Fall2016Newsle-er
This newsleJer summarizes current capabiliCes and includes graphs
showing usage trends to date for 2016. The NDIIF operates as a recharge
facilityandrevisesuserfeeseveryJuly1.
QuesCons about NDIIF policies should be directed to the Director or any
memberofthesteeringcommiJee.FortechnicalandschedulingquesCons,
seetherelevantNDIIFstaffmemberslistedonthefollowingpages.Contact
numbers and addiConal informaCon can be gained by
visiCnghJp://www.imaging.nd.edu.
Sincerely
ProfessorBradleySmith,DirectorofNDIIF
[email protected]
TableofContents
NDIIFNewsBriefs
p. 2
AdvancedElectronMicroscopyCore
p. 4
InVivoImagingCore
p. 11
OpCcalMicroscopyCore
p. 17
HistologyCore
p. 27
NDIIFPolicies
p. 28
1
NDIIFNewsBrief
1.  MidwestImagingandMicroanalysisWorkshopatNotreDame:The
thirdannualworkshop,heldMay2016,wasverysuccessfulwitha
focusonelectronbeamtechnologies.Theeventwillberepeatedmid
May2017.
2.  NDIIFAwardsforBestImagingPublicaIons2015:
•  TheBestElectronMicroscopyImagingPublicaConAwardfor2015
wasawardedtoSarahFathipour,agraduatestudentwith
ProfessorAlanSeabaughintheDepartmentofElectrical
Engineering.
•  TheBestBiologicalImagingPublicaConAwardfor2015was
awardedtoDr.ManuelaLahne,AResearchAssistantProfessor
andacollaboratorwithProfessorDavidHydeintheDepartment
ofBiologicalSciencesandtheCenterforZebrafishResearch.The
submissiondeadlineforthe2016awardsismidFeb2017.
3.  CTSICoreFacility:AffiliaConwithCTSIasacoreresearchfacilitywas
renewed.FormoreinformaConpleasevisit:
hJp://www.indianactsi.org
4.  InVivoImagingCore:acquiredaUniversalLaserCuJerVLS6.60–
LasercuJer/engraver.
5.  ElectronMicroscopyCore:acquiredaDENSheaCngandbiasingholder
foratomicresoluConimagingofin-situheaCng.
2
NDIIFMissionStatement
Established in 2008, the NDIIF provides an integrated suite of
sophis<cated microscopes and imaging sta<ons that enable the expert
userstoa@ackthemostcomplexmodernresearchproblemsand,equally
important, the resident professional staff (technicians and research
specialists)toguidethenon-expertusers.
NOTREDAMEINTEGRATEDIMAGINGFACILITY
ORGANIZATIONALPLANFY16
Director:Dr.BradleySmith,COS
SteeringCommittee:
Dr.KevinVaughan(Chair),COS
Dr.HollyGoodson,COS
Dr.CrislynD’Sousa-Schorey,COS
Dr.KenKuno,COS
Dr.GaryBernstein,COE
Dr.GlenNiebur,COE
Dr.PaulMcGinn,COE
Of1iceManager
TheresaBollinger
DirectorofBiologicalImaging
Dr.W.MatthewLeevy,
BiologicalImagingAssistantDirector
SarahChapman,
LabMangerCellMicroscopy
WilliamArcher,
DirectorofElectronMicroscopy
Dr.AlexMukasyan
LabManagerSEM,FIB
TatyanaOrlova
TEMProgramDirector
Dr.SergeiRouvimov
MicroprobeProgramManager
Dr.IanSteele
3
ADVANCEDELECTRON
MICROSCOPYCORE
AboutUs:
TheAdvancedElectronMicroscopyCoreintegratesasuiteofsophisCcated Dr.AlexanderMukasyan
ResearchDirector
devices that enable the expert users to aJack the most complex modern
research problems from micron to Angstrom spaCal resoluCon and the
residentprofessionalstafftoguidethenon-expertusers.Thecorecontains
auniquebundleofthreestate-of-the-artFEItoolsandincludes:
•  Magellan 400, Digital Field Emission Scanning Electron Microscope
(FESEM),recordhighspaCalresoluCon0.6nm;
•  Helios NanoLabTM DualBeam 600, SEM/Focus Ion Beam (FIB)
WorkstaCon that is capable of nano-prototyping, nano-machining, nanoanalysis,andadvancedTEMsamplepreparaCon;
•  EVO50Zeiss,environmental SEMwithPelCerStage,allowsoperaCon
underhighandlowvacuumcondiCons.
