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Preparation of biological specimensf o r E l e c t r o n M i c r o scopy TEM
for
Serving Advanced Technology
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2CONTENTS
Introduction 3
I Procedure of sample preparation of a biological specimen
I 1 Preparing a biological sample for ultramicrotomy 4
I 2 Fixation of biological tissue 5 2 1Extractionoftissue
2 2Fixation
2 3Dehydration
2 4Substitution
2 5Embedding
2 6Polymerization
Appendix:Preparingsolutionsusedinmicrotomy
I 3 Steps in sectioning with an ultramicrotome 8 3 1Trimming
3 2Sectioning
Settingaspecimen
Makingofultrathinsections
Thicknessofultrathinsection
Pickingupsections
Gridtypes
fHowtomakeglassknifes
gDiamondknife
I 4 Conventional staining of ultra-thin sections 12 4 1Variousstainingsolutions uranylacetate,leadcitrate 4 2Procedureofstaining
II Observation of single particles
and viruses by negative staining
II 1 Contrast in negatively staining 13
II 2 Staining procedures 13
III Preparation of supporting film III 1 Preparation of collodion supporting film (wet method) 14
III 2 Preparation of formvar supporting film (dry method) 14
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3Introduction
Thereareseveralmethods topreparebiologicalspecimensdependingon thenatureof the
specimenandthepurposeofthestudy.
Thispresentationintroducessomecommonlyusedmethods.
Please,becarefulinhandlingchemicalsusedinthesemethodsassomearetoxic.
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Fixation
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Embeddingcapsule
Embeddingplate
Diamondknife
Ultramicrotome
Ovenforpolymerization Knifemaker
I Procedure of sample preparation of a biological specimen
Toolsandchemicalslistedherearenecessaryforpreparation.
(Procedure) (Instruments/Chemicals)
Tweezers
Glassbottles
Razor
Glutaraldehyde
Osmiumtetroxide
Phosphatebuffer
Ethanol
Propyleneoxide
(Disposablesyringes)
(Disposablebeakers)
Epoxyresin
Embeddingplate(Topphoto), Embeddingcapsule(Middlephoto)
Ovenforpolymerization(Bottomphoto)
Ultramicrotome(Topphoto)
Grid
Diamondknife(Middlephoto)
(Glassknife)
(Knifemaker)(Bottomphoto)
Uranylacetate
Leadcitrate
Extractionoftissue1. Sectioning6.
Fixation2. Staining7.
Dehydration3. Observation8.
Embedding4.
Polymerization5.
I 1 Preparing a biological sample for ultramicrotomy
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I Procedureofsamplepreparationofabiologicalspecimen
5
A)Glutaraldehydesolution
Specimen
B) Razor
C)
Tweezers
Glutaraldehydesolution
Glassbottle
*Waterisremovedthroughamolecularsieve
I 2 Fixation of biological tissue
I -2-1 Extractionoftissue
I -2-2 FixationPre-fixation(fixesproteins)1~2hr.
2.5%glutaraldehydeinPhosphatebuffer
Post-fixation(Fixeslipids)1~2hr.(4C)1~2%OsO4inPhosphatebuffer
Washinginphosphatebuffer.10min
Washinginphosphatebuffer.10min2-3times(Liquidisexchanged2or3timesevery10minutes.)
A)Theextractedtissueisdippedinthechemicalfixationsolution.
B) Cuttingthetissuewithtworazorbladesasindicatedwillpreventunnecessarydeformationofthetissue.
C)Thesampleistransferredtoasecondglutaraldehydesolutionbykeepingthedropletwithtissueheldbetweenthetworazorsbladesbythebufferssurfacetension.
I -2- 3 Dehydration50%ethanol 10min.70%ethanol 10min.80%ethanol 10min.90%ethanol 10min.95%ethanol 10min.f100%ethanol* 20min.g100%ethanol* 20min.
Liquidisquicklyreplacedbeforethesamplecandry.
aspiration
specimen
Nextsolution
Methods of washing & dehydration
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6Siliconeembeddingplate
Gelatinouscapsuleembeddingcapsule Beamcapsule
I -2- 4 Substitution
Mixed-solutionofPOandresinPO:Epoxyresin=2:1 30min.
PO:Epoxyresin=1:1 1hr.
PO:Epoxyresin=1:2 1hr.
OnlyEpoxyresin2hr.orovernight
Onlypropyleneoxide(PO)10min2-3times(ChangeLiquid2or3timesevery20minutes.)
