ANATOMY12

HISTOLOGY1:THECELL
INTRODUCTION
I.ORGANIZATIONOFTHEHUMANBODY
II.THECELLCOMPOSITION
THECELLMEMBRANE
I.FUNCTIONSOFTHECELLMEMBRANE
II.ELECTRONMICROSCOPYOFTHECELLMEMBRANE
III.BASICSTRUCTURESOFTHECELLMEMBRANE
THECYTOPLASM
I.COMPOSITIONOFTHECYTOPLASM
II.CYTOPLASMICORGANELLES
III.FIBRILLARELEMENTS
IV.INCLUSIONS
NUCLEUS
I.FUNCTIONSOFTHENUCLEUS
II.COMPONENTSOFTHENUCLEUS
III.NUCLEARENVELOPEOFTHENUCLEUS
IV.THECHROMOSOMES
V.PROTEINS
MOVEMENTOFSUBSTANCESACROSSMEMBRANE
I.ENDOCYTOSIS
II.EXOCYTOSIS
HISTOLOGY2:LABORATORY
INTRODUCTIONTOHISTOLOGY
I.LIGHTMICROSCOPE
II.ELECTRONMISCROSCOPE
III.COMPARISIONBETWEENLIGHTANDELECTRONMICROSCOPE
HISTOTECHNIQUES
I.CONCEPTSINHISTOLOGY
II.IMPORTANTTERMINOLOGIES
III.STEPSINMICROSCOPY(PREPARINGSLIDES)
V.COMPARISONBETWEENLIGHTANDELECTRONMICROSCOPY
VI.DESCRIPTIONOFSTEPS
VII.STAININGSUSED
HISTOCHEMISTRY

INTRODUCTION
I.ORGANIZATIONOFTHEHUMANBODY
Cell
basicmorphologicalandfunctionalunitofalllivingthings
Tissue
combinationofcellswiththesamegeneralfunction
Organs
combinationoftissuesthatformamorecomplexfunctionalunit
Systems
organsthathaveinterrelatedfunctions
II.THECELLCOMPOSITION
CellMembrane
delimitsthecellfromitssurroundings
Cytoplasm
enclosedinthecellmembrane
Nucleus
separatedfromthecytoplasmbyanuclearenvelope

THECELLMEMBRANE
I.FUNCTIONSOFTHECELLMEMBRANE
Delimitscellfromsurroundings
Protectivecover
Determinessubstancesthatmoveinandoutofthecell
Attachmentforcytoskeleton
Formsspecializedjunctionswithmembraneofothercells
Receivesandgivesoutstimuli
Providesbindingsitesandreceptorsforenzymesandothersubstances
Allowsforcellrecognition
II.ELECTRONMICROSCOPYOFTHECELLMEMBRANE
810nmthick(notseeninLightMicroscope)
ElectronLucentLine(formedbytails)between2ElectronDenseLines(formedbyheads)

III.BASICSTRUCTURESOFTHECELLMEMBRANE
A.PhospholipidBilayer
containsaheadandtwotailswhichformsabilayer
headisthepolarandtailisnonpolar
1.Head

GlobularPolarandHydrophilic
GlycerolconjugatedtoanitrogenouscompoundbyaPhosphateBridge
Occupiestheoutersurfaceofbilayer
formsthe2electrondenselines

2.Tails

NonPolar/Hydrophobic
Straight,SaturatedFattyAcids
Maycontainunsaturatedfattyacids(withslightKinks)
occupiestheinnersurface
formstheelectronlucenttails

B.Proteins
halfofthemassofcellmembranes
hastwotypes:IntegralProteins+PeripheralProteins
1.IntegralProteins

formspartofthemembrane
alsocalledTransmembraneproteins
spanthewholethicknessofthemembrane
hydrophobicbecauseitinteractswithtails

2.PeripheralProteins

insertedon/looselyboundtoouterorinnersurfaces
hydrophilic/polar

C.Cholesterol
formspartofallcellmembranes
functionsforrigidity
canbesynthesizedbybodyfromothersubstances
D.Glycocalyx
presentinsomecells
thinlayerofamorphouselectrondensematerialonsurfaceofcell(outer)
functionsforCellRecognition,CelltoCellAdhesion,ImmunologicalResponse

consistsofGlycoproteinsandGlycolipids
Glycolipids=carbohydrates+lipids
Glycoprotein=carbohydrates+proteins

