Thisisusefulsinceirradiationbytheelectronbeamandthe
result-ingbeamdamageofthesamplecanresultinimagemovementand unpredictablelocaldistortionswithinthesampleitself.By record-ingmovies,thesemovementsanddistortionscanbecorrectedbya posteriorialignmentanddistortion-correctionoftheframes,which compensatesforsamplemovementandincreasestheresolutionof theimage.Anotheradvantageofthefastreadoutisseenwhenalow electrondoseisusedandthusindividualelectronscanbedetected. Infact,singleelectronscanbelocalizedwithsub-pixelaccuracy, so-calledsuper-resolutionmode,whichalsopositivelyaffectsthe DQEofthecamera(McMullanetal.,2014).InSPA,nownearatomic resolutionmapsareregularlyproducedofproteinandprotein com-plexes(Merketal.,2016).Intomography,theresolutionincrease resultingfromDEDshasenabledvisualizationofproteinsinside cellsandalsonear-atomicresolutionmapsusingsub-tomogram averaging(Schuretal.,2016).
3.5. Automateddatacollectionandstorage
Tomographicdatacanbeacquiredautomaticallybyseveral pro-grams,ofwhichserialEM(Mastronarde,2005),UCSFtomography (Zhengetal.,2004),TOM(Nickelletal.,2005),andXplore3D/Tomo4 (Thermo-Fischer,formerlyFEIcompany)aremostcommonlyused. Althoughthesearedevelopedfromdifferentperspectives(e.g.type ofsample,typeofmicroscope,typeofusers),theseprogramsin essenceperformthesameroutines(Kosteretal.,1992;Zieseetal., 2002).Withthecurrentimprovementsinhardware,tomographic acquisitionsoftwareistargetedtowardsautomatedrecordingof multipletilt-seriesoverseveraldays,withthesupportof movie-moderecordingbydirectelectrondetectorcamerasandtheVolta phaseplate.
Automateddatacollectionand movie-modesuper-resolution imaginghasfar-reachingconsequencesforimageprocessingand datastorage.Thenumberofdatasetsthatcanberecordedperday increasesonefactor,whilethesizeofasingletomogramtiltseries dataset,typically∼1Gbcaneffectivelyincreasebetweenoneortwo factors,witha4×increasegoingfromnormaltosuper-resolution mode and typically 7–30× increase due tothe frames used in movierecording.TheselargedatasetsneedCTFcorrection,movie alignmentandtomographicimageprocessing.Standarddesktop computershardlysuffice,andworkstationsareoftenemployedfor computation.
Fig.8.LayoutofsingleanddualaxistiltseriesinFourierspace.
Effectofangularsamplingandmissingwedgeontheresolutionintomograms.TheeffectoftheangularsamplingandthemissingwedgeintomographyshowninFourier spaceslices(a),surfacerenderingsofareconstructedthin-shelledsphere(b),andYZslicesthroughthecorrespondingtomogram(c).Theleftcolumnshowsthesituation whenthereisfullangularsamplingalongonetiltaxis(theX-axis).
ThetopimageshowsthatFourierspaceissampledequallyintheYZplane.Thesurfacerenderingandacentralsliceofthetomogramindicatenoanisotropicresolution inthereconstructedsphereinthosedirections.Themiddlecolumnshowsthesituationwhenthereislimitedtiltangle(from−60◦to+60◦)alongasingleaxis.Theresult
willbeamissingwedgeinFourierspacethatcanbeobservedinthesurfacerenderingandasdiminishingdensityatthetopandbottomofareconstructedsphere(b)and anelongationintheYZslice.Therightcolumnshowsthesituationwhentwotomogramsarecombinedfromtwoorthogonaltiltaxes(dual-axistomography)withlimited rotationbetween−60◦and+60◦.InFourierspace,themissingwedgeisreducedtoamissingpyramid,andtheanisotropyintheresolutionalongtheY-axisisreduced(from
G.vanTendeloo,D.vanDyckandS.J.Pennycook“HandbookofNanoscopy”,chapter36,page1313.2012.CopyrightWiley-VCHVerlagGmbH&Co.KGaA.Reproducedwith permission).
structuredatabases,suchastheproteindatabase(PDB)(Bernstein etal.,1977)andotherimagingmodalities(Patwardhanetal.,2014; Patwardhanetal.,2017).