I -2- 5 Embedding(Thefreshlymixedresinshouldbeused.)
Epoxyresin
[InthecaseofTAABEPON812resin]
EPON812 48gDDSA 19gMNA 33gDMP-30 2g
Thespecimenisplacedinawellofasiliconeembeddingplate.
Resinispouredoverthetissue.Ensurenoairbubblesare
trappedwithintheresin.*For a specimen with various orientations, use a silicone embedding plate.
[InthecaseofTAABEPON812resin]PutEPON812 DDSA MNAinbeaker.*Theuseofdisposablesyringesandbeakersfacilitatesthepostwashing.
Mixitwell.
AddDMP-30andstirwell.*Sinceanaddedvolumeissmall,theuseofsyringesfortuberculinisrecommended.
Specimen
Resin
SpecimenResin
Preparation of Epoxy resin
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I Procedureofsamplepreparationofabiologicalspecimen
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Ovenforpolymerization(3stagetype)Ovenforpolymerization(1 stagetype)
I -2- 6 Polymerization(35C 1day)45C 1day
60C 1day
Appendix Preparing solutions used in microtomy
1.Add40mLofdistilledwaterto10mLof25%glutaraldehyde
solution(commerciallyavailable:GA).(=>5%GAsolutionis
prepared.)
2.Add50mLof0.2Mphosphatebuffer.
=>100mLof2.5%GA-0.1Mphosphatebuffer(pH7.4)isprepared.
How to prepare 2.5% glutaraldehyde solution
SolutionA:Phosphatesodium(NaH2PO4)27.6g/L
SolutionB:Phosphatesodium(Na2HPO4)53.6g/L
A:B=28mL:72mL=>pH7.2
A:B=19mL:81mL=>pH7.4
How to prepare 0.2M phosphate buffer (Sorensens phosphate buffer)
1.Dissolve1gofosmiumcrystalin25mLofdistilledwater.
=>4%osmiumsolution(keptinadarkplaceandinarefrigerator)
2.Add5mLofdistilledwaterand10mLof0.2Mphosphatebuffer
to4%osmiumsolution(keptinadarkplaceandinarefrigerator)
preparedinStep1.
=>20mLof1%osmium-0.1Mphosphatebuffer(pH7.4)isprepared.*Preparationmustbeperformedinafumehood.
How to prepare 1% osmium solution
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8I 3 Steps in sectioning with an ultramicrotome
I - 3 -1 TrimmingExposethetissue.Trimthetipusingarazortoformapyramidalshape.
I 3 2 SectioningSettingaspecimen
Asampleblockandaknifearesetonamicrotome.
Ultramicrotome(LeicaEMUC6)
Examples of trimming
0.5mm0.5mm1mm
1mm
TrapezoidRectangleSquare
Acuttingsurfaceismadeintoasquare,rectangleortrapezoid.
Square :Idealforwhenultra-lowmagnificationimagesare
needed.Rectangle:Suitablewhensequentialsectionsarecutandwhenthe
blockcontainsbothhardandsofttissue.Trapezoid :Idealforkeepingtrackoftheorderofsectionsinaribbon.
Sampleblock
Diamondknife
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I Procedureofsamplepreparationofabiologicalspecimen
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A) The sections floated in the surface.
SectionsB) A actual section
Sections
Knifeboat(withdistilledwater)
Viewedfromtop
Daimondknife
MakingofultrathinsectionsSectionedthinspecimensfloatandareflattenedonwaterintheknifeboat.
PickingupsectionsPickupthinspecimensonwaterusingaTEMgrid.
A) Press MethodPlaceagridona thinspecimenwithasupport filmsidedown.
B) Pull up Method PlaceaTEMgridunderthinspecimenswithasupportfilmonthetop.Bringthinspecimensonthegridbyusinganeyelashprobe.
Advantage :SimpleandeasyDisadvantage:Thinspecimenmaybewrinkled Thinspecimensmaybeoverlaid
Advantage :Nowrinklesonspecimens Youcanplacespecimens asyoudesireDisadvantage:Skillneeded
Placeagridonathinspecimen
Immerseagridinwater.
Eyelashprobe(eyelashattachedtoprobehead)
The interference color and thickness of an ultrathin section
ThicknessofultrathinsectionThethicknessof thinspecimensmayvarydependingonthehardnessof
resinandroomtemperature.
Itisnecessarytojudgethethicknessbycolorofinterference.
Theoptimalthickness Thickness
Gray