THECYTOPLASM
I.COMPOSITIONOFTHECYTOPLASM(MATRIX+FORMEDELEMENTS)
A.MatrixoftheCytoplasm
Viscid,translucent,homogenous,colloidalsubstance
amorphousbutimportantcomplexdynamicpartofthecell
sitesofmanyimportantbiochemicalprocesses
providesmilieufortheorganellestoperformtheirfunctions
**CompositionoftheCytoplasmicMatrix
Water(70%ormorebyvolume)
InorganicIons
OrganicMolecules:Proteins,Lipids,Carbo,NucleicAcids,Enzymes,ProductofEnzymaticActivity
B.FormedElements
includesOrganelles+FibrillarStructures+Inclusions(discussedindetail)
CytoplasmicOrganelles:Mitochondria+Ribosomes+EndoplasmicReticulum+GolgiComplex+

Lysosomes+Peroxisomes(Microbodies)+CentrosomesandCentrioles
FibrillarStructures:Microfilaments+IntermediateFilaments+Microtubules
Inclusions:EndogenousInclusions+ExogenousInclusions
II.CYTOPLASMICORGANELLES
consistsofMitochondria,Ribosomes,ER,GC,Lysosomes,Peroxisomes,Centrosome
allorganelleshaveDeliminatingMembraneRibosomesandCentrosomes
organellesaremoreorlesspermanentstructureswhichperformfunctionsinthecell
A.TheMitochondria
**SizeandShape
0.51.0micradiameter;10micralong
hotdogshaped
**PropertiesofMitochondria
presentinallcellsexceptRBC
notvisibleinH&E;visibleinEM,specialstaining,PhaseContrastMicroscopy
motileandcanchangeshape
synthesizesallitsenzymesandcanselfreplicate
**StructureoftheMitochondria
STRUCTURE
WallofMitochondria

DESCRIPTION
*Portionsinvaginateintointercristalspace&formcisternae
*DoubleWalled:OuterMembrane+InnerMembrane

IntercristalSpace
IntermembranousSpace
IntracristalSpace

*Cavityenclosedbywallfilledwithmatrix
*Spacebetweenthetwowalls
*ProjectionsintoCristaeMitochondriales

**ContentoftheMitochondrialMatrix
granulesrichinMagnesiumandCalcium
enzymesoftheKrebsTricarboxylicAcidCycle
strandofDNA
ribosomes(RNAcontaininggranules)
messengerandtransferRNAs
**FunctionsoftheMitochondria
Powerhouseofthecell(sourceofenergy)
GenerateATP(principalsourceofenergyofvariousmetabolicprocesses)

B.TheRibosomes
**Size/Shape/Properties
smallgranules:1530nminsizeonlyseeninEM

Basophilic;numerousphosphategroupsintheirRNA
previouslyknownasErgastoplasmorBasophilicBodies
occursinglyorinclusters(PolysomesorPolyribosomes)
PolyribosomesareclustersofribosomesconnectedtoeachotherbymRNA
ribosomesandpolyribosomesoccerfreeincytoplasmorattachedtoRER
**FunctionsofRibosomes:
1.FreeRibosomes

synthesisofproteinsforcytoplasmonly(internal)
synthesisofproteins(assemblyofaminoacids)
synthesisofproteinsofstructuresthatarerenewed

2.AttachedRibosomes synthesisofproteinsthatareforexportbycell
alsosynthesizeproteinsforinternaluse
**CompositionofRibosomes
Ribosomes=LargeSubunit+SmallSubunit
bothsubunitsaredenseglobularstructuresw/rRNA&associatedproteins
largeandsmallunits(nottogetheryet)aretransportedtocytoplasmvianuclearpores;inthe
cytoplasm,UnionofLargeandSmallSubunittoformRibosomes
**RibosomesConsistof:
rRNAMolecules
ProteinsthatarelinkedtorRNA
C.EndoplasmicReticulum(ER)
ComposedofInterconnecting:Tubules,Vesicles,Cisternae(flattenedsacs)
theyaresupportingstructureforthecytoplasmandinvolvedintheproductionofcertainsubstances
**PropertiesoftheER
presentinallcellsbutseenonlyinEMandspecialpreparations
mostextensivemembranousstructureincytoplasm

dynamicorganellecapableofremodeling,disassembly/assembly&interactsw/otherorganelles

**MembranesoftheER
thinnerthantheplasmelemma(cellmembrane)
continuousw/nuclearmembrane;cavitycontinuousw/spacebetouter&innerlayersofnuclearmembrane
**RegionsoftheER(SmoothandRough)
1.RoughEndoplasmicReticulum
withribosomes(andpolyribosomes)
involvedinsynthesisandtransportofproteins
2.SmoothEndoplasmicReticulum(noribosomes)
siteforsynthesisofcholesterolandphospholipids
involvedintransportoffattyacidsandotherlipids
inmostcells,itslessdevelopedthanrERbutwelldevelopedinlivercells
instriatedmuscles,itismodifiedintoSarcoplasmicReticulum
D.GolgiComplex
alsocalledtheGolgiApparatusorGolgiBody
Function:involvedinproteinsynthesis(seebelow)
**PropertiesofGolgiApparatus
allcellshaveone;somemore
notseeninH&E,butlocationismarkedbyapaleregion(negativeGolgiImage)
seeninEMandcellsimpregnatedwithSilversaltsorOsmium
**GolgiComplexisaDynamicOrgan:
atFormingSurface,membraneisadded(transfervesicle)
atMaturingFace,membraneremoved(secretoryvesicle)
**FacesofGolgiComplex:
1.Trans/MaturingFace
2.Cis/FormingFace

