MICROSCOPY FOR MEDICAL AND LIFE SCIENCE STUDENTS

MICROSCOPY FOR MEDICAL AND LIFE SCIENCE STUDENTS

1. MICROSCOPY
Neethu soman
MSc Medical Biochemistry

2. INDEX
❑INTRODUCTION
❑HISTORY OF MICROSCOPE
❑PRINCIPLES OF MICROSCOPY
❑TYPES OF MICROSCOPE
❑APPLICATIONS OF MICROSCOPY

3. INTRODUCTION
➢Microscope(Greek:mikron=smallandScopes=tolook)
➢Itisanopticalinstrumentusedtomagnify(enlarge)minuteobjectsor
microorganismswhichcannotbeseenbynakedeye.
➢Microscopyisthescientificfieldthatinvolvestheuseofmicroscopesto
investigateobjectsanddetailsthataretoosmalltobeseenwiththenakedeye.
➢Microscopicmeansinvisibletotheeyeunlessaidedbyamicroscope

4. HISTORY OF MICROSCOPE
•ZachariasJansen(1580—1638)ofHollandinventedacompoundlight
microscope,onethatusedtwolenses,withthesecondlensfurthermagnifying
theimageproducedbythefirst.
•EnglishmanRobertHooke(1635—1703)furtherrefinedthecompound
microscope,addingsuchfeaturesasastagetoholdthespecimen,anilluminator,
andcoarseandfinefocuscontrols.until1800,compoundmicroscopesdesigned
byHookeandotherswerelimitedtomagnificationsof30xto50x,andtheir
imagesexhibitedaberrations.
•CarlZeiss(1816—1888)andErnstAbbe(1840—1905)addedthesubstage
condenseranddevelopedsuperiorlensesthatgreatlyreducedchromaticand
sphericalaberration,whilepermittingvastlyimprovedresolutionandhigher
magnification.

5. ➢PhysicistErnstRuskaandtheelectricalengineerMaxKnoll(1931)developed
thefirstprototypeelectronmicroscopewhichwascapableoffour-hundred-
powermagnification
➢ErnstLubkeofSiemens&Halske(1932)builtandobtainedimagesfroma
prototypeelectronmicroscope,applyingtheconceptsdescribedinRutenberg's
patent.
➢Ruska(1933)builtthefirstelectronmicroscopethatexceededtheresolution
attainablewithanoptical(light)microscope.
➢ManfredvonArdennes(1937)pioneeredthescanningelectronmicroscope.
➢Siemens(1939)producedatransmissionelectronmicroscope(TEM)in1939.

6. Father of Microscopy
•ANTONYVANLEEUWENHOOK was
aDutchscientist.In1674,viewinga
dropofrainwater,heobservedthings
movingwhichhecalled“Animalcules”
•Firsttoexperimentedwithmicrobes,
usingsinglelensedmicroscopesofhis
owndesigninventedin1670.
•Magnifiedupto200xandachieved
twicetheresolutionofthebest
compoundmicroscopes,mainly
becausehecraftedbetterlenses.

8. PRINCIPLES OF MICROSCOPY
MAGNIFICATION
RESOLUTION
NUMERICALAPERTURE
ILLUMINATION

9. MAGNIFICATION
❖Magnificationisthefactorbywhichanimageappearstobeenlarged.Itis
dependentuponthecurvatureandsizeofthelens.Theimageformedis
enlargedtoaparticulardegreecalledMagnifyingGlassthe“Powerof
Magnification”.
❖Whenlightpassesthroughtheobjectivelensandreachesyoureyethrough
theeyepiece,itappearslargerthanitsactualsize.
❖Thetotalmagnificationofanobjectistheresultofthemagnificationofthe
objectivelensmultipliedbythemagnificationoftheeyepiece.
❖Forexample,ifyouhaveamicroscopewitha10xeyepieceanda40x
objectivelens.
❖TotalMagnification=10x(eyepiece)×40x(objective)=400x

10. RESOLUTION
➢Resolutionorresolvingpoweristheabilityofalenstoshowtwoadjacentobjects
asdiscreateentities.
➢itisthemicroscope'sabilitytoshowfinedetailandclarityintheimagesit
produces.
➢Theresolvingpowerofamicroscopecanbecalculatedusingtheformula:
R=0.61λ​/NA
➢Where:Ristheresolvingpower,
λ(lambda)isthewavelengthoflightused,and
NAisthenumericalapertureofthelens.
➢Forexample,ifamicroscopeusesgreenlightwithawavelengthof500
nanometres(0.5micrometres)andhasanumericalapertureof1.4,theresolving
powerwouldbe: R=0.61×0.5μm/1.4​=0.22μm

