Transcript microscopy

ELECTRON
MICROSCOPY
Introduction
Principles of operation of TEM
Sample preparation for TEM
Advantages and disadvantages of TEM
Principles of operation of SEM
Sample preparation for SEM
Advantages and disadvantages of SEM
School of
Biological
Sciences
Investigating micrographs
INTRODUCTION
With the invention of the light microscope it was discovered
that plant and animal tissues were made up of aggregates of
individual cells. However, light microscopes are limited to
approximately x1000 magnification and have poor resolution.
Therefore not all the internal structures of a cell can be seen
with 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.
RESOLUTION
When there is sufficient light, two points 0.2mm apart or more
can 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 in
proximity and still be perceived as two separate objects.
Can’t see two
separate
objects
Can see two
separate
objects
The invention of the electron gun
led to the development of the
electron microscope.
Back to principles of operation
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.
Filament
Cathode
Electron beam
Anode
High
voltage
generator
Two main types of electron microscopes;
TEM Transmission
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.
SEM Scanning
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.
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 the
viewing screen and causing fluorescence.
That area will appear light. The image
comes from the arrangement of light
and dark patches on the screen.
PRINCIPLES OF
OPERATION OF TEM
Electron gun
Specimen holder
Projection chamber
The main components of a TEM are:
THE ELECTRON GUN – produces
an 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 a
fluorescent screen below which a
shutter and camera are located.
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Condenser lens
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Vacuum
Fluorescent
screen
Specimen
Objective lens
Projector lens
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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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.
Fixation for TEM
The tissue is cut into tiny pieces
It is then placed into fixing solution
Dehydration and embedding of TEM
Tissue is placed in final
embedding mixture and the
resin is polymerised in the
oven
Tissue is
dehydrated in
alcohol
It is then placed in a
dilute solution of resin
embedding media
Specimen vials
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
Advantages
TEM
Very good resolution
magnification
Can see sub-cellular
components and measure
them
Disadvantages
Thin sections are
effectively two dimensional
slices of tissue and do not
convey the three
dimensional arrangement of
cellular components
Artefacts may be created
PRINCIPLES OF
OPERATION OF SEM
Electron gun
Detector
Detector
Image viewing
Specimen chamber
Control panel
SEM uses electrons that are
emitted from the specimen
surface.
The specimen is scanned with
a very fine beam of electrons.
Electron source
Electron beam
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
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T.V. Monitor
Detector
Vacuum
Sample preparation for SEM
The preservation used will usually determine which
drying process to use.
There are two basic methods of drying the specimen:
Freeze-drying
– used after freezing
Critical point drying
– used after chemical fixation and dehydration
Freeze drying for SEM
Sample placed in
nitrogen slush to
maintain it’s
structure
Then placed in liquid
nitrogen to allow easy
handling
…..placed in
freeze drier
Sample placed
in copper holder
and……….
Sample is
mounted
on a stub
Critical point drying for SEM
Sample is
chemically fixed
Sample is placed in
critical point drier.
Here the sample is
flushed several times
with liquid CO2. The
pressure and
temperature is then raised which converts
the liquid CO2 to gas. The gas is then vented
off slowly.
Then dehydrated
with alcohol
The sample is
removed and
mounted on a stub
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
Sputter coating for SEM
ANODE
METAL
CATHODE
When power passes to the
anode, the noble metal
evaporates (called the plasma
effect) and the metal falls
onto and coats the specimen.
A sputter coater
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
Advantages
Disadvantages
SEM
Provides great depth of
focus
Only surface features seen
Micrographs show a 3D
image of specimen
Resolution attainable is not
very high (approx 10nmn)
Smaller and simpler in
comparison to TEM
Investigating Micrographs
Transmission Electron Micrographs
Scanning Electron Micrographs
Can you spot the differences between the two types of electron microscopy?
Transmission Electron Micrographs
Virus particles
Plasma membrane
Endoplasmic
reticulum
Golgi membranes
Mitochondrion
Section of mammalian cell
Scanning Electron Micrographs
Sample of geranium petal showing
the cone shaped projections and
the internal structure.
Measuring Micrographs
How to work out the size
of an organelle?
Measured size
Magnification
Measured size = 80mm
Convert to m = 80000
 80000 m
100000
= 0.8m or 800nm
Magnification of micrograph is X100000
for using this programme. We hope that it has
been useful!
This programme was developed as part
of a work placement project by
Sumerah Khan and Sheerin Dariani
THANKS TO:
Chris Gilpin, Ian Miller
Les Lockey, Samantha Newby
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/lifelonguniverse/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