related(faces)tothenucleus
concaveside
convexside

**ProteinSynthesis
Lysosomes
RibosomesrERGolgiComplexSecretoryVesicles

IntegralProteins
ExportoutsideCell

Proteinsaresynthesizedinattachedribosomes
FullyformedpolypeptidechainstraverserERmembraneandenterthelumen
WhileintransitinrER,proteinsareprocessed(newchemicalsareadded)

PolypeptidechainsarebroughttotheGolgiComplexinformofTransferVesicles
(BecausethereisnocontinuitybetweenERandGolgi)
AtCisFace(Formingface/Convex),transfervesiclescoalescew/membraneofGolgi
Complex
WhileintransitinGolgiComplex,proteinsareprocessed,concentrated,sorted,packaged
andlabeled
AtTransFace(Maturing/Concave)ofGolgiComplex,SecretoryVesicles(Condensing
VacuoulesBudoff

**SecretoryVesicles

someareincorporatedintodevelopinglysosomes
someincorporatedintointegralproteins
someconcentradedfurtherandbecomesecretorygranules,
laterreleasedatapicalregionofcellbyExocytosis

E.TheLysosomes
**PropertiesofLysosomes
heterogenousgroupofstructures
varyinshapeandsize(usuallyovoid:0.050.8micra)
notseeninH&E
seenbyhistochemicalmethodsthatidentifyenzymes
constituteanIntracellularDigestiveSystem:candigestanddegradenearlyallorganicsubstances
foundinthecells
**SimilaritiesofLysosomes
membranebound
containhydrolyticenzymes(hydrolases):morethan40identifiedtoday
**LysosomalEnzymes
synthesizedinrER
modifiedandpackagedinGolgiComplex
secretoryvesiclespinchofandfusewithdevelopinglysosomes
**FunctionsofLysosomes
Heterophagy digestionofsubstancesforeigntothecell(ex.Bacteria)
Autophagy
digestionofunneededcellorganelles
**LysosomesandPhagocytosis
lysosomesarenumerousinphagocytes(suchasNeutrophils)
1.Phagocytosis

bacteriaengulfed&broughttocytoplasminmembraneboundstructures
(phagosomesorphagocyticvacuole)

2.Phagosomes
**FateofEngulfedMaterial
1.DigestedNutrients

attackedbyprimarylysosomes
diffusefromlysosomeandrecycled

2.UndigestedMaterials alsocalledResidualBodies
keptwithinlysosome
coalescetoformLipochrome/Lipofuschinpigments(ex.Macrophages)
**TypesofLysosomes:
1.PrimaryLysosomes lysosomeswhichhavenotdigestedanythingyet
surroundsphagosome,fusemembranewithphagosome,andrelease
hydrolyticenzymes
2.SecondaryLysosomesreferstoprimarylysosomeanddigestedmaterial
alsocalledasPhagolysosome
**LysosomesinBoneResorption
Osteoclastsreleasehydrolyticenzymesextracellularly
digestbonematrix
F.Peroxisomes(Microbodies)
foundinallcells
simillartolysosomesmorphologically
**PropertiesofPeroxisomes
cantbedistinguishedinLMandEM
0.51.2Micra
distinguishedbyusingHistochemicalTechniques
containoxidasesandcatalasesinsteadofhydrolyticenzymes
**FunctionsofPeroxisomes
Enzymes(morethan40identified)catalyzemanymetabolicreactions
G.CentrosomesandCentrioles
1.Centrosome densesphericalareausuallynearnucleus
containsthecentrioles
2.Centrioles

pairoftubularorganelles,collectivelyreferredasDiplosome
lieperpendiculartoeachother
composedofelectrondensewallthatsurroundsanelectronluscent(hollow)spaceunderEM
**Wall Formedby9groupsofMicrotubules
eachgroup(Triplet)consistsofthreeMicrotubules

TripletObliquelyset
innermostmicrotubuleoftripletisconnectedtotheoutermostmicrotubuleof
adjacenttripletbyfinefilament
**FunctionsofCentrioles
o sourcesofmitoticspindlesthatappearduringmitosis
o produceciliaofciliatedcellsandflagellum(spermcells)
**ReplicationofCentrioles
o priortomitosis,bud(Procentriole)growsoutoflateralsurface
o budelongatesperpendiculartomothercentriole
o daughtercentrioleseparatesfrommothercentriole
o daughterandmothercentriolesmovetoonepoleofcell
o otherdaughter&mothercentriolesmovetooppositepoles