•  Titan 80-300, Transmission Electron Microscope (TEM) enables subAngstrom, atomic scale discovery and exploraCon in both TEM and STEM
modesoverawiderangeofmaterialsandoperaCngcondiCons.
•  JEOL2011,TransmissionElectronMicroscope(TEM)80-200kV,0.14nm
resoluCon,workhorseformaterialsandbiologicalsamples.
Researcherscanbetrainedtouseourdevices,orservicecanbeprovided
byexperiencedstaff.
ForgeneralquesConsabouttraining,orcontractedimageacquisiConand
analysis,contact,Dr.AlexanderMukasyan574-631-9825or
[email protected]
[email protected]
Mrs.TanyaOrlova
ResearchTechnician
!
Dr.SergeiRouvimov,
TEMProgramDirector
Dr.IanSteele,
MicroprobeProg.Director
SEM/FIBWorkstaOon:
HeliosNanoLabDualBeam600(FEI):
TheHeliosNanoLab™WorkstaIoniscapableof:
• nano-prototyping
• nano-machining
• nano-analysis
• advancedTEMsamplepreparaCon
SpecificaOons:
ElectronopOcs:
-Resolu<on:0.9nm@15kV,1.4nm@1kV
-Detec<on:in-lensSEandBSE
IonopOcs: Sidewinder™fieldemissionfocusedionbeamopCcs
withliquidGalliumionemiJer;
-Resolu<on:5.0nm@30kV
-Detec<on:CDEMdetector
Cross-secConofhighlyporous(93%)
metalbulkalloy(Prof.Mukasyangroup)
CrosssecConofamesasarrayforlayer
thicknessmeasurementandstructure
profilecontrol(Prof.TangfeiLuogroup)
ApplicaOon:
• PaWerning:Simultaneousimagingandpa@erning
withend-pointdetec<onthroughReal-TimeMonitor;
• AutoFIB;
• PlaOnumDeposiOon;
• SelecOveCarbonMill;
• EnhancedEtch;
• TEMsamplepreparaOon:
-AutoTEMG2;OmniprobeAutoProbe200.2
DiagnosOcs:-EDS&EBSD-PegasusPackage,EDAX
EBSDfortexturedAlalloy
(Prof.S.Songroup,PurdueUniversity)
FESEM:Magellan400(FEI)
TheMagellanXHRSEMallowssample
imagingatextremelylowbeamenergies
(<100eV),avoidingchargeeffectat
non-conducCvenano-scalesurfaces.
StaphylococcusaureusinteracCngwith
epithelialcells(TrevorKane)
Aring-diskelectrodearrayfabricatedon
glassbynanospherelithographyand
mulC-stepRIEetching(ChaoxiongMa)
TheMagellan400isafullydigitalFESEM
withSchoJkeyfieldemiJersourcemounted
ontheNGhot-swapgunmodulethatprovidesrecord
highspaCalresoluCon:0.6nm@15kV,0.9nm@1kV
FeaturesandspecificaOons:
-in-lensSEandBSEdetecConspeciallydesigned
forhigh-resoluConimagingatbothhighandlowkV’s;
-Everhart-ThornleySEdetectorforSEdetecCon.;
-AnintegratedIRCCDcameraforin-chamberviewing;
- RetractableAnnularSTEMDetectorenables
scanningtransmissionimaginginbrightfield,
darkfieldandhigh-angledarkfieldmodes.
EnergyDispersiveX-RaySpectrometer(EDS)Bruker
-SLEWWindow,detecConofBoronandup;
-energyresoluIon123eV(MnKα,0-100,000cps);
-Elementalmappingandmore
Asteelexposedto1.5MeVelectron
beamirradiaCon(SlavicaGrdanovska)
OlicellwithemergingM13bacteriophage.
