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E E LECTRON LECTRON M M ICROSCOPY ICROSCOPY Advantages and disadvantages of TE Advantages and disadvantages of TE Advantages and disadvantages of Advantages and disadvantages of SEM SEM Introduction Introduction Principles of operation Principles of operation of TEM of TEM Principles of operation Principles of operation of SEM of SEM Sample preparation for Sample preparation for TEM TEM Sample preparation for Sample preparation for SEM SEM Investigating Investigating micrographs micrographs School of Biological Sciences
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E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Apr 01, 2015

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Page 1: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

EELECTRONLECTRON

MMICROSCOPYICROSCOPY

Advantages and disadvantages of TEMAdvantages and disadvantages of TEM

Advantages and disadvantages of SEMAdvantages and disadvantages of SEM

IntroductionIntroduction

Principles of operation of TEMPrinciples of operation of TEM

Principles of operation of Principles of operation of SEMSEM

Sample preparation for Sample preparation for TEMTEM

Sample preparation for SEMSample preparation for SEM

Investigating micrographsInvestigating micrographsSchool of

BiologicalSciences

Page 2: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

INTRODUCTION

With the invention of the light microscope it was discoveredthat plant and animal tissues were made up of aggregates ofindividual cells. However, light microscopes are limited to approximately x1000 magnification and have poor resolutionresolution..Therefore not all the internal structures of a cell can be seenwith a light microscope.

In 1924 a French physicist by the name of de Broglie stated that a beam of electrons should behave in a similar way to a beam of

light i.e with wave properties the wavelength should be shorter. Therefore an electron

beam should give better resolution.

Page 3: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

RESOLUTIONWhen there is sufficient light, two points 0.2mm apart or

morecan be distinguished with the naked eye as being separate points. When this distance is less than 0.2mm, only one

point is seen. This distance is called the resolving power (or

resolution) of the eye.In other words resolution is the closeness two objects can be

inproximity and still be perceived as two separate objects.

Can’t see two separate objects

Can see two separate objects

Page 4: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

The invention of the electron gunled to the development of theelectron microscope.

The metal tungsten filament is heated to about 2500oC which causes it to release electrons.

Due to the large voltage difference between the

filament and the anode plate the electrons are forced to flow in the direction of the arrow.

The cathode shield increases the electron flow further and concentrates the electrons into a narrow beam. Anode

Electron beam

Cathode

Filament

High voltage generator

Back to principles of operation

Page 5: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

TEM Transmission SEM Scanning

TEM produces a high resolution image of the internal structures of cells. TEM uses the electrons that have passed through the specimen to form an image.

Two main types of electron microscopes;

SEM produces a three dimensional image of the specimen surface. A beam of electrons scans the whole specimen which then emits low energy, secondary electrons. This technique can be used to study whole cells.

Page 6: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

When an electron beam strikes a specimen a number of events occur. Electrons are scattered depending on the nature of the material.

If the electrons hit a dense array they are scattered out of the main beam and fewer electrons will reach the viewing screen. There is no fluorescence and that area appears dark.

If the electrons pass a scarcity of atoms they travel straight through, hitting theviewing screen and causing fluorescence. That area will appear light. The image comes from the arrangement of light and dark patches on the screen.

Page 7: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

PRINCIPLES OF OPERATION OF TEM

Specimen holder

Electron gun

Projection chamber

Page 8: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

THE ELECTRON GUN – producesan electron beam.

THE COLUMN – uses electromagnetic

lenses to control the beam and produce a magnified image

IMAGE VIEWING AND RECORDING

The image is produced on afluorescent screen below which a shutter and camera are located.

Condenser lens

The main components of a TEM are:

Objective lens

Projector lens

Specimen

Fluorescent screen

Vacuum

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Page 9: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Electrons only behave like light when they are manipulated in vacuum. Therefore the whole column is evacuated since atoms such as O2 and CO2 scatter the electrons.

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Page 10: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

SAMPLE PREPARATION FOR TEM

The aims of sample preparation are as follows:

To preserve the material in its natural state

To ensure that the material withstands changes which might occur on exposure to atmosphere, vacuum and electron beam.

Page 11: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Fixation for TEM

The tissue is cut into tiny pieces

It is then placed into fixing solution

Page 12: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Dehydration and embedding of TEM

Tissue is dehydrated in alcohol

It is then placed in a dilute solution of resin embedding media

Specimen vials

Tissue is placed in final embedding mixture and the resin is polymerised in the oven

Page 13: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Section cutting of TEM

Sections are cut on an ultramicrotome with a glass or diamond knife. The sections are floated off the edge of the knife onto the surface of a water trough.

