Transcript Introduction to TEM - School of Life Sciences

Electron Microscopy:
Lecture 1: Introduction
to the Transmission
Electron Microscope (TEM)
MSc Imaging in Biomedical Research, October 18th 2011
Julian Thorpe
The Sussex Centre for Advanced Microscopy
School of Life Sciences, University of Sussex
Why use electron microscopes (EMs)?
Transmission electron microscopes
(TEMs) utilise electrons as
their source of illumination
which gives much improved resolution over a light
microscope (around a thousand-fold better: c. 0.2nm
compared with 0.2mm)
this is mainly because the effective wavelengths
of accelerated electrons are extremely shorter
than those of light
Transmission EMs
As the name suggests, the electron beam is
transmitted through the sample
(normally a thin section of tissue or cells or a
particulate sample such as viruses or proteins)
so that the fine structure of the specimen may
be observed (e.g. cellular ultrastructure)
An electron beam has
wave-like properties
In 1923 de Broglie showed that an
electron beam has wave-like properties……
……thus pointing the way forward to
the possible development
of electron microscopes
Early development of EMs
1928-1931: Knoll and Ruska, in Berlin,
began development of electron lenses
and built a prototype EM
1937: Metropolitan Vickers Company
(Manchester, UK) supply first commercial EM (to
Louis Martin at Imperial College, London), but its resolution
was no better than that of a LM
Late 1930s: a resolution of about 7nm is achieved
Early development of EMs
1948-1953: The ultramicrotome was developed,
allowing cutting of ultrathin (60-100nm) sections
This was important, as electrons have
limited energy and cannot pass through sections
of more than a few hundred nm (except for high voltage TEMs)
Gas or water molecules would also obstruct the
passage of electrons down the ‘column’ of the TEM,
thus they operate under a high vacuum
Effective wavelengths in the TEM
l = (1.5/V)1/2 nm
where V = the accelerating voltage of the electron beam
Voltage
l
25,000 0.0077nm
50,000 0.0055nm
75,000 0.0045nm
100,000 0.0039nm
200,000 0.0027nm
1,000,000 0.0012nm
3,000,000 0.0007nm
Effective wavelength
decreases with
increased accelerating
voltage
Resolution in the TEM
Resolution (nm) = 0.61 X
l /N.A.
where N.A. = the numerical aperture of the objective lens
l
N.A.
Resolution
25,000 0.0077nm
0.01
0.47nm
50,000 0.0055nm
0.01
0.33nm
75,000 0.0045nm
0.01
0.27nm
100,000 0.0039nm
0.01
0.24nm
200,000 0.0027nm
0.01
0.17nm
1,000,000 0.0012nm
0.01
0.07nm
3,000,000 0.0007nm
0.01
0.04nm
Voltage
Resolution improves
with increased
accelerating
voltage (and
associated shorter
effective
wavelength)
Resolution in a ‘standard’ TEM vs LM
Comparing a ‘standard’ TEM of 100kV accelerating voltage
with a light microscope using UV illumination and optimal
objective lens numerical aperture
l
100kV TEM 0.0039nm
UV light
365nm
N.A.
Resolution
0.01
0.24nm
1.40
159nm
Although the wavelength of the illumination source in the TEM
is 5 orders of magnitude shorter, numerical apertures of LM
lenses are much greater
Life Sciences TEM: Hitachi-7100
(<125kV; resolution = 0.204nm)
A Million Volt TEM (resolution = 0.07nm)
3 Million Volt Hitachi: the most
powerful TEM ever made (resolution = 0.04nm)
3 Million Volt Hitachi: the most
powerful TEM ever made(resolution = 0.04nm)
Operator
The way TEMs are going?