(MicheleCostanCno,IUSB)
EnvironmentalSEM:EVO50LEO(CarlZeiss)
TheEVO50ZeissenvironmentalSEMequippedby
PelCerStage,whichallowstoworkbothunderhigh
andlowvacuumcondiCons,hasthefollowing
specificaCon:
Resolution
2.0nm@ 30kV (SE with LaB6 option )
Acceleration Voltage
0.2 to 30 kV
Magnification
35x to 1,000,000x
Field of View
6 mm at the Analytical Working Distance
(AWD)
EDX-ray Analysis
Oxford Instrument, resolution 133 eV
Available Detectors
• SE in HV - Everhart-Thornley
• SE in VPSE
• BSD in all modes - quadrant
semiconductor diode
Chamber
Approx. 365 mm (dia.) x 255 mm (h)
5-Axes
Motorized
Compucentric
Specimen Stage
Image Processing
Image Display
System Control
Element
Weight%
Atomic%
CK
OK
PK
Ga K
As L
In L
3.10
0.73
13.35
41.48
19.76
21.58
14.48
2.56
24.19
33.40
14.81
10.55
• X = 100 mm (+50mm, -50mm)
• Y = 125 mm (+65 mm, - 60 mm)
• Z = 55 mm (35 mm motorized)
• T = 00 - 900
• R = 3600 (continuous) Stage control by
mouse or optional joystick and control
panel
Resolution: Up to 2304x1024 pixel Signal
acquisition by integrating and averaging
Single flicker-free XVGA monitor with
SEM image displayed at 1024 x 768 pixel
Smart-SEM with Windows, operated by
mouse, keyboard and optional control
panel.
ApplicaIons:
UserfriendlyandwitharelaCvelylowuserfee,the
EVO50ZeissisanaJracCveopConforrapid
screeningofsamplesmicrostructures.Itisorenused
forEDXanalysiswithspaCalresoluConof1µm
Quantitative results
Weight%
50
40
30
20
10
0
C
O
P
Ga
As
In
EDXspectraanddataofelementalanalysisinthespotofGa-basedmonocrystal
TEM:Titan80-300(FEI)
TheTitan80-300microscopeincorporatesanovel
plasormthatallowsulCmatestability,performance
andflexibility:
• HighResoluCon(HR)TEMmode
• HRScanningTEM(STEM)mode
• HRElectronEnergyLossSpectroscopy(EELS)
• HREnergyDispersiveX-Ray(EDX)
FeaturesandspecificaOons(at300kV):
EnergySpread-0.7eV
PointResolu<on-0.2nm
Informa<onlimit-<0.1nm
STEMResolu<on-0.136nm
ApplicaOon
• TEMimagesandElectronDiffracOon
• HighResoluOonTEMimagesatAngstromresoluOon
• HighthroughputSTEMmodeatAngstromresoluOon
• EDX-composiOonalanalysisatnm-scale
• ElectronEnergyLossSpectroscopy
HAADFSTEMimagesofgradedAlGaNlayers(incross
secCon)showingbothplanarspontaneoussuper-lauces
and3DcomposiConalfluctuaConsintheAlGaNlayer.
InsertedisEDSscanlinesshowingvariaConofAlandGa
ingradedAlGaNlayer(lerimage).
S.Rouvimov,S.M.Islametal.“TEMAnalysisofStructure
andComposiConalFluctuaConsinMBEgrownAlGaN
StructuresforDeep-UVPhotonics”,EMC2015
V.Kanzyuba,S.Dong,etal(groupofProf.J.Furdyna),
“StructuralproperCesofSnMnSe:anew2DmagneCc
semiconductorwithpotenCalforspintronic
applicaCons”,MM2016;S.Dong,etal“Room
temperatureweakferromagneCsminSn1−xMnxSe22D
filmsgrownbymolecularbeamepitaxy”,APLMATERIALS
4,032601(2016)
EELSallowstoobtainelementalandchemical(suchas
valenceandthecoordina<onofspecificatoms)
informaOonwithnanometerresoluOon.
(a)SchemaCcand(b)falsecoloredTEMcross-secConoftheAg/HfO2
TSdevice.InsetshowsAg/HfO2/p+Siinterfacethatenablesthreshold
switchingacCon.Thecross-secConistakenalongx-y.(c)EDXscanof
theinterface.NikhilShukla,BenGrisafe,etal(groupofSuman
Da-a),“Ag/HfO2basedThresholdSwitchwith107SelecCvityand
100μAON-currentforCross-PointSelectorandSteep-slopePhase-FET
ApplicaCon”,2016IEEEInternaConalElectronDevicesMeeCng,San
Francisco,CA,2016.
TEM:JEOL2011
TechnicalSpecificaIons
•OperaCngvoltageof80–200kV
•HRPolePiecewithresoluConof0.14nm.
•MagnificaConof50x–1,500,000
•JEOLsingleCltholder
•Gatan636DoubleTiltholder
•Gatan622TVcamera
•CCDcamera*
•OxfordINCA30mm2LN2detector
!
