electron microscope

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Introduction

• Electron Microscope– Any class of

microscopes that use electrons instead of light to form images of very small objects such as individual parts of small living things

Electron Microscope

Uses a magnetic field to bend beams of electrons;– greater magnification

& resolving power than light microscope

The two types are: Scanning and Transmission

Which is the most powerful kind of microscope?

THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

fluorescent (TV) screen,photographic film

Human eye (retina), photographic film

Focussing screen

VacuumAir-filledInterior

MagnetsGlassLenses

High voltage (50kV) tungsten lamp

Tungsten or quartz halogen lamp

Radiation source

x500 000x1000 – x1500Maximum magnification

0.2nmFine detail

app. 200nmMaximum resolving power

Electronsapp. 4nm

Monochrome

Visible light760nm (red) – 390nm

Colours visible

Electromagnetic spectrum used

ELECTRON MICROSCOPELIGHT MICROSCOPEFEATURE

THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

Copper gridGlass slideSupport

Heavy metalsWater soluble dyesStains

Microtome only.Slices 50nm

Parts of cells visible

Hand or microtomeslices 20 000nmWhole cells visible

Sectioning

ResinWaxEmbedding

OsO4 or KMnO4AlcoholFixation

Tissues must be dehydrated

= dead

Temporary mounts living or dead

Preparation of specimens

ELECTRON MICROSCOPE

LIGHT MICROSCOPEFEATURE

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

particles• When the atoms of metal are excited by

sufficient energy in the form of heat, the electron leave their orbit, fly off from space & are lost in atoms

• Metal tungsten is commonly used as a source of electron

• 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 in image formation

• Electron interact with the atoms of the biological specimens to form the image

Electron beam

(A)

Transmitted electron

(B)Inelastically scatteredelectrons

(C)

Elastically scatteredelectrons

(D)

Back-scattered electrons

(E)Secondary electrons

X-rays

Visible light

1. Transmitted electrons (A) of the beam passes straight through the specimen on to the screen

2. Some electron (B) of the beam lose a bit of their energy while passing through the specimen & get deflected a little from their original axis of the beam inelastically scattered electrons

3. Some electron (c) interact with atoms of specimen & get elastically scattered without losing energy. Electron deviate widely

4. Some electron (D) get backscattered instead of getting transmitted through the specimen

5. In some cases the electrons get absorbed by the atoms of the specimen & instead low energy electron (E) are emitted. These electron are termed secondary electron. These are very useful for forming the image in the SEM

6. Some atom emit x-ray & light energy

Working & Image formation• Working of EM is based on same plan as that of light microscope

• Electron are used for magnification & image formation

• Image formation occurs by electron scattering

• Electron strike the atomic nuclei & get dispersed

•This dispersed electron form image

• The electron image is converted in to visible form by projecting on a fluorescent screen

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

• They get scattered & transmitted through the object & 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 visible light through excitation of the chemical coating of the screen

• Those electron which reach the fluorescent screen form bright spot while the area where the electron do not reach the screen form dark spot

• The varying degree of intensity of electrons form the image with varying degree of grey.

• Electron scattering, however, is due to the atomic nuclei which consist of protons & neutrons

• Higher the atomic number, greater the scattering

• Since biological materials generally have a low atomic number, the dispersion is poor

• Very poor dispersion means very poor contrast 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

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

Resolution

• The resolving power of a microscope is limited by wavelength of illumination forming the image

• Shorter wavelength, smaller detail can be resolved

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

Transmission Electron Microscope (TEM)

• Electrons are passed through very thin specimens to see what is inside!

• Invented in1933 • Magnification is

500,000x

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• The TEM is a complex viewing system equipped with a set of electromagnetic lenses used to control the imaging electrons in order to generate the extremely fine structural details that are usually recorded on photographic film.

• Since the illuminating electrons pass through the specimens, the information is said to be a transmitted image.

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

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Basic Systems Making Up a Transmission Electron Microscope

• The transmission electron microscope is made up of a number of different systems that are integrated to form one functional unit capable of orienting and imaging extremely thin specimens.

• The illuminating system consists of the electron gun and condenser lenses that give rise to and control the amount of radiation striking the specimen.

• A specimen manipulation system composed of the

specimen stage, specimen holders, and related hardware is necessary for orienting the thin specimen outside and inside the microscope.

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Basic Systems Making Up a Transmission Electron Microscope

• The imaging system includes the objective, intermediate, and projector lenses that are involved in forming, focusing, and magnifying the image on the viewing screen as well as the camera that is used to record the image.

• A vacuum system is necessary to remove interfering air molecules from the column of the electron microscope. In the descriptions that follow, the systems will be considered from the top of the microscope to the bottom.

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TABLE 6.3 Major Column Components of the TEM*

Component Synonyms Function of Components

Illumination System Electron Gun Gun, Source Generates electrons and provides first

coherent crossover of electron beam

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

Condenser Lens 2 C2, Brightness Varies amount of illumination on specimen—in combination with C1 (see Brightness in Table 6.4)

Condenser Aperture

C2 Aperture Reduces spherical aberration, helps control amount of illumination striking specimen

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Specimen Manipulation System

Specimen Exchanger Specimen Air Lock Chamber and mechanism for inserting specimen holder

Specimen Stage Stage Mechanism for moving specimen inside column of microscope

Imaging System Objective Lens — Forms, magnifies, and focuses first image (see

Focus in Table 6.4)

Objective Aperture — Controls contrast and spherical aberration

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

Intermediate Aperture Diffraction Aperture, Field Limiting Aperture

Selects area to be diffracted

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

Projector Lens 2 P2 Same as P1

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Observation and Camera Systems

Viewing Chamber

— Contains viewing screen for final image

Binocular Microscope

Focusing Scope Magnifies image on viewing screen for accurate focusing

Camera — Contains film for recording

Scanning Electron Microscopy (SEM) Visualizes Surface Features

• Extremely useful in studying surface structure

• The electron do not form the image by being transmitted, but by getting emitted from the surface of specimen

• Illuminating system of SEM similar to TEM

• The electron beam is, how ever compressed with 1 or more condenser coils, which result in the formation of an narrow pencil of electron

• The electron which falls on specimen are primary electron

• The electron probe is focused on the surface of 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

• The three dimensional image in SEM is formed by secondary electron

• But these electron does not have sufficient energy to excite the fluorescent screen and form the image as in TEM by primary electron

• In SEM secondary electron are first collected, amplified & then used for the formation of an image on the phosphor screen of cathode ray tube

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

Specimen Preparation

Specimens are coated with metals to deflect electrons from a beam scanned across the sample.

SEM of Stereocilia Projecting from a Cochlear (inner ear) Hair Cell

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

A microtome cutting sections of an embedded sample.

Fig. 3-22

electron microscope

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