III.FIBRILLARELEMENTS
formsthestructuralframeworkorSkeletonoftheCell(Cytoskeleton)
seenonlyinEMorusingspecialhistologicaltechniques
Types:Microfilaments,IntermediateFilaments,Microtubules
A.Microfilaments(Thinnest)
madeupofActinSubunits(FActinandGActin)
abundantinperipheralareasofthecell
presentinallcells(exceptRBC)
undergofrequentassemblyanddisassemblytoaccommodatechangesincellshape&movementofcell
**Actin(Monomer)
basicMonomerofMicrofilaments
comprises1015%oftotalproteinsinthecell
canbindwithATPandotherproteins
1.FActin

Filamentousform(50%ofactin)
formedasneeded
formedbytwostrandsofglobularGActin

2.GActin

Solubleform

**FunctionsofMicrofilaments:
provideinternalsupport
playsaroleinshapechangesandlocomotionofthecell
involvedinmovementoforganelles

B.IntermediateFilaments
sizebetweenmicrofilamentsandmicrotubuleinsize(1012nm)
unlikeActinfilaments,assembleanddisassemblyisnotfrequent
hasfivemajortypes:Keratin,Desmin,Vimentin,Neurofilament,GlialFilament
TYPE
Keratin

DESCRIPTION
*onlyinepithelialcells,numerousinkeratinocytes

Desmin(Skeletin)

*onlyinmusclecells(moreinsmooththanstriated)

Vimentin

*presentinallcellsfromMesenchyme(ex.Fibroblasts)
*scatteredallovercytoplasm

Neurofilament

*foundinNeuronsforneuralandinternalsupport
*incellbodyandprocesses

GlialFilament

*GlialFibrillaryAcidicProtein(GFA)
*foundinNeuroglialCellsforinternalsupport

C.Microtubules(Thickest:25nmindiameter)
PolymersofTubulin
hollowtubules
assembledanddisassembledasneeded(likemicrofilaments)
**Tubulin(Monomer)
monomerofMicotubules
formedbyPolymerizationof2subunits:AlphaTubulin&BetaTubulin
**StructureofMicrotubules
formedby13TubulinMoleculesarrangedaroundaLumen
stabilizedbyMicrotubuleAssociatedProteins(MAPs)
1Centriole=9Microtubules
1Microtubule=13TubulinMolecules
**FunctionsofMicrotubules
comprisethemitoticspindlesandciliaofciliatedcells
playaroleinmovementoforganelles
lendinternalsupportforthecell

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IV.INCLUSIONS
generallytemporaryandinertstructures
mostnotenclosedbymembranes
varyinsize,shapeandcontent
someareuseful,someharmfultocell
twoclassifications:EndogenousandExogenous
A.EndogenousInclusions
arisefromwithincell
ex)LipidDroplets,Glycogen,ZymogenGranules,PigmentGranules,andCrystals
1.LipidDroplets

inroutinepreparations,lipidextractedbyreagents;clearareasappear
intissuefixedwithGluteraldehydeandOsmicAcidgrayorblackglobules
inadiposecellshugeblob

2.Glycogen

storageformofcarbohydrates
seenonlyinspecialpreparationslikePASmethod

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sizes: LargeAlphaParticles(90nmdiameter)
SmallBetaParticles(2030nmdiameter)
EM: ElectronDense;notenclosedinmembrane
3.ZymogenGranules

insecretoryepithelialcells,ofteninapicalportionofcell
membranebound
secretoryproductsofprecursors

4.PigmentGranules

Melanin

inkeratinocytesofskin(synthesizedbymelanocytes)
cellsofsubstantianigrainbrain
cellsinpigmentepitheliumofretina

Hemosiderin brownpigment
seenbyselectivelystainingiron
productoflysosomaldigestionofhemoglobin
5.Crystals

infewcells(interstitialcells;sertolicellsoftestis)
notmembranebound
chemicalcompositionisunknown

B.ExogenousInclusions

originatefromoutsidethecell
ex)Liposome(Lipofuschin)Pigments,DustParticles
1.LipochromePigmentsLipofuschin
mostcommon;membranebound
undigestedremnantsofLysosomaldigestion(ResidualBodies)
2.DustParticles

inMacrophagesorPhagocyticCellsinlungs

NUCLEUS

largeststructureinsidethecell(310micraindiameter)whichoftenroundorspherical(butoccursinothershapes)
presentinallcells,exceptRBC
vitalstructure:removalofnucleusleadstodeathofcell