This “cerCfied” used instrument (2001) is
fully operaConal. For quesCons about TEM,
trainingandapplicaCons,please,contactDr
SergeiRouvimov,233SCnson-RemickHall
E-mail:[email protected]
Tel:574-631-0226(office)
TEMImagesofLargeUnilamellarvesicleswithand
withoutprotein.Doestheproteinbendmembranes?
KristenA.Johnson(groupofProf.R.V.Stahelin)
Low-andhigh-magnificaConTEMimagesof(a,c)
CdSand(b,d)CdS/NiNSs.Insetsgivethe
correspondingensembleSAEDimages(a,b)anda
high-resoluConTEMimageshowingbasalplane
laucefringes(toprightin(c))aswellasaNSside
view(boJomlerin(c)).EnsembleEDXSspectra
of2.16nm(e)CdSand(f)CdS/NiNSs.).
MaksymZhukovskyi,etal(groupofProf.Kuno)
“EfficientPhotocatalyCcHydrogenGeneraCon
fromNiNanoparCcleDecoratedCdS
Nanosheets”,ACSCatal.2015,5,6615−6623
Atomicmodels(top)andHRTEMimages(boJom)ofSnS2
toCu2SnS3.InsertedarediffracConpaJerns.Yuanxing
Wang,etal(groupofProf.K.Kuno),“Transforming
LayeredtoNonlayeredTwo-DimensionalMaterials:
CaConExchangeofSnS2toCu2SnS3”,ACSEnergyLeJ.
2016,1,175−181
ElectronProbeAnalyzer:CamecaSX-50
Chemicalanalysisandelementalimagingonamicronscale
TheCamecaSX-50canprovidehighaccuracy
chemicalanalysesofawidevarietyofpolished
samples(e.g.minerals,rocks,meteorites,metals,
glasses,cements,ceramics,etc.)withmicron
resoluCon.Elementalimagescanbeobtained
usingeitherwavelengthofenergydispersive
detectorsinaddiContoBSEimages.
Example:
ApplicaIontoba-eryfailure
Imaged areas
from different
batteries.
Polished section
Location of sulfate deposition during
discharge by imaging polished sections
of battery plates: Bright areas are
PbSO4 formed during discharge.
Sulfate interior – good
battery
Surface and interior
Surface only – prevents acid
reaction – battery fails
2 mm
INVIVOIMAGINGCORE
AboutUs:
TheNotreDameInVivoImagingCoreprovidesanon-invasiveapproachtoobservevariousdiseaseand
biologicalcondiConsinlivingmice.Thisfacilitycurrentlyhasteninstrumentsundermanagement,witheight
modaliCesavailablefordirectuseraccess.First,theIVIS®LuminaenablesthesensiCvedetecConof
bioluminescencefrommammalianandbacterialcellsthatexpressluminescentgeneCcreporterconstructs.
Next,theBrukerXtremeisuClizedtoimagecellsthatexpressfluorescentgeneCcreporterslikeGFPorRFP,
andalsodetectthebiodistribuConofnear-infrared(NIR)probesandnanomaterials.Further,theXtreme
offershighresoluCon,highspeedplanarX-rayforanatomicalandradiographicimaging.TheXtremealso
offersplanarscinCgraphicimagingofawiderangeofradiolabeledspecies.
InaddiContoplanaropCcalandX-rayimagingimaging,thefacilityoffersnuclearimagingwithPositron
EmissionTomography(PET),SinglePhotonEmissionComputedTomography,inaddiContoanatomical
imagingwithX-rayComputedTomography(CT).NuclearimagingmodaliCesareenabledbythestate-of-theartAlibraTrimodalPET/SPECT/CT.AsuiteofX-rayComputedTomographyequipmentincludestheScanCo
VivaCT80(smallFOV,highres),NeurologicaCereTom(largeFOV,lowres)andAlbira(medFOVandmedres).
NuclearprobesarecommerciallyavailablethroughlocalvendorslikeSpectronMRC(PET)andCardinal
Health(SPECT)toenableaccesstocriCcalapplicaConareasinheart,tumor,lung,andbone.Tomographic
anatomicalimagingofsorCssuesisprovidedbyanICONMRI.Finally,theInVivoCorealsooffersrapidbody
composiConanalysisofFatandLeanCssueweightsusinganEchoMRIsystem.