4. Imageprocessing
4.1. Tomogramreconstructionautomation
Typically, reconstruction of cryo-electron tomograms con-sists of four steps; pre-processing, tilt-series alignment, post-processing,andreconstruction.Preprocessinginvolvestruncation ofextremedatavaluescausedbyX-raysorocclusionbytheEMgrid. Tilt-seriesalignmentisnecessaryasthesampleisnotrotated per-fectlyduringacquisition.Obviouswhole-imagemovementscaused byimperfectstagebearingscanbealignedusingcross-correlation oftheimages.Smallerandlocaldistortionsarecaused bybeam inducedmovement,specimendriftanddefocuseffects.For low-contrastCET tilt series,fine alignment is usuallyperformed by trackinghigh-contrastfiducialmarkers,5–25nmgoldbeadsthat areaddedtothesample priortovitrification specificallytoaid alignment(Lutheretal.,1988;Walzetal.,1997).However, alter-nativemethods,suchascross-correlationandpatchtracking,are availablethatdonotrequiretheadditionoffiducialstothesample (Amatetal.,2010;Guckenberger,1982).Accuratefinealignment
iscritical,aspooralignmentresultsinprojectionmismatchand limitstheresolutionoftheresultingtomogram.Micrograph post-processing includes CTF correction and noise filtering. Finally, 3DreconstructionscanbeachievedbyeitherFourier-space algo-rithms,suchas weightedback-projection (WBP)(Radermacher, 1988),orreal-spacemethods,suchassimultaneousiterative recon-structiontechnique(SIRT)(Gilbert,1972).WBPismorecommon, and involves taking a Fourier transform of each image before placingtheseslicesintoa3DFouriervolumeattheangles spec-ifiedduringtiltseriesacquisition.TheFouriercomponentsofeach slicearethenweighteddependingontheirfrequencytoprevent oversamplingoflow-resolutiondetails.Next,aninverseFourier transform back-projects the volume into real-space, yielding a tomographicvolume.ReconstructionusingWBPisgenerallyfaster thanSIRT,althoughWBPtomogramscanbedominatedby high-frequencynoise,whereasreal-spacereconstructionmethods,such asSIRT,generallytakelongerdue totheiterativenatureofthe method,althoughnoiseissuppressed.
Fig.9. Sub-tomogramaveraging.
(A)Fromatomographicvolume,sub-volumescontainingsingleparticlesofthesamebiologicalstructureareextractedfromtomographicvolumes.Inthiscase,theparticle isamembraneattackcomplex(MAC)poreperforatingaliposome.Theparticlesarealigned(B)andaveraged(C)togenerateaninitialmap.(D)Particlesaretheniteratively re-alignedtothepreviousmapandaveragedtogenerateimprovedmaps(i–iv).(E)Thefinalmapwiththehighestresolutionisachievedwhenaniterationdoesnotgenerate anyimprovement,andcanbeinterpretedwiththeuseofcrystalstructures(EMD-3289(Sharpetal.,2016)).
manual processing to achieve the best quality. Reconstructing tomogramsfromatiltserieswasoriginallyaninteractive, labor-intensiveprocessinwhichmanystepsweremanuallyperformed andchecked,especiallygoldfiducial finealignment,inorderto generateahigh-qualitytomogram.Sincemanualtomogram recon-structionisratherinefficientandslowerthandataacquisitiontime, automatedtiltseriesimagealignmentandtomogram reconstruc-tionproceduresarehighlydesirable.Recently,severalautomated fiducial markedbased andmarker-free finealignment schemes havebeendeveloped(Castano-Diezetal.,2010;Hanetal.,2015; Sorzanoetal.,2009)andimplementedinseveralpackagessuchas IMOD(MastronardeandHeld,2017),Protomo(NobleandStagg, 2015)andUCSFtomo(Zhengetal.,2011)(foranextensivelistof tomographypackagesseehttps://en.wikibooks.org/wiki/Software ToolsForMolecularMicroscopy),aswellasindualaxistiltseries (Winkler andTaylor,2013).Furthermore,severalpackageswith additionaldatabasestorageareavailable(Dingetal.,2015;Zheng etal.,2007).