HT7700
120 kV
biomedical
TEM from
Hitachi
100%
integration of
all functions
into the
graphical user
interface
http://www.youtube.com/watch?v=h6VkvseFkzQ
Resolution in a ‘standard’ 100kV TEM
Optical Microscope
resolution c. 200nm
TEM
resolution c. 0.2nm
Resolution in the TEM
500nm
Resolution in the TEM
200nm
Resolution in the TEM
100nm
The electron beam source
The electron beam is routinely derived from a thin hairpin
filament of tungsten wire housed in a gun assembly
A high accelerating voltage is used to boil electrons off
the tip of the tungsten wire by thermionic emission and
these are fired down the column of the EM and
focused by electromagnetic lenses
The electron source
(Cathode Gun Assembly)
Figure c/o: http://en.wikipedia.org/wiki/Transmission_electron_microscopy
The electron source
A high accelerating
voltage is supplied
to the filament
Figure c/o: http://en.wikipedia.org/wiki/Transmission_electron_microscopy
The electron source
Electrons are
boiled off the tip
of the filament by
‘thermionic emission’
Figure c/o: http://en.wikipedia.org/wiki/Transmission_electron_microscopy
The electron source
The
‘Wehnelt cylinder’
has a higher –ve
charge than the
filament
Figure c/o: http://en.wikipedia.org/wiki/Transmission_electron_microscopy
The electron source
The
‘Wehnelt cylinder’
has a higher –ve
charge than the
filament…….
and thus
focuses the
electrons
Figure c/o: http://en.wikipedia.org/wiki/Transmission_electron_microscopy
The electron source
Electrons are
attracted to the
positively charged
‘anode plate’ and
pass through an
aperture within it
Figure c/o: http://en.wikipedia.org/wiki/Transmission_electron_microscopy
Transmission EMs
CrossSection
through
the
Column of
a TEM
(side view)
The TEM ‘column’
stands vertically with a
cathode gun assembly
at the top housing the
tungsten filament
Electrons are boiled
off the tip of this
filament by ‘thermionic
emission’ when a high
voltage is applied
Transmission EMs
CrossSection
through
the
Column of
a TEM
(side view)
Beneath this is an
anode plate, to which
electrons are attracted
and an aperture allows
their passage down the
TEM column
Transmission EMs
CrossSection
through
the
Column of
a TEM
(side view)
The TEM column
is maintained under a high
vacuum as electrons have
insufficient energy to pass
through gas and water
molecules
This vacuum is achieved
usually via oil diffusion
pumps, backed up by
rotary pumps
Transmission EMs
CrossSection
through
the
Column of
a TEM
(side view)
A series of leadshrouded and
water-cooled
electromagnetic lenses
make up the
bulk of the
TEM column
Transmission EMs
CrossSection
through
the
Column of
a TEM
(side view)
Condenser lenses
condense and focus the
electrons onto the
area of the specimen
being examined
Transmission EMs
CrossSection
through
the
Column of
a TEM
(side view)
An objective lens
surrounding the
specimen insertion area
primarily focuses
and initially magnifies
the image
Transmission EMs
CrossSection
through
the
Column of
a TEM
(side view)
Intermediate and
projector lenses
magnify and project
the focused image onto
the fluorescent screen
(converts electrons to
photons) at the base of
the column or to a CCD
camera beneath that
Transmission EMs
Electromagnetic lens defects
are similar to those of optical lenses…
…and these detract from achievement of the maximum
theoretical resolution. They are:
1. Spherical aberration:
Electrons passing through the lens periphery are
refracted more than those passing along the lens axis
and therefore do not have the same focal point.
Apertures are used in the TEM to limit the peripheral
electrons and minimise this aberration
Transmission EMs
Electromagnetic lens defects
are similar to those of optical lenses…
2. Chromatic aberration: electrons of different energies
converge at different focal points and this is essentially
equivalent to chromatic aberration in light microscopy
This can be minimised by:
• increasing the accelerating voltage
• an improved vacuum
• use of the thinnest possible specimen
Transmission EMs
Electromagnetic lens defects
are similar to those of optical lenses…
3. Astigmatism: occurs when the field within the
electromagnetic lens is not perfectly symmetrical.