OurgoalistotrainandempoweruserstoindependentlyuClizethefacilityanditsresources.Contactthe
DirectororAssistantDirectorofBiologicalImagingforassistance
Prof.MaJhewLeevy
Director,BiologicalImaging
[email protected]
SarahChapman
AssistantDirectorofBiologicalImaging
[email protected]
INVIVOIMAGINGCORE
KeyEquipment:
BrukerXtreme
(Twounitsoncampus)
InVivoFluorescence
PlanarX-ray
PanarScinCgraphy
IVISLumina
(Twounitsoncampus)
InVivoBioluminescence
ICONMRI
Demothru2016
Anatomicalimaging
ofsorCssue
EchoMRI
BodyComposiCon
Analysis
INVIVOIMAGINGCORE
KeyEquipment:
Neurologica
CereTom
LargeBoreX-rayCT
ScanCoVivaCT80
HighResoluConX-rayCT
MARSMedipix
BrukerAlbira
SpectralX-rayCT
PositronEmissionTomography(PET)
SinglePhotonEmissionComputedTomography(SPECT)
X-rayCT
ALBIRAPET/SPECT/CT
Tumor Imaging - Time-course of healthy lung volume
degradaIon during breast tumor metastasis. Calli Davison of
theSchaferLabisstudyingbreastcancermetastasisinlivingmice
using non-invasive X-ray computed tomography on the Albira
PET/SPECT/CT. Two cohorts of mice were injected with either
breast cancer cells, or saline (control), and were imaged over a
period of six weeks. The lung Cssue volume was segmented
(shownaboveinpurple)andquanCfiedforeachgroup.Thelung
destrucConcausedbytumorgrowthisreadilyapparentfromthe
invivoimages. TheSchaferlabiscurrentlyusingthistechnology
tostudythemetastaCcproperCesofbreastcancercelllineswith
alteredexpressionlevelsoftheenzymecatalase.
ImagingBrainStroke–Prof.RashnaBalsaraisusingPositronEmission
Tomography(PET)toimagebrainmetabolisminratswithcerebralischemia.The
imageshowstheCT(anatomicalmap,ler),PET(center)andPET-CToverlay(right)
ofarat24hoursarerastrokeevent.
TetramodalSPECT-PETCT-MRIofaSingle
Mouse–TheLeevylabhas
developedanewmethodfor
tetramodaltomographicimaging
ofmice.Hereweshowone
mouseimagedbyCT(X-ray),MRI
(SorTissue),PET(BoneScanwith
Na18F),andSPECT(Lungperfusion
scanwithMAA-99mTc.
WK2
WK4
WK6
ALBIRAPET/SPECT/CTcont.
EvanDoneyoftheLeevyLab
isdevelopingnovelmethods
thatcombinepreclinical
imagingandaddiCve
manufacturing.Ratsscanned
withtheAlbiraImageStaCon
X-rayCTmodality(top)were
renderedandeditedas
stereolithographsandprinted
asphysicalmodelsboJom
usinganumberof3DprinCng
plasorms,including
Shapeways.com(boJom)
SorCssuesegmentaCon,prinCnginmulCplecolorsandmodelcustomizaConarealso
possiblewiththisnewlydevelopedmethod.TheIntegratedImagingFacilitycurrently
hastwo3Dprintersandisimprovingandexpandingthesemethodstothe
microscopicleveltoincludehumandata,aswellascellsandorganelles.
SCANCOHIGHRESOLUTIONX-RAYCOMPUTED
TOMOGRAPHY(CT)
ImagingtheAnatomicalFeaturesofSpecimensRangingfrom
BeetlestoRats–X-rayCTmaybeusedtoimagetheinternalstructuralfeaturesof
ratsorother
specimenslike
beetles.
ApplicaConsinclude
bonedensity
imaging,anatomical
imaging,and
phenotyping.
BrukerMULTISPECTRALFXIMAGESTATION
FluorescenceImagingof
BoneRemodeling–The
MSFXspecializesinthedetecConof
fluorescentprobesinlivingmice.At
rightisamontageofX-ray,
fluorescence,andoverlayofamouse
injectedwithOsteosense-750,a
probeforboneremodeling.Image
generatedbyundergraduate
researchersintheLeevyLab.
