Microscopy

Living&Fixedcells
Lecture1

CellBiology
Cytologyinvestigatesthecellultrastructure, development,specialisation,operation, communication,andregulationofcellularactivities Definecellconcept

ScaleofLife
nm(nanometer,=109m)
~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 ) ~1 10m:thegeneralsizesforProkaryotes ~1m:Diameterofhumannervecellprocess ~9m:Humanredbloodcell ~10 30m:MostEukaryoticanimalcells ~10100m:MostEukaryoticplantcells ~90m:smallAmoeba ~100m:HumanEgg

Increasing size

m(micrometer,=106m)

1mm(1millimeter,=103m)

~1mm:Diameterofthesquidgiantnervecell ~120mm:Diameterofanostrichegg

3m:approx.lengthofanervecellofgiraffe'sneck

Collection&examinationofspecimens
Cellcollection&cultureusefulinstudyingcell characteristics:
DNAprofiling Metaboliteprofiling Cellultrastructure Cellfunction:cellidentification,reproduction, genetics,transformation,evolutionarystudies Tissueformationstudies

Cellsinculture(invitro) areausefulmodelfor studyingtheactivityofcellsinthewhole organism(invivo) Cellsneedtobecollectedandculturedcarefully

Collectionofspecimens
A. Microscopicorganisms
Wholeorganisms
1. 2. 3. 4. 5. 6. Scrapings Chemicalmeans Filtration/Collectingnets Sonication Microscopeslides Soilwatermedium

B. Macroscopicforms:removearepresentative portionin ordertodescribe: 1. organproperties 2. describedifferentsystems

Examinationofspecimens
TemporaryMounts
Examiningfreshmaterial
Thesimplestmethodistoplacejustenoughofthe waterormediumsampleontoamicroscopeslide Carefullyloweracoverglassontoit Placetheslideonstageofthebinocularorcompound lightmicroscope Startbyobservingthespecimenatlowermagnification

SamplePreparation
Oneormoreofthefollowingproceduresmay berequiredtoprepareasample:
Fixation Permeabilization Staining Dehydration Clearing Mounting

Examinationofspecimens
Stains
Iodine Methylene blue Haematoxylin Eosin

Uses
Starchindicator;starchandiodineturnadarkbluecolour Stain nucleiandmakethemmorevisible anuclearstainthat,withamordant,stainsnucleibluevioletorbrown acounterstain tohaematoxylin,cytoplasmic material,cell membranes,andextracellularstructurespinkorred aphysiologicaldyetoshowwatermovementinxylemtissue stainscollagen,smoothmuscle,ormitochondria. stainsDNA colours unhealthycellsinthefinalstagesofapoptosis,ordeliberate celldeath stainslignininsclerenchyma ofplantcells stainscellulose;greatervisibilityofcellwalls stainscellwallspurplewhencombinedwithamordant.Thisstainis usedinGramstaining

Fuschia Ethidium bromide

PhloroglucinolHCl Toluidine blue Crystalviolet

SamplePreparation:
Embeddingtissuesectionsformicroscopicexamination
Apieceoffixedtissueis dehydratedthrough
successivealcohol concentrations purealcohol xylene

Thespecimenisnextplaced inwarmliquidparaffin, whichisallowedtoharden Apieceoftheencased specimenismanually sectionedorpreferably mountedonthearmofa microtome Thearmmovesupanddown asametalorglassbladecuts thespecimenintosectionsa fewmthick

LightMicroscopy

o o o o

Mostbasicformofmicroscopicexamination
Lightiscausedtopassthroughanobjectandisthenfocusedbythe primaryandsecondarylens. Thecompoundmicroscopeconsistsofthreelenssystems:
TheCondenserlenssystem TheObjectivelenssystem TheEyepiece(ocular)lenssystem Thesethreelenssystemsarecomposedofglasslenses.

Types:
1. 2. 4. 5. 5. Compoundlightmicroscope/brightfield Dissectingmicroscope: Darkfield PhaseContrastmicroscope Fluorescencemicroscope

Light Microscope

LightMicroscopy

Light/Brightfield

Darkfield

Principleofdarkfieldmicroscopy

PhaseContrastMicroscopy

Resolutionenhancement
Resolutionmaybeoptimised throughanyorallof thefollowingmeans:
1. Increasingtheangleoflightincidence,byalteringthe position/designofthecondenserlens. 2. Maximisation oftherefractiveindex.Maybedoneby 3. Decreasethewavelengthoflightused

ElectronMicroscopy
Lightisreplacedbyabeamofelectrons Specimensforelectronmicroscopygenerallymustbefixed,sectioned,and dehydrated,andthenstainedwithelectrondenseheavymetals Vastlyimprovedresolvingpower electronbeamshaveveryshortwavelengths.
electronmicroscopesalsouseelectromagneticlenses.

Electronmicroscopesarealsousedmainlyforresearchpurposes. 1. TransmissionElectronMicroscope(TEM):electronspassthroughthe object(cf. compoundlightmicroscope) 2. ScanningElectronMicroscope(SEM): electronsarebouncedoffof thesurfaceofthespecimen(cf. binocularlightmicroscope) 3. Cryoelectron microscopy: unfixed,unstainedfrozenspecimens 4. ScanningTunnelingMicroscopy(STM): scanningaverysharpmetal wiretipoverasurfaceandbyapplyinganelectricalvoltagetothetip orsample,wecanimagethesurfaceatanextremelysmallscale downtoresolvingindividualatoms. 5. AtomicForceMicroscopy(AFM): hastheadvantageofimaging almostanytypeofsurface,includingpolymers,ceramics,composites, glass,andbiologicalsamples.AnimprovementonSTM

ElectronMicroscopy

Studyingcellswith theEM
Cellfractionation:
Disruptcellboundary Differential centrifugation Biochemical& physiologicalanalyses arethencarriedout onisolatedorganelles

Light microscopy techniques

Electron microscopy techniques