lec 2 Types of the Microscop.pdf Microscopy The science of investigating small objects and structures using a microscope

Microscopy
The science of investigating small objects and
structures using a microscope

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Dr. HalahKamal
1stClass
College of Medical Sciences Technology/ University of Al-
Mashreq
Lecture 2:
Types of the microscopes

2. MicroscopeClassification:Basedonhowtheimageiscreated,
Typesofthemicroscopes:
1.LightMicroscope:Thetypesoflightmicroscope:
A.Brightfieldmicroscope.
B.Darkfieldmicroscope.
C.Phasecontrastmicroscope.
D.Fluorescencemicroscope.
E.Ultravioletmicroscope.
2.ElectronMicroscope(EM):ThetypesofEMmicroscope:
1.Scanningelectronmicroscope(SEM).
2.Transmissionelectronmicroscope(TEM).

3. 1-Lightmicroscope:
•Useslighttocreatespecimen
image(usesunlightor
artificiallight).
•Lensesareglassorplastic.
•Magnificationandresolution
aregood(1000X).
Advantage:
•Smaller→portable.
•Cost-effective.
•Easy specimen preppers.
•Training simple/ user friendly.
•Can see microscopic items.
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4. ApplicationofLightMicroscope
•Observationofmorphologymicroorganisms.
•Detectionofcellstructures.
•Observationofintracellularstructures.
•Observationofmotility.
•Measurementofsize.
•Observationofbloodsmears.
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5. A. Bright fieldMicroscope
•Itisusedtoviewstainedornaturallypigmentedspecimens
thatproduceadarkimageagainstabrighterbackground.
•Hasseveralobjectivelenses.
•2types:SimpleandCompound.
•Simple:Hasasinglemagnifyinglens.ex:magnifyingglass,
Leeuwenhoek’smicroscope.
•Compound:Has2ormoreobjectivelenses(morelenses
createabetterimageandbetterresolution).
•Lightpassesthroughspecimenintoobjectivelens.
•Seriesoflensesformagnification.
•Oilimmersionlensincreasesresolution.
•Resolution=200nm.
Types of the Light microscopes:

6. The Effects of Immersion Oil on Resolution

7. Advantage:
•Used to view live and immobile
specimens such as bacteria and tissues
or stained cells.
•Simple setup with very little preparation
required.
Disadvantage:
•Biological specimens are often of low
contrastand need to be stained.
•Staining may destroyor introduce
artifacts.
•Resolution is limited to 200nm.
•Light dust is easily observe, in cheek
cells views with bacteria.

8. B.DarkfelidMicroscope
•Theopticalsystemenhancesthecontrast
ofunstainedbodies.
•Thespecimenappearsgleamingbrightly
againstadarkbackground.
•Itisusedinmicrobiologyandin
autoradiography.
•RequisitesforDark-felidMicroscopy
•Dark-felidcondenser.
•High-intensitylamp.
•Funnelstop.
Dark field microscopy

9. Advantages
•Simplesetup
•Providescontrasttounstainedtissue.
•Examinationoflivebloodsamples,tissues,bacteria
(Treponemapallidum,Leptospira,Campylobacterjejuni,
Endospore)andfungi.
Disadvantages
•Thespecimenneedstobestronglyilluminatedwhichcan
damagedelicatesamples.
Treponema pallidum

10. C.PhaseContrastMicroscope
•Firstdescribedin1934byDutchphysicistFritsZernike.
•Produceshigh-contrastimagesoftransparentspecimens
PrincipleofPhaseContrastMicroscopy
•Unstainedbacteriahaveconstituentsofdifferent
refractiveindexes(Diffractionoflight).
•Aphase-contrastmicroscopeemploysanoptical
mechanismtotranslateminutevariationsinphaseinto
correspondingchangesintheintensityofanimage.
Requisites for Phase contrast Microscopy
•Annular Diaphragm
•Phase Plate

11. Advantage
•Phase-contrastenablesvisualizationofinternalcellular
componentswithoutstanning.
•Diagnosisoftumorcells.
•Idealforstudying&interruptingthinspecimens.
•Examinationofawidevarietyoflivingcellsintheir
naturalstategrowth,dynamics,andbehaviorincell
culture.
Disadvantage
•Annuliorringlimitstheaperturetosomeextentwhich
causesadecreaseinresolution.
•Notidealforthickspecimens.
•ShadeoffandHaloeffectmayoccur.