DynamicImagingofP.aeruginosa
SwarmBehavior-Researchersfromthe
labofProf.JoshuaShroutareperformingCme
lapsefluorescenceimagingGFP-expressing
bacteriatomonitortheirgrowthandswarm
kineCcs.Atlerisasingleframefromathreeday
Cmecourse,inwhicha“fire”colorscaleisusedto
indicatedtheintensityofGFPsignalcomingfrom
differentregionsontheplate.
OpIcalImagingofRFP
OvarianCancerMets–The
Cowden-DahllabisusingRFPexpressingovariancancercellsto
trackandquanCfytumormetastasis
duringexvivoimaging.Theimageat
rightshowsaphotoofanIPregion
ofamouse(ler),theRFP
fluorescenceimage(center),andan
overlayofboth.
IVISLUMINA
InVivoImagingofaBioluminescent
SalmonellaInfecIon–ResearchersfromtheSmith
LabhavedevelopedinvivomodelsofinfecConusing
bioluminescentbacteria.Thesebacteriaareengineeredto
emitlight,thusfacilitaCngtheirdetecConusingalight
sensiCveCCDchip.BLIemissionreportsonthehealthof
theseinvadingcells,anddrugtherapymaybenon-invasively
monitoredasadecreaseinlightemanaCngfrominfected
Cssue.
MulImodalProstateTumorImaging–AcollaboraConbetween
researchersfromtheSuckowLab,LeevyLab,andDr.BrianRabinovichfrom
MDAndersen,hasproducedaprostatetumorcelllinewithdualbioluminescent
andfluorescentgeneCcreporters.Fromlertorightisasub-Qtumorwith
signalcapturedinphotographic,mCherryfluorescence,exogenous
MMPSense750probefluorescence,Cerenkovluminescence(FDG),andPET
(FDG)modaliCes.
Photo
mCherry
MMPSense750 Cerenkov
PET
OPTICALMICROSCOPYCORE
AboutUs:
TheOpCcalMicroscopyCore(OMC)givesresearchersaccesstohigh
endresearchmicroscopesallowingthemtoimage,eitherfixedorlive
(inenvironmentallycontrolledchambers),fluorescentlylabeledcells
andCssuesthataremountedonslidesorinpetridished.Oursystems
letsusersworkwithuptofour(separatechannels)fluorescent
markersintheemissionspectrarangingfromDAPIintheUV
wavelengthstothefar-redfluorophoresnearingtheIRwavelengths.
Thecontrollingsorwaresgivethemtheopportunitytoacquiresingle
informaCveimagesaswellasz-seriesimagesforthree-dimensional
reconstrucCons.
BillArcher
ResearcherscanbetrainedtouseourdevicesinasliJleastwo
hours.TheCoreislocatedinSuite007inthebasementofGalvinLife
LabManager
SciencesBuilding.ForconsultaCons,schedulingoftraining,orfor
OpCcalMicroscopyCore
contractedimageacquisiCon,contacttheLabManager,BillArcher
574.631.5443([email protected]).
ApplicaIons:geneCcallyencodedfluorescentproteins–anCbodies-fluorescentmolecular
probes–singlecellsandmulClayeredCssues–changesinCssuearchitecture–Cme-lapsemovies–
movementandgrowthofsub-cellularorganellesandmacromolecules–fluorescentionicgradient
reporters–imagingoffixedandlivingsystems:bacteria,proCsts,Drosophila,zebrafish,allculturedcell
types–transparentnon-livingsamples
Equipment:
NikonAZ100Marco/ZoomScope
TheNikonAZ100isamulC-purposemacrozoommicroscopethat
hasmulCplemethodsforonetoobservesamples(fixedslides,to
smallorganisms)eitherinbrighsield(toplitorbacklit),Nomarski
DIC,orepi-fluorescence.ItcoversawiderangeofmagnificaCons,
from10xto320xallowingtheusertoimagethesamesamplefrom
macrotomicroobservaConsaswellascreatemoviesoflive
samples.
Brighlield/Backlit
NomarskiDIC
Brighlield/Toplit
Above:ImagesbyJackieinDr.MichaelPfrender’slablookingat
developmentalstagesinthewaterfleaDaphnia
Below:ImagesbyChrisKegelmanworkinginDr.JoelBoerckel’slab
Mouseembryo
ribcage
Mouseembryo
skull
Mouseembryo
vertebra
NikonEclipse90iWidefieldFluorescentMicroscope
TheNikonEclipse90iisaneasy-to-usestandardupright
brighsield/fluorescentresearchmicroscopeequipped
withtwocameras(onecolorandonemonochromaCc)
andiscapableofcapturingsingleimagesormovies,of
wholeCssues,smallorganisms,etc.witharangeof
objecCvelensesfrom4xupto100x.