11. NUMERICAL APERTURE
•TheNumericalAperture(NA)ofamicroscopeisacriticalparameterthat
determinesitsabilitytogatherlightandresolvefinedetailsinthespecimen
beingobserved.
•Itisadimensionlessnumberthatdescribesthelight-gatheringabilityofthe
objectivelensofthemicroscope
•NumericalAperture(NA)isdefinedastheproductoftherefractiveindexofthe
mediumbetweenthelensandthespecimen(n)andthesineofthehalf-angleof
themaximumconeoflightthatthelenscangather(θ).
•NA=n⋅sin(θ)

12. IILUMINATION
➢Effectiveilluminationisrequiredforefficientmagnificationandresolving
power.Artificiallightfromatungstenlampisthemostcommonlyusedlight
sourceinmicroscopy.Illuminationinmicroscopyisacrucialaspectofobtaining
clear,high-qualityimagesofspecimens.
➢Itinvolvesprovidingtherightamountandtypeoflighttoilluminatethe
specimenforobservation.Properilluminationenhancescontrast,resolution,and
overallimagequality.
1.TransmittedIllumination:Lightpassesthroughthespecimenfrombelow,transmitted
throughthesample.Commonlyusedincompoundlightmicroscopesforobservingthin,
transparentspecimenslikebiologicalsamplesonglassslides
2.ReflectedIllumination:Lightisdirectedontothespecimenfromabove,reflectingoffthe
surface.Suitableforopaqueorthickspecimens,suchasmetals,ceramics,orthickbiological
samples.

13. ABERRATION
•Aberrationisapropertyofopticalsystemsuchaslensesthatcauseslighttobe
spreadoutoversomeregionofspaceratherthanfocusedtoapoint.
•Anaberrationinthecontextofmicroscopesreferstoanydeparturefromideal
imagingconditions,resultingindistortionorblurringoftheobservedimage.
•Theseaberrationscanbecausedbyimperfectionsinthelensesorotheroptical
componentsofthemicroscope.
•Aberrationcausestheimageformedbyalenstobeblurredordistorted.
•Aberrationassociatedwithmicroscopearesphericalaberrationandchromatic
aberration.

14. SPHERICAL ABERRATION
•Sphericalaberrationisacommon
opticalaberrationthatoccurswhen
lightrayspassingthroughthe
peripheryofalensormirrorare
focusedatadifferentpointthanthose
passingthroughthecentre.
•Inmicroscopes,sphericalaberration
candegradeimagequalitybycausing
blurringanddistortion,particularlyat
highmagnifications
•Createsacurvedimageratherthan
flat.

15. CHROMATIC ABERRATION
•Createsablurryimagelikearainbow,causedbythelensactingasaprism
•whichoccursduetodifferencesinthecurvatureofalensormirror,chromatic
aberrationarisesfromthedispersionoflight,wheredifferentwavelengthsof
lightarerefracteddifferentlyastheypassthroughalens
•AchromaticobjectiveandApochromaticobjectives.
1.Apochromaticlenssystems:Thesearedesignedtominimizebothspherical
andchromaticaberrationsbycombiningmultiplelenselementsmadefrom
differenttypesofglasswithvaryingdispersionproperties.
2.Achromaticlenses:Thesearedesignedtoreducechromaticaberrationby
combiningtwoormorelenselementsmadefromdifferenttypesofglassto
bringtwoormorewavelengthsoflightintofocusatthesamepoint

16. PARTS OF MICROSCOPE

17. MICROSCOPE

18. 1.Eyepiece(Ocular):Theeyepieceisthelensatthetopofthemicroscopethat
youlookthrough.Typicallyprovides10xmagnification.Somemicroscopes
haveadjustableeyepiecestoaccommodateuserswithdifferentvision.
2.ObjectiveLenses:Theobjectivelensesarelocatedontherevolvingnosepiece
beneaththeeyepiece.Theselensesareresponsibleformagnifyingthespecimen.
•Microscopesusuallyhavemultipleobjectivelenseswithdifferent
magnificationpowers(e.g.,4x,10x,40x,100x).
•High-qualitymicroscopesmayfeaturespecializedobjectivessuchasoil
immersionlensesforhigh-resolutionimaging.