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13. •CondenserAnnulus
•Thecondenserannulusorannulardiaphragmisan
opaqueflat-black(light-absorbing)platewitha
transparentannularring.
•Produceshollowconeoflight.
•PhasePlate
•Placedinthebackfocalplaneoftheobjective.
•Function
•Enhancesphasedifferencebyretardingdiffractedwavefront
byone-quarterofwavelength.
•Reducestheintensityofdirectraysandequalizesitwith
diffractedrays'intensity.

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Image of Phase contrast microscope

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D.FluorescenceMicroscopy
•Specimensareusuallystainedwithafluorescentdye
(fluorochromes).
•Examinedunderthemicroscopewithultravioletlightareseen
asabrightobjectsagainstadarkbackground.
•Whenthedyemoleculesreturntotheirnormalstate,they
releaseexcessenergyintheformofvisiblelight
(fluorescence).
UseofFluorescenceMicroscopy
-AuramineRhodamine
-YellowfluorescenceTuberclebacilli.
-AcridineOrangeR-givesorange-redfluorescencewith
RNAandyellow-greenfluorescencewithDNA.
-Immunofluorescence.

16. •Principle

17. E-UltravioletMicroscope
•Ithasquartzlenses.
•Slidesareusedultravioletradiationas
theillumination.
•Theuseofshorterwavelengthsthanthe
visiblerangeenablestheinstrumentto
resolvesmallerobjectsandtoprovide
greatermagnificationthanthenormal
opticalmicroscope.
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18. 5
1
2.ElectronMicroscope(EM)
•Co-inventedbyMaxKnolland
ErnstRuskain1931.
•ElectronMicroscopesuseabeamof
highlyenergeticelectronsto
examineobjectsonaveryfine
scale.
•Magnificationcanupto2million
timeswhilethebestlight
microscopecanmagnifyupto2000
times.
•Thisexaminationcanyieldinfo
aboutTopography,Morphology,
Composition,Crystallographic
structure.

19. •Scanagold-platedspecimentogivea
3-Dviewofthesurfaceofanobject
whichisblackandwhite.
•Usedtostudysurfacefeaturesofcells
andviruses.
•Itisusedinphysics,biologyand
chemistryandexaminestheinsectto
bonethroughSEM.
•ThescanningElectronmicroscopehas
resolution1000timesbetterthanthe
Lightmicroscope.
Types of Electron Microscope (ME)
A. Scanning Electron Microscope (SEM)

20. SEMIMAGES
Vibrio cholerae with polar flagellaTreponemapallidum

21. •Acceleratedusingapositiveelectrical
potential.
•Astreamofelectronsisformed
focusedbymetallicapertureand
Electromagnets.
•Interactionsoccurinsidethe
irradiatedsamplewhichisdetected
andtransformedintoanimage.
•ProjectorLensformsanimageona
Fluorescentviewingscreen.
•2DImage.
•Magnification10,000Xto100,000X.
B. Transmission Electron Microscope(TEM)

22. •Itisusedtostudycells.Ultrathinslicesofmicroorganisms
likevirusesareplacedonawiregrid,thenthesecellsare
stainedwithgoldorpalladiumandthenusedtoobserveunder
atransmissionelectronmicroscope.

23. Light Microscope
•Resolutiontoapproximately
0.2micrometers.
•Thislimitsthepractical
magnificationlimitto
~1500x.
•Out-of-focuslightfrom
pointsoutsidethefocalplane
reducesimageclarity.
•Theinternalstructureis
difficulttoobserve.
Electron Microscope
•Large
•Anexpensivepieceofequipment.
•Theneedforaccuracyand
experiencewhenpreparingthe
sampletobestudied.
•Theneedtoapplyathinlayerof
metaltothesampleasgold;To
allowelectronstobereflected.
•Theinabilitytouseitto
monitorlivecells;isbecausethe
samplemustundergodrying,and
ahighdoseofradiation,which
leadstoitsdeath.
Disadvantages

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