19
Below:ImagesbyTsuyoshiTokusumiworkinginDr.RobertSchulz’slab
Drosophilaembryo
DAPIwavelength
BloodvesselsseeninasecIon
ofahumanuterus
Drosophilaembryo
FITCwavelength
DiatomstakenwithmonochromaIc
camera
Above:ImagesbyBillArcher
GEHealthcareDeltaVisionDeconvoluIon
Microscope
TheDeltaVisionimagingsystemisaninvertedmicroscopethatallows
theusertocapturewidefieldepi-fluorescenceimages,andwiththe
deconvoluConalgorithmredirectalloutoffocuslighttobecomein
focusforsharpimages.Itcandothiswithfixedcellsand/orCssues,
butitsgreateststrengthisitsabilitytoobtainandanalyzelong-term
Cme-lapsethree-dimensionalimageswithaminimumofphotodamageonlivecellsorsmallorganismswiththeaidofits
environmentalchamber.Theimagescanbecapturedwitha
CoolSnapHQ2cameraforhighresoluConimagesoranEMCCD
CascadeII/512high-sensiCvitycameraforsampleswithlowlight.
20
Chromosomalarchitecture–offluorescently
markedchromosomesfromthegenomeof
Malpighiantubulecellsofthemosquito
Culex.ThisworkwasdoneinDr.FrankCollins
Laboratory
ImagingofSubcellularStructures–Theimagesbelowshowcellsduringthe
metaphaseperiodofcelldivisiontoexaminethestructure,assembly,andbehaviorof
kinetochoresinliveculturedCOS-7cells,researchbeingdonethelaboratoryofDr.
KevinVaughan.
Andor/NikonSpinningDiskConfocalMicroscope
AndorTechnologyandNikonInstrumentsworkedto
producethisSpinningDisk,livecellconfocal
microscope.Thissystemisdesignedtoimagethin
sampleswithlowemissionfluorescenceaswellas
capturecellulareventswithminimalphoto-damage
tothecells.Thissystemisalsocapableofpreforming
themodaliCesofFRET,FRAP,andTIRF.
Forareadablesummaryofspinningdiskmicroscopy,see:
hJp://cshprotocols.cshlp.org/cgi/content/full/2010/11/pdb.top88
21
Recordingcytoskeletalbehavior–Dr.HollyGoodsonobtainsimagesofliveCOS-7
cellsoverCme.UsingFluorescenceRecoveryArerPhoto-bleaching(FRAP)(red
circle)modalityshestudiestherealCmebuildingofthefluorescentlylabeled
microtubulecytoskeletonaswellasthemovementofintracellularorganelles
associatedwiththecytoskeleton(greenarrowhead).
ObservaConofmembraneandnear
membranephenomenawithTIRFmodality
(CourtesyofD.ToomteoftheYaleUniversity
ofMedicineanAndorUSA)
NikonC-2LaserScanningConfocalMicroscope
The Nikon C-2 confocal microscope is a simple
operaConal confocal setup on a Nikon Ni-E upright
research microscope and can be considered the liJle
sister of the Nikon A1-R/MP confocal system. This
allowsresearcherstoimageandanalyzetheirfixedcells
or Cssues from above, when an inverted scope is not
pracCcalfortheirsamples.
22
MolecularProbeslideofBPAEpreparedcellslabelledwithMito-TrackerRed,Alexa
Fluor488phalloidinandDAPItakenbyBillArcher.Aboveler20xmag,aboveright
40xmag.
A3-DmodelrenderedfromaCmelapse,
z-seriesacquisiConlookingforcellmovement
betweenlayersofalivezebrafishreCnaby
Dr.ManuelaLahneworkinginDr.David
Hyde’sLaboratory.
NikonA1-R/MulI-PhotonLaserScanningConfocalMicroscope
TheNikonA1-RConfocalisapowerful,fully-automatedconfocal
imagingsystemallowinguserstocapturehigh-qualityconfocal
imagesofcellsaswellasmoleculareventsathighspeedswithits
ResonantScannerorwithenhancedsensiCvityusingitsSpectral
DetecCon.Withtheuseofanenvironmentalchamberyoucan
imageinrealCmelivecellsandCssuesthus,makingitidealfora
broadrangeofuses.Finally,withthebenefitsthatcomewithtwo
photonimaginginMulC-PhotonmodeonecanworkwithliveCssues
fordeepimaging,lowphoto-damagingandhighspeedacquisiCons
notpossibleinnormalconfocalimaging.