I.FUNCTIONSOFTHENUCLEUS
DataBankofthecell
GenesinitsChromosomescontaininformationneededforsynthesisofproteinsandnucleicAcids
II.COMPONENTSOFTHENUCLEUS(MATRIX+CHROMATIN+NUCLEOLUS):
A.NuclearMatrix(AmorphousSubstance+NuclearScaffolf)
1.AmorphousSubstance=water,proteins,metabolites,ion
2.NuclearScaffold(NuclearSkeleton;NuclearMatrix)
filamentousproteinnetworkseeninInterphaseNucleus

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anchoredoninnersurfaceofnuclearmembrane
supportingframeworkthatmaintainsoverallsizeandshapeofnucleus
B.Chromatin(ChromatinMaterial;ChromatinThreads)
chromosomesatinterphasewhicharefinethreadsthatareentangledwitheachother
beforecelldivision,theycondensetoformbasophilicrodlikestructures(chromosomes)
**DispersalPatternsofChromatin:
1.Heterochromatin
collectivetermforcondensedareasofchromatinswhichtakeupstains
clumporgranules(ChromatinGranules)
portionsofchromosomesNOTactivelyproducingRNA
2.Euchromatin

extendedareasofchromatinwhichDONTtakeupstains
composedofportionsofchromosomesproducingRNA

C.Nucleoli(oneormore)
spherical,highlybasophilic,usuallyeccentricallylocatedinnucleus
nodeliminatingmembrane
presentonlyinInterphase(disappearduringearlymitosis/reappearduringlatetelophase)
Function:synthesizeRibosomalSubunits
**NumberofNucleoli
usuallyonepernucleusbutmaybemorethan1andlargerincellsthatactivelysecreteprotein
absentincellsthatdontsynthesize(orlittle)proteins(musclecells)

**ThreeRegionsofNucleolus(UnderEM)
1.NuclearOrganizingRegion
severalpernucleolus
circularpaleareasurroundedbyParsFibrosa
areawherechromosomesw/NucleolarOrganizersgettogethertotranscriberRNA
resultingrRNAformParsFibrosa
*NuclearOrganizers

sequenceofbasesthatcodeforrRNA
inhumans,5pairsofchromosomesareknowntohavethese

2.ParsFibrosa
ElectronDenseareasurroundingnucleolarregion
rRNAmoleculesformedinNucleolarOrganizingRegion

3.ParsGranulosa
granularappearance
accumulationofRibonucleoproteins(ribosomalsubunits)

III.NUCLEARENVELOPEOFTHENUCLEUS
twomembranes(outerandinnernuclearmembrane)
bothmembranesaresimilarandthinnerthanCellMembrane
78nmthick(cellmembraneis810nm)
seeninEM,notinLM
canberegardedasaspecializedportionofendoplasmicreticulum
o PerinuclearspaceiscontinuouswithcavityofER
o OuternuclearmembraneiscontinuouswithmembraneofER
o alsohasattachedRibosomes
A.PerinuclearSpace

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alsocalledPerinuclearCisternaorIntermembranousSpace
separatesthetwomembranesandcontinuouswithcavityofER
size:1030nm
B.NuclearPores
perforatestheNuclearEnvelope
circularopenings(7075nmdiameter);hundredstothousands
inpores,innerandoutermembranesarecontinuouswitheachother
channelforexchangeofsubstancesbetweencytoplasm&nucleus
stabilizedbyFibrousLamina
1.FibrousLamina

associatedwithinnernuclearmembrane
stabilizesnuclearpore;fibrilarprotein(30100nmthick)
clumpsofChromatinattached,formingoutlineofn.envelope

2.NuclearPoreComplex electrondensestructuresurroundingthepore
3.PoreDiaphragm

thindiaphragmthatcoverstheporew/electrondensegranuleinthecenter

IV.THECHROMOSOMES
23Pairs(22SomaticChromosomes+1PairofSexChromosome)
seenduringcelldivision
consistofNucleoproteins+DNAMolecules
A.Nucleoproteins
attachedproteins
has2majortypes:Histones+NonHistones

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B.DNAMolecule
twistedladderw/twostrandswrappedaroundeachother
rungsconnectstrandswithHbonds
Strands=SugarandPhosphatemolecules
Rungs=Nitrogencontainingbases(Bases:Adenine,Thymine,Guanine,Cytosine)
**DNABases DNAsequencereferstoorderofbasesalongdoublestrand
thereare3billionnitrogencontainingbasesinchromosomes
itaccountsforuniquenessofeachprotein
**Genes

asegmentofDNAstrandofvariablelength
containsthecodeforproductionofspecificproteins
numerousperchromosomes
humanshavebetween30,00040,000genes
HumanGenomeisthecollectivetermforallDNAinallchromosomes(databasewithall
neededinstructionsforsynthesisofallproteins)
NucleusChromosomesDNAGenes