4.2. Themissingwedgelimitsinterpretationoftomographic volumes
Duetothegeometryofboththespecimen/EMgridand objec-tivelensofthemicroscope,tiltseriesaretypicallycollectedover anangularrangebetween±60◦(Fig.1).Thisresultsina“missing wedge”ofdata(inFourierspace),causinganisotropicresolutionin thetomogram,whichisseenaslossofresolutionintheaxial direc-tion(Fig.8)(Diebolderetal.,2015;Sharpetal.,2017).Anisotropic resolutionisalsoinfluencedbythemissinginformationbetween sequentialtiltimages, whichhaveangularstepsof2–3◦ (Koster etal.,1997).Anisotropycanbereducedusingdual-axis tomogra-phy,wherebytwotiltseriesareacquiredwitharelative90◦inplane rotation(Penczeketal.,1995).Thetworesultingtomogramsare thenalignedandcombinedintoasingletomographicvolume.This procedurereducesthemissingwedgeintoa“missingpyramid”, whicheliminatesanisotropyespeciallyintheplaneorthogonalto
thetiltaxis(Mastronarde,1997),butdoesnotremoveblurringof thereconstructionalongthedirectionoftheelectronbeam. Dual-tilttomographyrequiresthatthesameregionisimagedtwiceand thereforeitisnecessarytofractionatetheelectrondoseoverboth tiltseriesinordertopreventincreasedradiationdamage. There-fore,eachofbothdualaxistiltseriesreceiveshalfofthetotaldose comparedtoasingleseriesandthustheseeitherhavevery poor-contrastimagesorhalfthenumberofimages.
Onemethodtoeliminatethemissingdataentirelyisimaging arod-shapedsampleinaholderthatcanberotatedbyafull180◦ (PalmerandLowe,2014).Alternatively,incasethetomogram con-tainsmanyidenticalcopiesofaproteincomplexorparticle,the missingdataoftheseparticlescanbefilledinbyvolumeaveraging, knownassub-tomogramaveraging.
4.3. Sub-tomogramaveragingandtemplatematching
meth-Fig.10.Surfacerenderingofcellularcryoelectrontomograms.
(A)Aslicethroughacryo-electrontomogramofacellshowsallkindsofstructuresin2D.(B)Somestructuraldensitiescanbesurfacerenderedbytemplatematching,e.g. actin(blue),whileothersaredrawninbyhand:lipidmembranes(yellow),microtubules(green),mitochondria(purple),andintermediatefilaments(red).(Forinterpretation ofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
ods,evennearatomicresolutionstructurescanbeachieved(Schur etal.,2016).
Apartfromaveragingsub-volumesfromatomograminorder toincreaseresolution,onecanlookforknownstructuresinside a tomogram. The structure of many molecular machines from cellsaredetermined(e.g.fromX-raydiffraction,cryoEMandNMR techniques)andavailable(e.g.fromtheProteinDataBank(PDB); www.rcsb.org(Bermanet al.,2000)or theElectronMicroscopy Database(EMDB)(Lawsonetal.,2016)).Byutilizinga template-matchingapproach, an extensivesix-dimensional(three spatial dimensionsandthree rotationaldimensions)cross-correlational searchbetweenthemolecularvolumeandthecellulartomogram canbeperformedtofindthepositionandorientationofcertain proteinmoleculesinsidepartsofacell(Nickelletal.,2006). Molec-ularcrowdingandlowtomogramresolutionlimittheapplicability ofthistechnique(Becketal.,2009),buthugeimprovementshave beenmadeusingthestate-of-the-artimagingtechniquesdescribed herein(Mahamidetal.,2016).
