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

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

3Introduction

Thereareseveralmethods topreparebiologicalspecimensdependingon thenatureof the

specimenandthepurposeofthestudy.

Thispresentationintroducessomecommonlyusedmethods.

Please,becarefulinhandlingchemicalsusedinthesemethodsassomearetoxic.

Fixation

4

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

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

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

I Procedureofsamplepreparationofabiologicalspecimen

7

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

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

I Procedureofsamplepreparationofabiologicalspecimen

9

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