V.PROTEINS
nucleuscontainsthecodefortheirproduction
synthesisoccursinthecytoplasm
codeforaparticularproteinisfirsttranscribedfromDNAofgenetoanmRNA(carriescodetocytoplasm)
asidefrommRNA,twootherRNAsalsotranscribedinNucleus&broughttothecytoplasm:
transferRNA(tRNA)
ribosomalRNA(rRNA)
messengerRNA(mRNA)
allproteinsinnucleusareimportedfromcytoplasm
allRNAproducedinnucleusdestinedforcytoplasm

MOVEMENTOFSUBSTANCESACROSSMEMBRANE
I.ENDOCYTOSIS
fromextracellularspaceintothecell
phagocytosisandpinocytosis
A.Phagocytosis

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ifsubstanceissolidlikebacteriaordust
usesreceptors
ingestionofphagocytesof:Bacteria,Exogenousparticulatematter
ex)Neutrophils;Macrophages
**StagesofPhagocytosis:
ReceptorsofPhagocytestarttobindparticulatematerial
Pseudopodiastarttoform(cellprocesses)
Pseudopodiaencirclesparticulatematerialandfuse
MembraneboundparticulateisnowaPhagocyticVacuole
PhagocyticVacuoleisattackedbyLysosomes
B.Pinocytosis
ifsubstanceisliquid
commontoallcells
noreceptorsnecessary
cellmembraneinvaginatestoenclosefluid
**PinocyticVesicle

resultingmembraneboundstructurethathasbuddedoff
attackedbylysosomes

**Trancytosis

transportofpinocyticvesicleacrosscell

**FormsofPinocytosis:
a.Macropinocytosis:largeamountsofliquid
b.Micropinocytosis:minuteamountsofliquid
II.EXOCYTOSIS
frominsidethecelltoextracellularspace(mostlysecretoryproducts)
membraneofsecretoryvesiclefuseswithplasmalemma
secretionflowsoutintoextracellulararea
excesscellmembraneisgeneratedbyremovedwhenplasmalemmainvaginatestoformvesicle
thatispinchedoffandbroughttoGolgiComplex
**SecretoryVesicle

secretoryproductanditsmembrane
oftenappearsasagranule(SecretoryGranule)

**ConstitutiveSecretion exocytosiswheresecretoryvesiclesarenotvisibleasgranules(verysmall)

HISTOLOGY2:LABORATORY

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INTRODUCTIONTOHISTOLOGY

studyoftissueswhicharecomposedofcellsthatcarrythesameofsimilarfunction(s)
usuallywiththeaidofmicroscopes
Objective:Toidentifythefunctionofthetissue
**Cytology

studyofcellsandcellularpartsthatmakeupthetissue
Objective:Toidentifythefunctionofthecellthroughthepartspresent
TwoTypesofMicroscope:LightMicroscopeandElectronMicroscope

I.LIGHTMICROSCOPE
A.ComponentsoftheLightMicroscope
LightSource=Bulbordirectsunlight
MicroscopeLenses:
a.CondenserLens
betweenlightsourceandspecimen
collectslightfromsource;projectsitthroughspecimen
b.ObjectiveLens

oneormoreionrotatingturretlocatedbetweenspecimenandocularlens
enlarges&resolvesspecimensimageandprojectsimagetoocularlens

c.OcularLens

furtherenlargesimageandprojectsontotheobserversretina

B.TypesofLightMicroscopes
1.CompoundBrightField

usesaseriesoflens
entirefieldidilluminatedbylightthroughacondenser
specimentranslucentandneedstobestained

2.DarkFieldMicroscope

needsspecialcondenserforcontrast
specimenunstained

3.PhaseContrastMicroscope

withspeciallenssystem
basedonthedifferencesinthelightspeedretardationbydifferentstructures
inspecimengivingdiff.lightintensity
usedforlivespecimens

4.PolarizingMicroscope

allowsforvisualizationofrepetitive/crystallinestructures(birefringent)
suchascollagenfibersormyofibrils
stainnotnecessary

5.FluorescenceMicroscopes

allowedlocalizationofsubstanceslabeledwithfluorescingcompounds
suchasFluoresceinorRhodamine

6.InterferenceMicroscopes

combinedfeaturesofPhaseContrast+Polarizing
providescontrastonunstainedmaterial
canbeusedtocalculatemassofcellularcomponents

7.ConfocalScanningMicroscope allowsvisualizationof3Dstructuresw/ocuttingsections
useslaseropticsandcomputerizedimaginglightisfocusedat
specificdepthandspecimenisscannerpointbypoint
II.ELECTRONMISCROSCOPE
electronsdeflectedorabsorbeddonotreachthescreen