19. 3.Stage:
•Thestageistheplatformwherethespecimenisplacedforobservation.
•Itoftenincludesmechanicalcontrols(e.g.,knobsorcontrols)forprecise
movementofthespecimeninboththeXandYdirections.
4.Condenser:
•Thecondenserislocatedbeneaththestageandhelpsfocuslightontothe
specimen.
•Itmayhaveadjustablediaphragmstocontroltheamountoflightreaching
thespecimen,improvingcontrastandresolution.

20. 5.IlluminationSystem:Microscopesfeaturevariousilluminationmethods,including:
a)Brightfieldillumination:Themostcommonmethodwherelightpassesthroughthe
specimenandiscollectedbytheobjectivelens.
b)Darkfieldillumination:Illuminationfromthesides,allowingobjectstoappearbright
againstadarkbackground.
c)Phasecontrastanddifferentialinterferencecontrast(DIC):Techniquesusedforobserving
transparentorunstainedspecimens.
d)Fluorescenceillumination:Excitesfluorescentmoleculesinthespecimentoproduce
fluorescentsignals.
e)Lightsourcescanincludehalogenbulbs,LEDlights,orspecializedlampsdependingon
theilluminationtechnique.

21. 6.FineandCoarseFocusAdjustments:
•Theseknobsorcontrolsareusedtobringthespecimenintosharpfocus.
•Thecoarseadjustmentmovesthestageupanddownrapidlyforrough
focusing,whilethefineadjustmentallowsforprecisefocusing.
7.Base:
•Thebaseprovidesstabilityandsupportfortheentiremicroscope.
8.BodyTube:
•Thebodytubeconnectstheeyepiecetotheobjectivelenses,maintaining
thecorrectdistanceandalignmentbetweenthem.

22. 9.Diaphragm:Thediaphragmislocatedbelowthecondenserlensandcontrols
theamountoflightthatreachesthespecimen.Itcanbeadjustedtochangethe
brightnessandcontrastoftheimage.
10.StageClips:Theseareusedtoholdthespecimenslideinplaceonthestage

23. TYPES OF MICROSCOPE

24. COMPOUND LIGHT MICROSCOPE
Principle:Compoundlightmicroscopesusevisiblelightandaseriesoflenses
tomagnifysmallspecimens.Theyworkbypassinglightthroughthespecimen
andmagnifyingitwithobjectiveandeyepiecelenses.
Applications:Widelyusedinbiology,medicine,education,andresearchfor
viewingcells,tissues,microorganisms,andothersmallobjects.Theyare
versatiletoolsforstudyingbiologicalsamples,includinglivespecimens.
Advantages:Relativelysimpletouse,cost-effective,andsuitableforobserving
livespecimens.Theyprovidehigh-qualityimageswithsufficientmagnification
formostbiologicalstudies.
Limitations:Limitedresolutioncomparedtoelectronmicroscopes,typicallyupto
around0.2micrometres.Theyarenotsuitableforobservingstructuressmallerthan
thewavelengthofvisiblelight

25. BRIGHT FIELD MICROSCOPE
•BrightfieldMicroscopeisalsoknownastheCompoundLightMicroscope.
•Itisthesimplestofalltheopticalmicroscopyilluminationtechniques.
•Sampleilluminationistransmitted(i.e.,illuminatedfrombelowandobserved
fromabove)whitelight,andcontrastinthesampleiscausedbyattenuationOf
thetransmittedlightindenseareasOfthesample.
•Itisanopticalmicroscopethatuseslightraystoproduceadarkimageagainsta
brightbackground.
•ItisusedinBiology,CellularBiology,andMicrobiologicalLaboratorystudies.
•Thismicroscopeisusedtoviewfixedspecimens,thathavebeenstainedwith
basicstains,givesacontrastbetweentheimageandtheimagebackground.
•Itisspeciallydesignedwithmagnifyingglassesknownaslensesthatmodify
thespecimentoproduceanimageseenthroughtheeyepiece.

26. PRINCIPLE
•InBrightfieldMicroscope,thespecimenmustpassthroughauniformbeamofthe
illuminatinglighttobethefocussedandproduceanimage.
•Themicroscopewillproduceacontrastingimagethroughdifferentialabsorptionand
differentialrefraction
•Thespecimensusedarestainedtointroducecolourforeasycontracting
characterization.
•Thecolouredspecimenswillhavearefractiveindexthatwilldifferentiateitfromthe
surrounding,presentingacombinationofabsorptionandrefractivecontrast.
•Themicroscopefunctionisbasedonitsabilitytoproduceahigh-resolutionimage
fromanadequatelyprovidedlightsource,focusedontheimage,producingahigh-
qualityimage.
•ThespecimenwhichisplacedonamicroscopicslideisviewedunderOilimmersionor/and
coveredwithacoverslip.Oilimmersionimproveresolutionbyreducinglightscatter.

