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MicroscopyLiving&Fixedcells

Lecture1

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CellBiology

Cytologyinvestigatesthecellultrastructure,

development,specialisation,operation,

communication,

and

regulation

of

cellular

activities

Definecellconcept

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ScaleofLife

nm

(nanometer,

=

10

9

m)

~0.1nm:diameterofahydrogenatom

~10nm:thicknessofcellmembranes

~11nm:Ribosome

~25nm:Microtubule

~50nm:Nuclearpore

~100nm:LargeVirus~150250nm:smallbacteriasuchasMycoplasma

~200nm:Centriole

~200nm:(200~500nm)Lysosomes

~200nm:(200~500nm)Peroxisomes

~800nm:giantvirus(Mimivirus )

m(micrometer,=106m)

~1 10m:thegeneralsizesforProkaryotes

~1m:Diameterofhumannervecellprocess

~9m:Humanredbloodcell

~10 30m:MostEukaryoticanimalcells

~10 100m:MostEukaryoticplantcells~90m:smallAmoeba

~100m:HumanEgg

1mm(1millimeter,=103

m)~ 1mm:Diameterofthesquidgiantnervecell

~120mm:Diameterofanostrichegg

3m:approx.lengthofanervecellofgiraffe'sneck

Incre

asing

s

ize

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Collection&examinationofspecimens

Cellcollection&cultureusefulinstudyingcellcharacteristics:

DNAprofiling Metaboliteprofiling

Cellultrastructure

Cellfunction:cellidentification,reproduction,genetics,transformation,evolutionarystudies

Tissueformationstudies

Cellsinculture(invitro) areausefulmodelforstudyingtheactivityofcellsinthewholeorganism(invivo)

Cellsneedtobecollectedandculturedcarefully

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Collectionofspecimens

A. Microscopicorganisms

Wholeorganisms

1. Scrapings2. Chemicalmeans

3. Filtration/Collectingnets

4. Sonication5. Microscopeslides

6. Soilwatermedium

B. Macroscopicforms:removearepresentative portioninordertodescribe:

1. organproperties

2. describe

different

systems

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Examinationofspecimens

TemporaryMounts

Examiningfreshmaterial

Thesimplestmethodistoplacejustenoughofthe

waterormediumsampleontoamicroscopeslide

Carefullyloweracoverglassontoit

Placetheslideonstageofthebinocularorcompound

lightmicroscope

Startbyobservingthespecimenatlowermagnification

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SamplePreparation

Oneormoreofthefollowingproceduresmay

berequiredtoprepareasample:

Fixation

Permeabilization

Staining

Dehydration

Clearing

Mounting

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Examinationofspecimens

Stains UsesIodine Starchindicator;starchandiodineturnadarkbluecolour

Methylene blue Stain nucleiandmakethemmorevisible

Haematoxylin anuclearstainthat,withamordant,stainsnucleibluevioletorbrown

Eosin acounterstain tohaematoxylin,cytoplasmic material,cell

membranes,andextracellularstructurespinkorred

a

physiological

dye

to

show

water

movement

in

xylem

tissue

Fuschia stainscollagen,smoothmuscle,ormitochondria.

Ethidium bromide stainsDNA

colours unhealthycellsinthefinalstagesofapoptosis,ordeliberate

celldeath

PhloroglucinolHCl stainslignininsclerenchyma ofplantcells

Toluidine blue stainscellulose;greatervisibilityofcellwalls

Crystalviolet stainscellwallspurplewhencombinedwithamordant.Thisstainis

usedinGramstaining

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SamplePreparation:Embeddingtissuesectionsformicroscopicexamination

Apieceoffixedtissueisdehydratedthrough successivealcohol

concentrations purealcohol

xylene

Thespecimenisnextplacedinwarmliquidparaffin,whichisallowedtoharden

Apieceoftheencasedspecimenismanuallysectionedorpreferably

mountedonthearmofamicrotome

Thearmmovesupanddownasametalorglassbladecuts

thespecimenintosectionsafewmthick

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LightMicroscopy

Mostbasicformofmicroscopicexamination Lightiscausedtopassthroughanobjectandisthenfocusedbythe

primaryandsecondarylens.

Thecompoundmicroscopeconsistsofthreelenssystems:o TheCondenserlenssystem

o TheObjectivelenssystem

o TheEyepiece(ocular)lenssystem

o Thesethreelenssystemsarecomposedofglasslenses.

Types:1. Compoundlightmicroscope/brightfield

2. Dissectingmicroscope:

4. Darkfield

5. PhaseContrastmicroscope

5. Fluorescencemicroscope

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Light

Microscope

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LightMicroscopy

Light/Brightfield Darkfield

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Principleofdarkfieldmicroscopy

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PhaseContrastMicroscopy

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Resolutionenhancement

Resolutionmaybeoptimised throughanyorallof

the

following

means:1. Increasingtheangleoflightincidence,byalteringthe

position/designofthecondenserlens.

2. Maximisation oftherefractiveindex. Maybedoneby3. Decreasethewavelengthoflightused

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ElectronMicroscopy Lightisreplacedbyabeamofelectrons Specimensforelectronmicroscopygenerallymustbefixed,sectioned,and

dehydrated,andthenstainedwithelectrondenseheavymetals

Vastlyimprovedresolvingpower

electronbeamshaveveryshortwavelengths. electronmicroscopesalsouseelectromagneticlenses.

Electronmicroscopesarealsousedmainlyforresearchpurposes.

1. TransmissionElectronMicroscope(TEM):electronspassthroughthe

object(cf. compoundlightmicroscope)2. ScanningElectronMicroscope(SEM): electronsarebouncedoffof

thesurfaceofthe specimen(cf. binocularlightmicroscope)

3. Cryoelectron microscopy: unfixed,unstainedfrozenspecimens

4. Scanning

Tunneling

Microscopy

(STM): scanning

a

very

sharp

metal

wiretipoverasurfaceandbyapplyinganelectricalvoltagetothetiporsample,wecanimagethesurfaceatanextremelysmallscaledowntoresolvingindividualatoms.

5. AtomicForceMicroscopy(AFM): hastheadvantageofimaging

almostanytypeofsurface,includingpolymers,ceramics,composites,glass,andbiologicalsamples.AnimprovementonSTM

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ElectronMicroscopy

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Studyingcellswith

theEM

Cellfractionation: Disruptcellboundary

Differentialcentrifugation

Biochemical&physiologicalanalysesarethencarriedoutonisolatedorganelles

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Light

microscopy

techniques

Electron

microscopy

techniques