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electronspassthroughthespecimenreachthescreen
electronstravelthroughvacuum
A.ComponentsofElectronMicroscopes
1.Cathode(Negative)
metallicfilm
emitselectronswhenintenselyheatedinavacuumwithelectriccurrent
2.Anode(Positive)
positivelychargedmetalplatewithsmallholeinthecenter
voltagedifferencebetweenCathode&Anode(60100kV)acceleratesthepassageofelectrons
towardstheanode
Electronspassthroughtheholetoformtheelectronbeam
3.CondenserElectromagnet
deflectsandfocusestheconeofbeantowardsthespecimen
4.ObjectiveElectromagnet
deflectstheelectronbeamthatpassedthroughthespecimen
magnifiestheimage
5.ProjectorElectromagnet
furtherenlargesimageandprojectstofluorescentscreenorphotographicemulsion
6.FluorescentScreen
PlatecoatedwithmaterialsthatFluorescenceaselectronsstrikeit
B.TypesofElectronMicroscopes
1.TransmissionElectronMicroscopes(TEM)
allowsvisualizationoftheinternalultrastructureofcellsandtissuesandminutestructuresinside
thecellorintercellularspaces
resolution0.2nm
2.ScanningElectronMicroscopes(SEM)
visualizationoftheSurfaceStructuresonly
allows3Drepresentationofthespecimen
resolution2nm
III.COMPARISIONBETWEENLIGHTANDELECTRONMICROSCOPE
LIGHTMICROSCOPE
ELECTRONMICROSCOPE
UsesLightpickedupbymirror
UsesElectronsemmitedfromametallicfilament(Cathode)
Focusedbycondensertospecimen
Electronspassthroughpositivelychargedmetalplate(Anode)
withsmallholeinthecenter
Condenser:Lens
Condenser:Electromagnet
MediumisAir
WithProjectorElectromagnet
Visualizeddirectlythroughocularlenstoretina Usesfluorescentscreenorphotographicemulsion;Noocularlens
Specimen:38um
Specimen:0.080.1um

18

HISTOTECHNIQUES

involvesaseriesofstepsfortissuepreparationforsubsequentviewingunderthemicroscope
differentfromHistochemistry
Objective:Tovisualizemorphologyoftissuesandcells

I.CONCEPTSINHISTOLOGY
**UnitsofMeasure:
Millimeter mm
Micrometer um
Nanometer nm

10m
10m
10m

**ImportanceofHistotechnique:
Neededinpreparationoftissuesformicroscopicviewing
Tounderstandrationalebehindthestepsinpreparationofslidesandhowtheyaffecttheoutcomeof
tissuesontheslide
II.IMPORTANTTERMINOLOGIES
A.Magnification
increasesapparentsizeofthespecimen(makesimageappearlarger)
functionoftheobjectiveandocularlenses
Magnification=(PowerofObjective)X(PowerofOcularLens)
B.Resolution
measureshowclosedistancebetween2pointsarewherethese2pointsarestillconsideredasseparate
thesmallerthevalue,thegreatertheresolution
independentofmagnification
dependentontheNumericalApertureofObjectiveLensandthewavelengthofillumination
NumericalAperture:widthoflensopeningofobjective

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III.STEPSINMICROSCOPY(PREPARINGSLIDES)
LIGHTMICROPSCOPY
Fixation

Dehydration

Clearing

Infiltration

Embedding

Microscopy
Staining

Rehydration

Sectioning
ParaffinRemoval

Mounting

ELECTRONMICROSCOPY
(NOParaffinRemovalandRehydration)
Fixation

Dehydration

Clearing

Infiltration
Embedding

Microscopy
Staining

Mounting

V.COMPARISONBETWEENLIGHTANDELECTRONMICROSCOPY
STEP
LM
Fixation
YES
Dehydration
YES
Clearing
YES
Infiltration
YES
Embedding
YES
Sectioning
YES
Mounting
YES
RemovalofParaffin
YES
Rehydration
YES
Staining
YES

Sectioning

TEM
YES
YES
YES
YES
YES
YES
YES
XXX
XXX
YES

SEM
YES
YES
XXX
XXX
XXX
XXX
YES
XXX
XXX
Sputter
Coating

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VI.DESCRIPTIONOFSTEPS(INDETAIL)
STEPS
1.Fixation

RATIONALE
*Preservationofstructuralorganization
*PreventsBacterialandEnzmyaticDigestion
*InsolubilizestissuecomponentstopreventDiffusion
*Protectsdamagefromsubsequentstepsintissueprocessing
*Actsasmordant

SUBSTANCEUSED
LM:Formalin
EM:Glutaraldehyde

*Easespenetrationoftissuebyclearingagent
*Preparesfixedtissueforinfiltrationwithembeddingmedium
*Fixedtissueimmersedinalcoholconcentrationtoreplacewater