27. APPLICATIONS
1.UsedtounderstandcellstructuresincellBiology,Microbiology,
BacteriologytovisualizingparasiticorganismsinParasitology.
2.MostOfthespecimenstoviewedarestainedusingspecialstainingto
enablevisualization.Examples:NegativestainingandGramstaining.
3.Someofitsapplicationsinclude:
•Tovisualizeandstudytheanimalcells
•Tovisualizeandstudyplantcells.
•Tovisualizeandstudythemorphologiesofbacterialcells
•TOidentifyparasiticprotozoanssuchasParamecium.

28. DARK FIELD MICROSCOPY
•Microscopesaredesignatedaseitherlightmicroscopesorelectron
microscopes.
•Lightmicroscopesusevisiblelightorultravioletraystoilluminatespecimens.
•Thisissimilartotheordinarylightmicroscope;however,thecondensersystem
ismodifiedsothatthespecimenisnotilluminateddirectly.
•Thecondenserdirectsthelightobliquelysothatthelightisdeflectedor
scatteredfromthespecimen,whichthenappearsbrightagainstadark
background.
•Livingspecimensmaybeobservedmorereadilywithdarkfieldthanwith
brightfieldmicroscopy.

29. PRINCIPLE
❖Indarkfieldmicroscope,thelightsourceisblockedOff,causinglighttoscatterasit
hitsthespecimen.
❖Thisisidealformakingobjectswithrefractivevaluessimilartothebackground
appearbrightagainstadarkbackground.
❖WhenlighthitsanObject,raysarescatteredinallazimuthsOrdirections.
❖ThedesignOfthedarkfieldmicroscopeissuchthatitremovesthedispersedlight,or
zerothorder,sothatonlythescatteredbeamshitthesample.
❖TheintroductionOfacondenserand/orstopbelowtheStageensuresthattheselight
rayswillhitthespecimenatdifferentangles,ratherthanasadirectlightsource
above/belowtheobject.
❖Theresultisa"coneoflight"whereraysarediffracted,reflectedand/orrefractedoff
theobject,ultimately,allowingtheindividualtoviewaspecimenindarkfield.

30. USES
•Demonstrationofverythinbacterianotvisibleunderordinaryilluminationsince
thereflectionofthelightmakesthemappearlarger.
•FrequentlyusedfordemonstrationofTreponemapalliduminclinicalspecimens.
•Demonstrationofthemotilityofflagellatedbacteriaandprotozoa.
•Darkfieldisusedtostudymarineorganismssuchasalgae,plankton,diatoms,
insects,fibbers,hairs,yeastandprotozoaaswellassomemineralsandcrystals,
thinpolymersandsomeceramics.
•Usedtostudymountedcellsandtissues.
•Usefulinexaminingexternaldetails,suchasoutlines,edges,grainboundaries
andsurfacedefectsthaninternalstructure

31. COMPOUND LIGHT MICROSCOPE

32. STEREO MICROSCOPE
(DISSECTING MICROSCOPE)
Principle:Stereomicroscopesprovideathree-dimensionalviewoflarger
specimensbyusingtwoseparateopticalpaths.Theyuseincidentlighttoilluminate
thespecimenfrommultipleangles,allowingfordepthperception.
Applications:Idealfortaskssuchasdissecting,examiningsurfaces,electronics
inspection,materialsciences,andotherapplicationswheremanipulationor
dissectionofsamplesisrequired.
Advantages:Offerawidefieldofview,longworkingdistances,andalargedepth
offield.Theyarewell-suitedforviewingopaqueobjectsandspecimensthatrequire
manipulation.
Limitations:Lowermagnificationcomparedtocompoundmicroscopes,typically
uptoaround100x.Limitedresolutioncomparedtoelectronmicroscopes.