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ConfocalImagingofRhodopsin:Workdoneby
studentsdoinganUndergraduateResearch
coursetaughtbyDr.MichelleWhaley.
TopRightImage:Studentswereimaging
rhodopsinproteinintheeyesofmosquitos.
SlideswerepreparedwithcareintheHistology
Facility.
BoJomRightImage:AlexMetoxen,
undergraduateresearcherinDr.J.O’Tousa’slab,
wasexaminingrhodopsin(green)migraConto
lightsensiCvephotoreceptormembranes(red)
intheeyeofthefruitfly.
DeepimagingofliveIssue:
Theinfraredlaseremployedin
themulC-photonexcitaConmodalityof
theNikonA1-R/MPisbestsuitedfor
long-termlivescanningdeepintothe
Cssue.
Atthelerareimagesofa3Dcultured
tumortakenbyAmyLeliaertfromthe
labofDr.ZachSchafer.Thetwo-photon
capabilityallowsimagingwithmulCple
filtersetsthroughtheenCrecyst,to
generatethreedimensionaldatasetsof
theseclustersoftumorcells.
24
Whichdevicewillbestservetheneedsofyourexperiment?
Device
Live-cell
environmental
chamber?
Approximate
sample
penetraIon
depth
(micrometers
)
Blur
minimizaIon
mechanism
Unique
modaliIes
NikonAZ100Macroscope
no
1,000+
none
8xzoom
NikonEclipse90i
no
1,000+
none
-
GEHealthcareDeltaVsion
YES
400
deconvoluIon
image
deconvoluIon
Andor/NikonSpinningDisk
YES
200
spinning-disk
confocality
FRAPPA,TIRF,
FRET
NikonC-2
no
200–300
laser-scanning
confocality
-
NikonA1-R
YES
400–600
laser-scanning
confocality
FRET
NikonA1R-MP
YES
600-800
mulIphoton
excitaIon
infrared-laserinduced
mulIphoton
excitaIon
InaddiIon:
Off-lineImageProcessingStaIonwith
AutoQuantDeconvoluIonSoqware
(Bright-fieldandConfocal)
…becauseyoushoulddeconvolveeverything!
25
AboutUs
HISTOLOGYCORE
TheNotreDameHistologyCore(NDHC)
providesameanstoexaminebiological
processesinmice,rats,frogs,zebra
fish,fruitflies,sheepbone,andeven
humanCssuebyuseof
immunohistochemicaltechniquesand
pathology.
SarahChapman
BiologicalImagingAssistant
Director
[email protected]
Equipment
ThefacilityislocatedinFreimannLifeSciencesanditisequippedwithaShandonCitadel
TissueProcessor,Leicamicrotome,Tissue-TekIIIembeddingstaCon,andanewLeica
CryostatwithaspecialTungstenbladeaJachmentcapableofslicingthroughbone.
Services
INFLAMMATION
HematoxylinandEosininLiver
ANGIOGENESIS
VEGFinKidney
APOPTOSIS
Caspase-3inOvary
ServicesincludeCssuefixaCon,processing,embedding,
secConing,andstainingofparaffinorfrozenCssue
secCons.
StainingofCssuesecConsrangesfromrouCneH&E’s
(Hematoxylin-Eosin)tospecialstainsdemonstraCng
specificCssuestructures.
TheCoreoffersimmunohistochemicalstainingwith
anCbodiessuppliedbyinvesCgators.
PROLIFERATINGCELLS
PCNAinBone
26
FABRICATIONLABORATORY
Nowopenforbusinessacrosscampus!
AboutUs:
Thisfacilitymaintains3DprinCng
andlasercuungequipmentforrapid
prototypingofpartsforresearch,
entrepreneurship,orother
applicaCons.Availablefor
producConanddesignconsulCng.
Locatedin010GalvinHall.
TonyVanAvermaete
FabLabCoreManager
[email protected]
Equipment:
Objet30Prime
Usefulwithhard(Vero)andsor
(Tango)seriesliquidresinsfrom
Stratasys
TazFDM3DPrinter
Filamentbasedprinter
LowerresoluCon,lowercost
UniversalVLS6.60
LaserCu-er/Engraver
65WaJlaserwith18”x32”
workingarea
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