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Introduction


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Introduction


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Introduction


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Introduction


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Introduction


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Introduction


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Introduction


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Introduction


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Introduction


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Electron Microscope Any class of

microscopes that useelectrons instead of light to form imagesof very small objectssuch as individualparts of small livingthings


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Electron Microscope

Uses a magnetic field tobend beams of electrons;

greater magnification& resolving power than light microscope

The two types are: Scanning and Transmission


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Which is the most powerful kind of microscope?


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THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

fluorescent (TV) screen, photographic film

Human eye (retina),photographic film

Focussingscreen

Vacuum Air-filled Interior Magnets Glass Lenses

High voltage (50kV)

tungsten lamp

Tungsten or quartz

halogen lamp

Radiation

source

x500 000 x1000 x1500 Maximummagnification

0.2nmFine detail

app. 200nm Maximumresolving power

Electrons app. 4nm

Monochrome

Visible light 760nm (red) 390nm

Colours visible

Electromagneticspectrum used

ELECTRON MICROSCOPE LIGHT MICROSCOPE FEATURE


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THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

Copper grid Glass slide Support

Heavy metals Water soluble dyes Stains

Microtome only.

Slices 50nmParts of cells visible

Hand or microtome

slices 20 000nm Whole cells visible

Sectioning Resin Wax Embedding

OsO 4 or KMnO 4 Alcohol Fixation

Tissues must bedehydrated

= dead

Temporary mountsliving or dead

Preparation of specimens

ELECTRONMICROSCOPE

LIGHT MICROSCOPE FEATURE


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Properties of electron

Electron are used as a source of illumination They are negatively charged subatomic

particles

When the atoms of metal are excited bysufficient energy in the form of heat, theelectron leave their orbit, fly off from space& are lost in atoms

Metal tungsten is commonly used as a sourceof electron


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The electron are readily absorbed &scattered by different form of matter

So a beam of electron -> produced &sustained only in high vacuum

Electron are like light waves-> So used inimage formation

Electron interact with the atoms of thebiological specimens to form the image


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Electron beam

(A)

Transmittedelectron

(B)Inelastically scattered

electrons

(C)

Elastically scatteredelectrons

(D)

Back-scattered electrons

(E)Secondary electrons

X-rays

Visible light


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1. Transmitted electrons (A) of the beam passesstraight through the specimen on to the

screen2. Some electron (B) of the beam lose a bit of

their energy while passing through the

specimen & get deflected a little from theiroriginal axis of the beam inelasticallyscattered electrons

3. Some electron (c) interact with atoms of specimen & getelastically scattered withoutlosing energy. Electron deviate widely


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4. Some electron (D) getbackscattered insteadof getting transmitted through the specimen

5. In some cases the electrons get absorbed bythe atoms of the specimen & instead lowenergy electron (E) are emitted. Theseelectron are termed secondary electron .These are very useful for forming the image inthe SEM

6. Some atom emit x-ray & light energy


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Working & Image formation Working of EM is based onsame planas that of light microscope Electron are used for magnification &image formation Image formation occurs by electronscattering Electron strike the atomic nuclei & getdispersed This dispersed electron form image The electron image is converted in tovisible form by projecting on afluorescent screen


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Electron in the form of a beam pass throughthe condenser coil & fall on the object

They get scattered & transmitted through theobject & pass through the objective coil,which magnifies the image of the object

The projector coil further magnifies the image& projects on the fluorescent screen/film

The image formation occurs when the energy

of the electron is transformed in to visiblelight through excitation of the chemicalcoating of the screen


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Those electron which reach the fluorescentscreen form bright spot while the area where

the electron do not reach the screen formdark spot The varying degree of intensity of electrons

form the image with varying degree of grey. Electron scattering, however, is due to theatomic nuclei which consist of protons &neutrons

Higher the atomic number, greater thescattering


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Since biological materials generally have a lowatomic number, the dispersion is poor

Very poor dispersion means very poorcontrast in the image formation

In order to increase contrast, a number of salts with high atomic number are used.

Such salts can be used during the process of fixation or staining


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Magnification Objective & projector coil help in magnifying the

image formed in EM. In order to have maximum magnification, an

intermediate coil is fitted between the objective& projector coils.

This coil further increase the magnification Magnification > objective coil 100, projector

coil - 200, so net magnification= 20,000 EM fitted with intermediate coil can achieve

magnification as high as 1,60,000


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Resolution

The resolving power of a microscope is limited bywavelength of illumination forming the image

Shorter wavelength, smaller detail can beresolved

This concept led to discovery of EM Resolution power of good EM is 4-10


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Transmission Electron Micr oscope (TEM)

Electrons arepassed through very thinspecimens to seewhat is inside!