33. ELECTRON MICROSCOPE:
Principle:Electronmicroscopesuseabeamofelectronsinsteadoflightto
illuminatethespecimen.Theyoffermuchhigherresolutionthanlightmicroscopes
duetotheshorterwavelengthofelectrons.
Types:
1.TransmissionElectronMicroscope(TEM):allowsonethestudyoftheinner
surface
2.ScanningElectronMicroscope(SEM):.usedtovisualizethesurfaceof
objects.
•Three-dimensionalimaging

34. ELECTRON MICROSCOPE
The main components of an electron microscope are:
•An electron gun
•Electromagnetic lens system
•Vacuum system
•Camera/detector
•Computer

35. Basic Principles
•Thegunconsistsofanelectronsource,electrode,Wenheltassemblyandanode.
•Acurrentisrunthroughthefilament/crystaltoheatit,resultingintheemission
ofelectronsfromthetip.Thehighvoltagedifferencebetweenthecapandthe
anodecausestheelectronstoaccelerateandformabeam
•TEMlensesareelectromagnetic,creatingprecise,circularmagneticfieldsthat
manipulatetheelectronbeam,muchthesamewaythatopticallensesfocusand
directlight.
•similarlytoopticallenses,electromagneticlensesarealsosusceptibleto
aberrations

36. ELECTRON MICROSCOPE:
Applications:Used in materials science, nanotechnology, biology, and other
fields where high-resolution imaging is necessary. TEM is suitable for studying
internal structures, while SEM is ideal for surface imaging.
Advantages:Exceptional resolution, capable of imaging structures at the atomic
level. Can achieve magnifications exceeding 1,000,000x.
Limitations:Requires extensive sample preparation, including dehydration and
coating with conductive materials. Expensive equipment and specialized training
are needed for operation and maintenance.

37. TRANSMISSION ELECTRON
MICROSCOPE (TEM)

38. TRANSMISSION ELECTRON
MICROSCOPE (TEM)
•TEM is a microscopy technique where a beam of electrons is transmitted through
an ultra thin specimen.
•An image is formed from the interaction Of the electrons transmitted through the
specimen;
•The image is magnified and focused onto an imaging device, such as a
fluorescent screen, on a layer of photographic film, or to be detected by a sensor
such as a CCD camera.
.

39. HOW IT WORKS
•The condenser lens system focuses the emitted electrons into a coherent beam.
•The first condenser controls the spot size of the beam. This is controlled by the spot size
setting in the TEM software.
•The second condenser focuses the beam onto the sample (this is controlled by the
‘brightness’ knob on the microscope). The condenser aperture restricts the beam by
excluding high angle electrons. Usually a middle sized condenser aperture is suitable.
•The objective lens focuses the electrons transmitted through the sample into a magnified
image.

40. •The objective aperture can be used to increase contrast by excluding high angle
transmitted electrons.
•The intermediate and projection lenses enlarge the image. When the electrons hit
the phosphorescent screen, it generates light which allows the human eye to view
it.
•Images can be acquired using a high resolution

41. SAMPLE PREPARATION FOR TEM

42. SAMPLE PREPARATION FOR
ELECTRON MICROSCOPY
•TEM specimens must be:
•Very thin ,Well preserved ,Electron dense and Stable in the vacuum
•The degree of specimen preparation for biological TEM depends on the
specimen
•Particulate samples (eg: protein and viruses) can be stained and viewed quickly
•Cells and tissue samples require extensive preparation for TEM

43. SAMPLE PREPARATION FOR ELECTRON
MICROSCOPY
1.DEHYDRATION : The wet sample is dehydrated by keeping in increasing
concentration of ethanol or acetone
2.FIXATION
❖Fixation is done by immersing the specimen in chemical preservatives
called fixative.
❖Osmium tetroxide, glutaraldehyde, potassium permanganate, formalin, etc.
are common fixatives.
❖These fixatives form covalent bond with biological molecules like proteins
and lipids.
❖They stabilize the structural organization in the specimen.

44. •Fixation stops cellular processes and
aims to preserve the specimen as close as
possible to its natural state.
Characteristics of a good fixative:
•Permeates cells readily and acts quickly
•Is irreversible
•Does not cause fixation artifacts
Methods of fixation include:
•Chemical fixation with aldehydes
•Cryo-fixation with liquid nitrogen
•Microwave fixation

45. •Tissue can be cry-fixed using LN
2 in the High Pressure Freezer and then further processed for
TEM (adds 1 week)
•Specimens are mounted into specimen carriers and cryo-fixed with LN
2 under high pressure
(~2000 bar) to prevent damaging ice crystal formation up to 200 μm into the tissue
•Samples are then carefully transferred to the AFS and freeze-substituted with solvent (+
osmium and/or glutaraldehyde or uranyl acetate) at sub-zero temperatures.
•Cons of cryofixation: time consuming, finicky and restrictions on sample size, possible ice
crystal issues
•Pros of cryo-fixation: best possible ultrastructural preservation, maintains fluorescence and
antigenicity