Ethanol

*Preparestissueforinfiltration
*DehydratingagentreplacedwithClearingagent

LM:Xylene
EM:Propyleneoxide

4.Infiltration

*Preparesclearedtissueforembedding
*Tissueimmersedinaseriesofclearingagentembeddingmedium

LM:Xyleneparaffin
EM:PropyleneOxide/
Plastic

5.Embedding

*Preparestissueforsectioning
*Makestissuefirm(forsectioning)
*Allowsthinsectioning

2.Dehydration
3.Clearing

*ForEM:tissueblockneedstobehardtoallowverythinsections

LM:Paraffin
EM:Plastic

*Thinsectionsallowlightorelectronstopenetratespecimenand
formimage:
LMsize:38um
EMsize:0.080.1um

Equipment:
*RotaryMicrotome
*UltraMicrotome

7.Mounting

*Foreaseofhandlingandtopreventdamage

8.ParaffinRemoval
(LMonly)
9.Rehydration
(LMonly)

*Preparationforstaining
*Paraffinisdissolved
*H&Earewatersoluble

LM:GlassSlide
EM:CopperGrid
WarmWaterBath

10.Staining

*Tissuestructurescannotbedistinguishedw/ostain

6.Sectioning

IncreasinglyDiluteAlcohol
H&E
LeadCitrate

**LimitationsandAssociatedArtifactsintheSteps:
1.Fixation
induceschangeinchemicalcompositionandmayproducestainingartifacts

21

2.Dehydration alcoholmaydenatureproteins
waterlosscausesunevenshrinkageofcomponentswithdiffwatercontent
unnaturalspacesbetweencellsandtissuelayers
3.Clearing
maydenatureproteinsandcauseunevenshrinkageoftissuecomponents
4.Infiltration heatmaydenatureproteinofinterest
bubblesmaybeleftbypoorinfiltration
5.Sectioning dullknifecancrushofpinchtissue
vibrationcanleadtovaryingthicknessintissue
6.Mounting
tissuesectionmaydevelopfolds
7,Staining
multiplestainingmaybeneededtocharacterizestructures
VII.STAININGSUSED
A.StainingforLightMicroscopy
1.Hematoxylin
bluebasicdye:BasophilicStructures(BLUE)
attractsacidiccomponentsofcell(RNA,DNA)whichappearblueontheslide
2.Eosin

acidicreddye:EosinophilicorAcidophilic(RED)
attractsbasiccomponentsofcellsuchasbasiccomponentProteinorZymogen
Granules,Cytoplasm

3.SilverStain

forCollagenIII,ReticularFibers

4.ResorcinFuschin

forElasticFibers

5.PeriodicAcidSchiff forCarbohydrates
**LipidsareusuallydissolvedduringpreparationinvolvingParaffinMethod
TYPES
BasicDyes

STAINS
Hematoxylin
ToluidinBlue
AlcianBlue

AFFINITY
BasophilicTissue

Eosin
OrangG
AcidFuschin

AcidophilicTissue

LipidSolubleDyes

OilRed
SudanBlack

LongchainHydrocarbons(Fats,oils,
waxes)

Multicomponent
HistochemicalReaction

PAS

ComplexCarbohydrates(Glycogen,
Glycosaminoglycans)

FeulgensReaction

NuclearChromatin

AcidicDyes

Ex.DNA,RNA,RibosomesSulfated
Glycosaminoglycans

Ex.BasicProteins

22

B.StainingforElectronMicroscopy(HeavyMetal:ElectronDense)
1.UranylAcetate,LeadCitrate Nonspecific;adsorbtosurfacesandenhancecontrast
2.OsmiumTetroxide

fixativethatbindstophosphategroupsofmembrane
phospholipids,enhancingcontrast

3.RutheniumRed

Polyanions;complexcarbohydrates
ex)OligosaccharidesofGlycocalyxandGAG

HISTOCHEMISTRY

usedfordetectingions,lipids,nucleicacids,proteinsandaminoacids,carbohydrates,
catecholamines,enzymes,antibodies,andantigens
Objective:Torevealthechemicalcompositionoftissuesandcellsbeyondtheacidbasedistribution
shownbystandardstainingmethods
A.Ions

usingchemicalreactionstoidentify
ex.Ironcontainingtissuesisincubatedw/potassiumferrocyanideandHCl

B.Lipids
osmiumtetroxidereactswithlipidtoformaprecipitate
C.NucleicAcid
FeulgenreactionforDNA
AcridineOrangeFluorescence(yellowgreenforDNAandredorangeforRNA)
ToluidineBluestainsforRNAblue
D.Proteins
SakaguchireactionforArginine
MillionreactionforTyrosine
E.Carbohydrate
PeriodicAcidSchiffforpolysaccharideslikeglycogen
AlcianblueforGlycoseaminoglycan
F.Cathecholamines
throughFluorescenceinpresenceofdryformaldehydevapors

23

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