4.4. Tomogramsegmentation
Asinmedicaltomographicreconstructions,theinterpretation of3Dvolumedataisdependentonaspecialistwitha familiar-itywiththe specimenand knowledgeof thepotentialartifacts presentintomograms.Interpretationisdifficultfortheuntrained eyebecauseoneisaccustomedtolookingatsurfacesandnotat noisyslicesfromvolumes.Therefore,surfacerepresentationofthe volumesintobiologically-relevantsegmentsgreatlyaidsthe inter-pretationand 3D viewof the volumefor both the trained and untrained eye(Fig.10).A particularproblem with(automated) segmentationofcryo-electrontomogramsisthatthevolumesare noisy(duetothelimitedelectrondose),anisotropicallydistorted (duetothetiltgeometry/missingwedge), andthat density val-uesarenotlinearwiththeoriginalsignal(duetotheCTF,phase contrastandreconstructionalgorithms).Inpractice,structuresof interestareoftendrawnbyhandintothevolume,whichisboth highlytime-consumingandsubjective.Automatedsegmentation isdesirablebecauseitislessuserintensiveand,moreimportantly, itprovidesanobjective,reproducible waythatallows interpre-tationandquantificationofresults.Theseobjective,reproducible segmentedtomogramscanalsobedepositedin databasessuch asEMDB(Lawson etal.,2016).Identifyingparticularstructures withincellularvolumesislimitedbytheresolutionofthe tomo-gram.However,certainlargerstructures,suchasactinfilaments andmicrotubules,weresuccessfullytracedandsegmented auto-maticallybytemplatematchingofrod-likeshapes(Rigortetal.,
2012b;Rusuetal.,2012).Also,someinitialattemptson segmen-tationofsurfacesofliposomesweremade(Koningetal.,2013). Eventemplatematchingofspecificproteinsinsidecellsis possi-ble(Asanoetal.,2015).TheincreasingqualityofCETimagingand theresultingtomograms,combinedwithdevelopmentand imple-mentationofbetterandfastertemplatematching,neuralnetwork algorithmsandincreasingcomputationalresourcesmakesit possi-bletoautomaticallysegmentand/ortemplate-matchmanyofthe largerstructuresinsidecellulartomograms(Chenetal.,2017),and developmentsinrelatedfields,suchasmulti-dimensionaltransfer functionvolumerendering(Knissetal.,2002),arealsoexpectedto yieldimprovementsintomogramvisualization.
5. Relatedtechniques
AsidefromtheCETtechniquedescribedabove,whichisbased on 2D imaging at different angles, there are several related newly-developingcryotechniquesthatareworthmentioningin the context of three-dimensional imaging of cells and tissues inan anatomicsetting:cryoserial block-facescanningelectron microscopy(cryoSBF-SEM),whichcombinesserialsectioningand imaging of surfaces with scanning electron microscopy (SEM), cryosoftX-raytomography(cryoSXT),inwhichX-raysareused for imaging,and cryocorrelativelight and electronmicroscopy (cryoCLEM),which combinesfluorescentlightmicroscopy(fLM) imagingfortargetingspecificsiteswithCET.
5.1. CryoSBF-SEM
Fig.11.Cryo-CLEM.Cryocorrelativelightandelectronmicroscopy.
(A)Cryo-fluorescentlightmicroscopy(cryo-fLM)imagingoffluorescentlytaggedStreptomycesbacterialcellscanaidthetargetingofcertainstructures.(B)ImagingcryoEM ofthesameareaallowstargetingoflabeledstructuresintheelectronmicroscope.(C)HighermagnificationcryoEMimagingandaccurateoverlaydeterminesthespecific siteforCETimaging.(D)Overlayofthecryoelectrontomogramsliceandthecryo-fLMlightsupthelabeledstructuresinsidethecell.
5.2. CryoSXT
Incryosoft X-raytomography (cryoSXT),likeCET,a3D vol-umeofbiologicalmaterialisalsoreconstructed fromaseriesof projectionviews,thoughwithseveraldifferences.X-raysareused forimaging,andsincetheyhavelargerpenetrationdepthfor vit-rified biological samples than electrons, complete cells can be imaged.ThesoftX-rayshaveatunedenergythatfallswithinthe so-called‘waterwindow’(between284–543eV)whichoptimizes theabsorptiondifferenceof X-raysbetweenoxygenandcarbon elements,which isthecontrastingmechanism.TheX-raybeam isfocused ontothesample andthedetector byzone-plates, X-raydiffractingelements.Thecurrentresolutionisintheorderof 25–35nm.CorrelativecryofLM/cryoSXTtechniquesarealso devel-oped and typicalexamples of cryo-SXTimaging are wholecell reconstructionsoftheyeastSaccharomycescerevisiae.Foramore in-depthreview,seeCarzanigaetal.(2014).