46. secondary fixation
•Osmium tetroxide (very toxic!) is a heavy metal that fixes
unstaturatedlipids and is also electron dense.
•Used as both a secondary fixative and an electron stain and
significantly improves specimen preservation (especially
membranes) and contrast.
Microwave processed liver tissue, E Johnson

47. •Dehydration is the process of gradually replacing water in the sample
with a solvent (usually acetone or ethanol).
•The solvent is then gradually replaced with resin. This process can
be lengthy and depends on both the sample and type of resin used.
Resin blocks Poor resin infiltration

48. EMBEDDING
•The specimen is embedded in a hard embedding medium like
araldite vestopaI-W or Epson-812 or plastic medium
•Thickness using a glass or diamond knife fixed in an
ultramicrotome, The thin section is mounted on a copper grid Of
3mm diameter and covered With parlodoan-

49. Leica Ultracut7ultramicrotome, Dunn School Introduction to ultramicrotomy video, University of Sydney

50. NEGATIVE STAINING
•Contrast can be increased by post-staining sections with salts of heavy metals,
specifically uranyl acetate and lead citrate solutions.
•Uranyl acetate stains protein and DNA and also acts as a mordant for lead citrate,
which is a more general stain.
•Coat grids with plastic film and carbon
•Apply the particulate specimen
•Stain with heavy metal solution, eg: uranyl acetate, phosphotungstic acid,
sodium silica tungstate
•Blot dry and view in the TEM

51. SCANNING ELECTRON
MICROSCOPE (SEM)

52. SAMPLE PREPARATION FOR SEM
•well preserved with no surface contamination or damage
•Stable in the vacuum ,Conductive , Composed of high atomic number elements
•The conventional preparation for SEM samples is similar to that for TEM, although the
resin and sectioning steps are omitted.
•There are less size restrictions on SEM samples compared to TEM.
•Itisusedforstudyingsurfacesandrevealingsurfacemorphologyatnanoscaleresolutions
•This microscopy technique involves using a focused beam of electrons to scan across the
surface of a specimen

54. •Once the dehydration series is complete, the solvent itself must be removed from the tissue
without introducing surface tension/drying artifacts into your sample. This is achieved through
the use of a transitional fluid, most commonly hexamethyl disilazane (HMDS) or liquid CO
2.
Air drying is not recommended, as ethanol evaporation generally causes severe surface tension
artifacts.
•Liquid CO
2 can be used to flush the solvent from tissue using a technique called Critical
Point Drying (CPD).
•if a biological specimen is not mounted and coated correctly, it will react to the electron beam
(an effect called charging), resulting in sample damage and/or image distortion.
•Mounting immobilizes the sample on a conductive backing, grounding it. Ensure that your
sample is in full contact with the conductive backing; if not, use conductive glue (eg: carbon
and silver) to ensure conductive continuity.
•

55. •Sputter coating with metal ions deposits a thin continuous conductive layer over the sample,
such that charge from the electron beam flows to the ground and does not build up on the
sample.
•Sputter coating also increases the SE signal (and therefore contrast), high Z elements have a
higher yield of SEs than low Z elements (biological material!).
•Variable pressure and environmental SEM (ESEM) allows untreated, hydrated specimens to
be imaged at high resolution.
•Utilises a specialised detector and vacuum system that enables imaging under low pressure
conditions (ie; not a vacuum!).

56. CONFOCAL MICROSCOPE:
➢An optical imaging technique for increasing optical resolution and contrast of a
micrograph.
➢Radiations emitted from laser cause sample to fluoresce
➢uses pinhole screen to produce high resolution images.
➢Eliminates out of focus.
➢So images have better contrast and are less hazy.
➢A series of thin slices of the specimen are assembled to generate a 3 dimensional
image.
➢Is an updated version of fluorescence microscopy.

57. ❖In confocal microscopy two pinholes are typically used:
❖A pinhole is placed in front of the illumination source to allow transmission
only through a small area
❖This illumination pinhole is imaged onto the focal plane of the specimen, i.e.
only a point of the specimen is illuminated at one time.
❖Fluorescence excited in this manner at the focal plane is imaged onto a
confocal pinhole placed right in front of the detector
❖Only fluorescence excited within the focal plane of the specimen will go
through the detector pinhole.
❖Scanning of small sections is done and joined them together for better view.

58. WORKING MECHANISM
•Confocal microscope incorporates 2 ideas:
1.Point-by-point illumination of the specimen.
2.Rejection of out of focus of light.