Can be due to imperfect boring of the lens polepieces
or contamination of the column, specimen or apertures
.....TEMs have astigmatism controls to correct for this
Preparation of biological
samples for TEM
• Fixation: ‘Greater care’ needed for samples
prepared for TEM, owing to the improved resolution
and the higher magnifications possible
• Samples have to be dry* (as the TEM operates under a high
vacuum): therefore samples are dehydrated (* the
exception to this are frozen-hydrated samples viewed by cryo-TEM)
• Samples have to be ultrathin: this is because of
the limited energy of the electron beam
Therefore:
• special resins designed to allow cutting of ultrathin (c.50100nm) sections are used to infiltrate and ‘embed’ samples
• Or small particulates may be viewed (after air-drying)
Preparation of biological
samples for TEM
• Fixation: Normally a double-fixation in buffered
glutaraldehyde (c.2-5%; cross-links proteins) and
subsequently osmium tetroxide (1%; imparts electrondensity to lipidic components)
• Dehydrate: in an ethanol series
• Resin embedding: infiltration with epoxy resin for a
few days and heat-polymerised
• Thin sectioning: must be ‘ultrathin’ to allow the
electron beam to transmit through the section
Thin sectioning/’Ultramicrotomy’
• Sections of c. 60-100nm are cut on an
ultramicrotome and collected on TEM support
‘grids’
<<< 3mm >>>
Staining to Achieve Contrast
Contrast in thin sections examined in the TEM is
facilitated by the use of heavy metals that can occlude
and absorb electrons. These are routinely:
• Osmium tetroxide: when used as a secondary fixative
imparts electron-density to the lipidic component,
especially membranes
• Uranyl acetate and lead citrate: are used as ‘poststains’. The former binds nucleic acids and proteins and
the latter subsequently enhances the contrast
‘Negative staining’ of
particulate samples
• A very simple but effective method to examine
particulate samples at high resolution
• A drop of the sample is aliquotted onto a coated
TEM support grid
• A drop of heavy metal stain (e.g. uranyl acetate) is
then dropped onto the sample and allowed to dry
down around it
• When viewed under the TEM the sample appears
to be negatively-stained as the heavy metal
creates an electron-dense background
Negative Staining
bacteriophage
Life Sciences TEM: Hitachi-7100
(<125kV; resolution = 0.204nm)
High voltage
supply cable
Electron gun region
TEM column
Specimen airlock
Viewing screen area
plus binoculars
Hitachi-7100 TEM
Specifications:
• Accelerating voltage range: 25 – 125kV
• Magnification ranges:
• 50 – 1,000X in ‘low mag’ mode
• 1,000 – 600,000X in ‘zoom’ mode
• Resolution: 0.204nm (lattice)/0.45nm (particle)
• Motorized and tilting specimen stage with ‘memorise’
and ‘relocate’ specimen positions facilities
TEM image formation
Image and contrast formation results from electrons
that are:
• non-transmitted
(occluded by the heavy metal stains)
• scattered
(elastic and inelastic)
• unscattered
(transmitted)
TEM image formation
Elastically scattered electrons
(by the nucleus of an atom)
contribute mostly to
image contrast
TEM image formation
Inelastically scattered electrons
are concentrated within
smaller scattering angles
TEM image formation
Unscattered (or transmitted)
electrons will pass through
the specimen to form the
electron-lucent regions of
the image
TEM image formation
Higher accelerating voltages result in:
• Increased electron speed and a concomitant decrease
in the incidence of inelastic scattering
• So, although resolution is improved (because of the
shorter effective wavelengths), contrast is lowered
This can be redressed by the use of smaller apertures
in the objective lens, but at the expense of some lowering
of the resolution
TEM image formation
Condenser lens aperture
Objective lens aperture
Or CCD camera
TEM imaging
CCD
camera
Gatan Ultrascan 1000 CCD Camera
Gatan Ultrascan 1000 CCD Camera
Specifications:
• CCD active area 28.7mm X 28.7mm
• 2048 X 2048 pixels (14mm each)
• 16-bit digitization
• Binning 1,2,3,4,6 and 8X
• CCD readout: full or sub area
• Readout speed: 4MPix/sec (4-port
parallel)
• Scintillator: standard phosphor
• Coupling: fibre optic (1:1)
• Mounting position: on axis bottom port
• Peltier cooling –25deg C regulated
Gatan Ultrascan 1000 CCD Camera
Camera workstation
alongside TEM
Camera controller
(with low noise electronics
and high speed read-out at
4 megapixels per second)
TEM imaging
TEM imaging
TEM imaging to reveal ‘ultrastructure’
of cells & particulates
Useful Links
My TEM Website (includes information on sample preparation,
methodologies, TEM instructions, image galleries, etc.):
University of Iowa ‘Central Microscopy Research Facility’
(excellent site for background on TEM):
University of Liverpool ‘Matter’ (excellent site for background on
electron optics, with many interactive features):
Reimer and Kohl (2008) (online book ‘Transmission electron microscopy:
physics of image formation’)
University of Georgia ‘Centre for Advanced Ultrastructural
Research’ (excellent powerpoint on intermediate to high voltage TEM):
Videos
Structure and Function of the TEM
The TEM: part 1
The TEM: part 2