Invented in1933 Magnification is

500,000x
http://physics.nist.gov/GenInt/STM/fig1.htmlhttp://physics.nist.gov/GenInt/STM/fig1.html


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The TEM is a complex viewing systemequipped with a set of electromagnetic lenses used to control the imaging electrons in orderto generate the extremely fine structuraldetails that are usually recorded onphotographic film.

Since the illuminating electronspass through the specimens, the information is said to be atransmitted image.

The modern TEM can achieve magnificationsof one million times with resolutions of 0.1nm.


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Basic Systems Making Up a TransmissionElectron Microscope

The transmission electron microscope is made up of a number of different systems that are integrated toform one functional unit capable of orienting andimaging extremely thin specimens.

The illuminating system consists of the electron gunand condenser lenses that give rise to and controlthe amount of radiation striking the specimen.

Aspecimen manipulation system composed of thespecimen stage, specimen holders, and relatedhardware is necessary for orienting the thinspecimen outside and inside the microscope.


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Page 167

TABLE 6.3 Major Column Components of the TEM*

Component Synonyms Function of Components

IlluminationSystem

Electron Gun Gun, Source Generates electrons and provides firstcoherent crossover of electron beam

Condenser Lens 1 C1, Spot Size Determines smallest illumination spot sizeon specimen (see Spot Size in Table 6.4)

Condenser Lens 2 C2, Brightness Varies amount of illumination onspecimen in combination with C1 (seeBrightness in Table 6.4)

Condenser Aperture

C2 Aperture Reduces spherical aberration, helps controlamount of illumination striking specimen


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SpecimenManipulationSystem

Specimen Exchanger Specimen Air Lock Chamber and mechanism for inserting specimenholder

Specimen Stage Stage Mechanism for moving specimen inside column of microscope

Imaging System

Objective Lens Forms, magnifies, and focuses first image (seeFocus in Table 6.4)

Objective Aperture Controls contrast and spherical aberration

Intermediate Lens Diffraction Lens Normally used to help magnify image fromobjective lens and to focus diffraction pattern

Intermediate Aperture Diffraction Aperture,Field LimitingAperture

Selects area to be diffracted

Projector Lens 1 P1 Helps magnify image, possibly used in somediffraction work

Projector Lens 2 P2 Same as P1


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Observationand CameraSystems

ViewingChamber

Contains viewing screen for final image

Binocular Microscope

Focusing Scope Magnifies image on viewing screen for accurate focusing

Camera Contains film for recording


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ScanningElectronMicroscopy(SEM) VisualizesSurface Features


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Extremely useful in studying surface structure

The electron do not form the image by beingtransmitted, but by getting emitted from thesurface of specimen

Illuminating system of SEM similar to TEM The electron beam is, how ever compressed

with 1 or more condenser coils, which resultin the formation of an narrow pencil of electron


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The electron which falls on specimen areprimary electron

The electron probe is focused on the surfaceof specimen

The electron are emitted as secondary

electrons from the surface The specimen is kept at inclined angle As the electron does not have to pass through

specimen, its thickness is not important


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Layout and performance of SEM

1-3 Electron gun

4, 10 Aperture

5-6 Condenser lenses

7 Scanning coils

8 Stigmator

9 Objective lens

11 X-ray detector

12 Pre-amplifier

13 Scanning circuits

14 Specimen

15 Secondary electron detector

16-18 Display/Control circuits


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Specimen Preparation

Specimens arecoated withmetals todeflectelectrons froma beam

scanned acrossthe sample.


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SEM of StereociliaProjecting from a Cochlear(inner ear) Hair Cell
http://micro.magnet.fsu.edu/primer/java/electronmicroscopy/magnify1/index.htmlhttp://micro.magnet.fsu.edu/primer/java/electronmicroscopy/magnify1/index.html


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Copper grid slides

2007 Paul Billiet ODWS
http://www.saburchill.com/IBbiology/bio_hp.htmlhttp://www.saburchill.com/IBbiology/bio_hp.html


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Higher Resolution Is Achieved by Viewing Sections of Fixed, Stained, andEmbedded Samples

A microtome cutting sections of an embedded sample.


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Microtome knife

2007 Paul Billiet ODWS
http://www.saburchill.com/IBbiology/bio_hp.htmlhttp://www.saburchill.com/IBbiology/bio_hp.html


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Fig. 3-22

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