59. •Light source of very high intensity is used—zirconium arc lamp in Minsky's
design & laser light source in modern design.
a)Laser provides intense blue excitation light.
b)The light reflects off a dichroic mirror, which directs it to an assembly of
vertically and horizontally scanning mirrors.
c)These motor driven mirrors scan the laser beam across the specimen.
d) The specimen is scanned by moving the stage back & forth in the vertical &
horizontal directions and optics are kept stationary.

60. Dye in the specimen is excited by the laser light & fluoresces.
• The fluorescent (green) light is descanted by the same mirrors that are used
to scan the excitation (blue) light from the laser beam then it passes through the
dichroic mirror then it is focused on to pinhole.
• the light passing through the pinhole is measured by the detector such as
photomultiplier tube.
• For visualization, detector is attached to the computer, which builds up the
image at the rate of 0.1-1 second for single image

61. ADVANTAGES
The specimen is everywhere illuminated axially, rather than at different angles,
thereby avoiding optical aberrations.
Entire field of view is illuminated uniformly.
The field of view can be made larger than that of the static objective by
controlling the amplitude of the stage movements.
Image formed are of better resolution.
Cells can be live or fixed.
Serial optical sections can be collected.
Taking a series of optical slices from different focus levels in the specimen
generates a 3D data set.

62. DRAWBACKS
•Resolution : It has inherent resolution limitation due to diffraction. Maximum
best resolution of confocal microscopy is typically about 200nm.
• Pin hole size : Strength of optical sectioning depends on the size of the pinhole.
• Intensity of the incident light Fluorophores :
a)The fluorophore should tag the correct part of the specimen.
b)Fluorophore should be sensitive enough for the given excitation wave length.
c) lt should not significantly alter the dynamics of the organism in the living
specimen.
•Photobleaching: photochemical alteration of a dye or a fluorophore molecule
such that it permanently is unable to fluoresce

63. CONFOCAL MICROSCOPE:

64. PHASE-CONTRAST MICROSCOPE
•The phase contrast microscope is a light microscope.
• It is modification of compound microscope.
• It contains all the component of a compound microscope in addition to an
annular ring and a phase plate.
• It magnifies not only object but also changes in brightness.
•Phase -contrast microscope enhance contrast in transparent specimens By
exploiting differences in refractive index
.
.

65. Principle
❑The phase contrast microscope separates the illuminating background light
and the specimen scattering light.
❑Phase contrast microscope is used to visualize transparent, colourless,
unstained, living biological specimens. These objects are called phase objects.
❑Light is bend (diffracted) and retarded based on the refractive index of the
object.
❑Highly refractive structures bend and retard light much.
❑This principle is used in phase contrast microscope.

66. PHASE-CONTRAST MICROSCOPE
Principle: Phase-contrast microscopes enhance the contrast of transparent and
colourless specimens by exploiting differences in refractive index. They convert
phase differences in light passing through the specimen into brightness
variations.
Advantages: Allows for the visualization of live, unstained specimens without
the need for special preparation techniques such as fixing and staining. Suitable
for observing cellular structures and dynamics.
Applications: Commonly used in cell biology, microbiology, and other fields
where live-cell imaging is required, such as observing cell division and motility.
Limitations: Limited to transparent specimens with subtle density variations.
Lower resolution compared to electron microscopes

68. FLUORESCENCE MICROSCOPE
Principle: Fluorescence microscopes excite fluorescent dyes in the specimen with
specific wavelengths of light, producing fluorescent emissions that are captured to
create an image.
Advantages: Enables the visualization of specific molecules or structures labelled
with fluorescent markers, allowing for precise localization and tracking within cells
and tissues.It is commonly used for observing living cells in real-time.
Applications: Widely used in cell biology, immunology, genetics, and other fields
for visualizing specific molecules and structures within cells and tissues, such as
studying protein localization and dynamics.
Limitations: Requires fluorescent labelling of the specimen, which can affect
sample integrity and may introduce artifacts. Limited to specimens with suitable
fluorescent markers.

69. ATOMIC FORCE MICROSCOPY
•AFM is a type of scanning probe microscopy(SPM), with demonstrated
resolution on the order of fractions of a nanometre, more than 1000 times better
than the optical diffraction limit.
•The information is gathered by "feeling" or "touching" the surface with a
mechanical probe
•AFM provides a 3D profile of the surface on a nanoscale, by measuring forces
between a sharp probe and the surface.
•The AFM has three major abilities: force measurement, imaging, and
manipulation.
•It is powerful because an AFM can generate images at atomic resolution with
angstrom scale resolution height information, with minimum sample
preparation.