The colour of the sections vary with thickness. When the sections are gold they are picked off the surface with a copper grid.

The section on the copper grid is now ready for staining and viewing in the electron microscope.

3.05mm

Page 14: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Advantages Disadvantages

Thin sections are effectively two dimensional slices of tissue and do not convey the three dimensional arrangement of cellular components

Very good resolution

Can see sub-cellular components and measure them

magnification

Artefacts may be created

TEM

Page 15: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

PRINCIPLES OF PRINCIPLES OF OPERATION OF SEMOPERATION OF SEM

Control panel

Image viewing

Specimen chamber

Electron gun

DetectorDetector

Page 16: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

SEM uses electrons that are emitted from the specimensurface.

The specimen is scanned with a very fine beam of electrons.

These are scattered as they hit high and low points in the specimen.

The scattered electrons are measured by a detector and used to control a second beam which forms an image on a TV screen

Specimen

Vacuum

Detector

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Electron source

Electron beam

T.V. Monitor

Page 17: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Sample preparation for SEM

The preservation used will usually determine which

drying process to use.

There are two basic methods of drying the specimen:

Critical point drying – used after chemical fixation and dehydration

Freeze-drying – used after freezing

Page 18: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Freeze drying for SEM

Then placed in liquid nitrogen to allow easy handling

Sample placed in nitrogen slush to maintain it’s structure

Sample is mounted on a stub

Sample placed in copper holder and……….

…..placed in freeze drier

Page 19: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Critical point drying for SEM

Sample is chemically fixed

Then dehydrated with alcohol

Sample is placed in critical point drier. Here the sample is flushed several times with liquid CO2. The pressure andtemperature is then raised which converts the liquid CO2 to gas. The gas is then vented off slowly.

The sample is removed and mounted on a stub

Page 20: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Coating the specimen for SEM

Most biological specimens are poor conductors and poor

emitters of secondary electrons therefore the surface of the

sample needs to be coated with a thin layer of a conducting material.

There are two ways to do this:

Sputter coating

Evaporation of carbon

Page 21: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Sputter coatingSputter coating for SEMWhen power passes to the anode, the noble metal evaporates (called the plasma effect) and the metal falls onto and coats the specimen.METAMETA

LL

ANODE

CATHODE

A sputter coater

Page 22: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Evaporation of carbon for SEM

Two carbon rods are placed end to end. One of the rods is

sharpened to a point. These are placed in a vacuum and the

specimen is placed below them. When electricity passes

through the carbon rods, the carbon tip evaporates and the

carbon falls onto and coats the specimen.

CARBON RODS

CARBON RODS

Page 23: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Advantages Disadvantages

Provides great depth of focus

Micrographs show a 3D image of specimen

Smaller and simpler in comparison to TEM

Only surface features seen

Resolution attainable is not very high (approx 10nmn)

SEM

Page 24: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Investigating MicrographsTransmission Electron Micrographs Scanning Electron Micrographs

Can you spot the differences between the two types of electron microscopy?

Page 25: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Transmission Electron Micrographs

Mitochondrion

Plasma membrane

Endoplasmic reticulum

Golgi membranes

Virus particles

Section of mammalian cell

Page 26: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Scanning Electron Micrographs

Sample of geranium petal showing the cone shaped projections and the internal structure.

Page 27: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

Measuring MicrographsMagnification of micrograph is X100000 How to work out the size

of an organelle?

Measured size Magnification

80000 m 100000

= 0.8m or 800nm

Measured size = 80mm

Convert to m = 80000

Page 28: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

for using this programme. We hope that it has been useful!

THANKS TO:Chris Gilpin, Ian MillerLes Lockey, Samantha Newby

This programme was developed as part of a work placement project by Sumerah Khan and Sheerin Dariani

Page 29: E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

References

1) B. Schotanus (1980) Electron microscopy, what is it ? Marketing electron optics. Philips Export B.V. Eindhoven.

2) Dr Yvonne Miller (1998) Preparation of specimens for TEM and SEM.

3) Mike Mahon, Chris Gilipin, Ian Miller (2000) Microscopy and analysis University of Manchester - School of Biological Sciences.

4) Sam Newby (2000) Freeze drying and critical point drying EMPGU.

5) Specimen preparation (1991) (21/1/00) http://www.lifelong.com/lifelong-universe/Academic world/SEM/ specimenprep.html pages 1-2.

6) Dr. Ron Butler (1980) Transmission electron microscopy, What an SEM is ?, Aims of specimen preparation and Electron microscopy unit. EMPGU