5.3. Cryo-correlativelightandelectronmicroscopy
TherecentdevelopmentsinCETenableimagingof macromolec-ularandproteinstructuresinsidecryogenicallypreservedcellular landscapes(Mahamidetal.,2016).Suchhigh-resolutiondatasets relyonhigh-magnificationtomograms,whichlimitstheareathat canbeimaged.Specificpositionsorareasofinterestcantherefore bedifficulttoidentifywithinthecell.Onesolutionistoemploy cryo-correlativelightandelectronmicroscopy(cryoCLEM),which combinesCET withcryo-fLMof thesame sample(Fig.11).This ispossibleby usinglabelling techniquesforfLM,eithergenetic tagging(suchasGFP)orbychemicallabelingusingspecificdyes totargetandidentifystructuresofinterest,eitherinatwo-step
LM/EMapproach(Celleretal.,2016;Hamptonetal.,2017)oran integratedsetup(Faasetal.,2013;Wangetal.,2017).Currently withcryo-CLEM,theresolutiongapbetweenfLM(∼400nm)and CET(∼4nm)istwoordersofmagnitude,althoughlocalizationcan beperformedwitha precisionof∼40nmwithfLM(Schorband Briggs,2014).Nevertheless,targetingspecificmoleculesinsidea crowdedlandscaperequiredhighercryo-fLMimagingtechniques, suchassuper-resolutionfLM(Wolffetal.,2016)foraccurate local-ization,andfuturedevelopmentsaretargetedtowardsintegrating theseintocryo-CLEM. Thiswillneed adaptationofLMsystems and thedevelopmentof probesspecificallyfor super-resolution cryoCLEM,butwillallowmultidimensionalfunctionalimagingof cellularlandscapesatnanoscaleresolution.
6. Conclusionsandperspectives
Despite these recent advances, CET is still relatively labor-intensiveandslowcomparedtoSPAcryo-EM.Datacollectionof cryo-EMislargelyautomated,aftersettingimagingparametersand semi-automatedtargetingofimagingpositionsontheholey sup-port.CETshouldbemadefasterby(i)automatedtargetingusing CLEMbytheintegrationwithfluorescentlightorramanmicroscopy techniques,and(ii)fastertiltseriesacquisition,whichnow typ-icallytakesapproximatelyanhour, byimplementationof more stablestagetiltbehavior,adaptedacquisitionsoftwareandfaster cameras,inordertoallowaspeedincreaseofcircaafactorof100 (Migunovet al.,2015).Furthermore,tomographicimage recon-structionshouldbefullyautomatedandintegratedwithacquisition andalsorobustinordertopreventlargedatastoragerequirements ofintermediaterawfiles.Automaticsegmentation,quantification andvisualizationby3Dsurfacerenderingshouldbedevelopedto aidinterpretationofdata.InordertomakeCETmorewidely appli-cable,designatedCETmicroscopesystems,comparabletomedical imagingdevices(CAT,MRI,CT)mightbethewayforward.
Dueto the advanceswe have described, the ability to per-formmolecularimagingofsubcellularstructuresatnanoscalelevel invivoandinsitumeansthattheapplicabilityofCETtobiologyand medicinehasneverbeenhigher.Futuredevelopmentswillonly increasetherelevancyofthistechniquetodiagnostics.Wewilluse thesetechniquestoimagebiologicalprocessesintheirnative envi-ronment,suchascellular,viralandbacterialinfectionsandimmune complexesbindingtocells,toelucidatethemolecularmechanisms ofdisease.
Acknowledgements
This work has been supported by iNEXT, project number 653706,fundedbytheEuropeanUnion’sHorizon2020research andinnovationprogramme undergrantagreement759517,the DutchfoundationSTWProjects13711,NanosurfAGandSmartTip B.V.,and12713,MicroscopyValley,theCouncilforChemical Sci-ences(CW)oftheNetherlandsOrganizationforScientificResearch (NWOgrant700.57.010).Theauthorsacknowledgethesupportand theuseofresourcesofInstruct,aLandmarkESFRIproject.Wethank RobHoeben,TonRabelink,TonRaapandPeterNibbering(LUMC) forcriticalreadingofthemanuscript.
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