70. •The main principle behind atomic force microscopy is measuring the forces between a
sharp probe tip and a sample surface.
•Mode of operation in atomic force microscopy are Contact Mode , Tapping Mode and
Non-contact Mode
•The typical range of resolution achievable with atomic force microscopy is Sub-
nanometre.
•The material is commonly used for the probe tip in atomic force microscopy is Silicon.
•The primary advantage of dynamic or tapping mode AFM over contact mode AFM is
to reduce sample damage.
•"force curve" refer to the curve representing the force between the tip and the sample as
a function of distance

72. APPLICATION
❖The AFM has been applied to problems in a wide range of disciplines of the natural sciences,
including solid-state physics, semiconductor science and technology, molecular engineering,
polymer chemistry and physics, surface molecular-biology, cell-biology, and medicine.
❖It gives information about the toughness, roughness and smoothness value of surface.
❖Applications in the field of solid state physics include (a) the identification of atoms at a
surface, (b) the evaluation of interactions between a specific atom and its neighbouring atoms.
❖In molecular biology, AFM can be used to study the structure and mechanical properties of
protein complexes and assemblies. For example, AFM has been used to image microtubules
and measure their stiffness.
❖In cellular biology, AFM can be used to attempt to distinguish cancer cells and normal cells
based on a hardness of cells, and to evaluate interactions between a specific cell and its neigh
boring cells in a competitive culture system.
❖Soft surfaces are analysed by this technique without damaging it like Lipids.
❖Covalent bond strength is measured by this Technique

73. LIMITATIONS OF AFM
•Slow Imaging Speed: AFM typically operates at slower imaging speeds
compared to other microscopy techniques like scanning electron microscopy
(SEM) or optical microscopy.
•Complexity of Operation: Operating an AFM requires a certain level of
expertise and training due to its complex setup and delicate probe-sample
interactions. This can result in longer learning curves for users new to the
technique.
•Surface Sensitivity: AFM is highly sensitive to surface conditions, including
sample preparation, cleanliness, and even atmospheric conditions. Variations in
these factors can affect imaging results and introduce artifacts.
•Probe Wear and Damage: The sharp probe tip used in AFM can experience
wear or damage during imaging, especially in contact mode AFM where the tip
physically interacts with the sample surface. This can lead to decreased imaging
quality and increased experimental variability over time.

74. LIMITATIONS OF AFM
•Limited Imaging Range: The imaging range of AFM is typically limited to the
micrometer scale, which may not be suitable for studying larger samples or
structures. Additionally, AFM may struggle with imaging samples that have
significant height variations or roughness beyond its range.
•Sample Compatibility: AFM is not always compatible with all types of samples
or environments. For example, samples that are soft, sticky, or highly mobile may
pose challenges for AFM imaging, and certain environmental conditions (such as
high humidity) can affect imaging results.
•Cost: While the cost of basic AFM systems has decreased over the years, high-
resolution and specialized AFM setups can still be relatively expensive, making
them less accessible for some researchers or laboratories.

75. REFERENCES
•Text Book Of Microbiology By Surinder Kumar
•Prescott’s Microbiology , 10
th
Edition , Willey Sherwood Woolverton
•Microbiology 5th Edition Lansing M. Prescott.
•Essential Microbiology By Stuart Hogg
•Laboratory Exercises In Microbiology 5
th
Edition, Harley
•Principle and Techniques of Biochemistry and Molecular Biology. Keith Wilson and John Walker (Eds). 6th Edition.
Cambridge University Press.
•Physical Biochemistry (Application to Biochemistry and Molecular Biology).David Frei Felder (Ed.) WH Freeman and
Company, San Francisco.
•Parija S.C. (2012). Textbook Of Microbiology ed.), India: Elsevier India.
•Cappuccino, J. and Welsh. C. (2014). Microbiology: A Manual, Global Edition. 1st Pearson Education.
•Sastry A.S. & Bhat SK. (2016). Essentials of Medical Microbiology. New Delhi : Jaypee Brothers Medical pub I IS hers.
•Trivedi P.C., Pandey S, and Bhaduria S. (2010). Textbook Of Microbiology. Pointer Publishers; First edition
•Patskovsky; et al. (2014). "Wide-field hyperspectral 3D imaging. of functionalized gold nanoparticles targeting cancer cells by
reflected light microscopy". Bioptomes. 8