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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
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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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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
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Copper grid slides
2007 Paul Billiet ODWS
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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
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Fig. 3-22
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