electron microscopy: lecture 1: introduction to the transmission electron microscope (tem)

Electron Microscopy: Lecture 1: Introduction

to the Transmission Electron Microscope (TEM)

School of Life Sciences, University of SussexSchool of Life Sciences, University of Sussex

Julian ThorpeJulian ThorpeThe Sussex Centre for Advanced MicroscopyThe Sussex Centre for Advanced Microscopy

MSc Imaging in Biomedical Research, October 18th 2011

Why use electron microscopes Why use electron microscopes (EMs)?(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 0.2m)

this is mainly because the effective wavelengths of accelerated electrons are extremely shorter

than those of light

As the name suggests, the electron beam is transmittedtransmitted through the sample

(normally a thin sectionthin section of tissue or cells or a particulate sampleparticulate sample such as viruses or

proteins)

so that the fine structurefine structure of the specimen may be observed (e.g. cellularcellular ultrastructureultrastructure)

Transmission EMsTransmission EMs

An electron beam has An electron beam has wave-like propertieswave-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 EMsEarly development of EMs

1928-1931: Knoll and RuskaKnoll and Ruska, in Berlin, began development of electron lenses

and built a prototype EM

1937: Metropolitan Vickers Company (Manchester, UK) supply first commercial EMfirst 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 7nmresolution of about 7nm is achieved

Early development of EMsEarly development of EMs1948-1953:1948-1953: The The ultramicrotomeultramicrotome was developed, was developed, allowing cutting of ultrathin (60-100nm) sectionsallowing cutting of ultrathin (60-100nm) sections

This was important, as This was important, as electrons have electrons have limited energy and cannot pass through sections limited energy and cannot pass through sections

of more than a few hundred nm of more than a few hundred nm (except for high voltage TEMs)(except for high voltage TEMs)

Gas or water molecules would also obstruct the Gas or water molecules would also obstruct the passage of electrons down the ‘passage of electrons down the ‘columncolumn’ of the TEM, ’ of the TEM,

thus they operate under a thus they operate under a high vacuumhigh vacuum

Effective wavelengths in the TEMEffective wavelengths in the TEM = (1.5/V)1/2 nm

where V = the accelerating voltage of the electron beam

Voltage

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 wavelengthdecreases with

increased acceleratingvoltage

Resolution in the TEMResolution in the TEMResolution (nm) = 0.61 X /N.A.

where N.A. = the numerical aperture of the objective lens

Voltage 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

Resolution improves with increased

acceleratingvoltage (and

associated shortereffective

wavelength)

Resolution in a ‘standard’ TEM vs LMResolution in a ‘standard’ TEM vs LM

N.A. Resolution

100kV TEM 0.0039nm 0.01 0.24nm

UV light 365nm 1.40 159nm

Comparing a ‘standard’ TEM of 100kV accelerating voltagewith a light microscope using UV illumination and optimal

objective lens numerical aperture

Although the wavelength of the illumination source in the TEMis 5 orders of magnitude shorter, numerical apertures of LM

lenses are much greater

Life Sciences TEM: Hitachi-7100Life Sciences TEM: Hitachi-7100(<125kV; resolution = 0.204nm)

A Million Volt TEMA Million Volt TEM(resolution = 0.07nm)

3 Million Volt Hitachi: the most 3 Million Volt Hitachi: the most powerful TEM ever madepowerful TEM ever made (resolution = 0.04nm)

Operator

(resolution = 0.04nm)

3 Million Volt Hitachi: the most 3 Million Volt Hitachi: the most powerful TEM ever madepowerful TEM ever made

The way TEMs are going?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

Optical MicroscopeOptical Microscoperesolution c. 200nmresolution c. 200nm

TEMTEMresolution c. 0.2nmresolution c. 0.2nm

Resolution in a ‘standard’ 100kV TEMResolution in a ‘standard’ 100kV TEM

Resolution in the TEMResolution in the TEM

500nm


200nm


100nm

The electron beam sourceThe electron beam source

TheThe electron beam electron beam is routinely derived from a thin hairpin filament of tungsten wire housed in a gun assembly

A high accelerating voltageaccelerating voltage is used to boil electrons off the tip of the tungsten wire by thermionic thermionic emissionemission and these are fired down the columncolumn of the EM and focused by electromagnetic lenseselectromagnetic lenses

The electron sourceThe electron source(Cathode Gun Assembly)(Cathode Gun Assembly)

Figure c/o: http://en.wikipedia.org/wiki/Transmission_electron_microscopy

The electron sourceThe electron source


A high acceleratingA high acceleratingvoltage voltage is supplied is supplied to the filamentto the filament



Electrons Electrons areareboiled off the tipboiled off the tipof the filament byof the filament by‘‘thermionic emission’thermionic emission’



TheThe ‘‘Wehnelt cylinder’Wehnelt cylinder’has a higher –vehas a higher –vecharge than the charge than the filamentfilament



TheThe ‘‘Wehnelt cylinder’Wehnelt cylinder’has a higher –vehas a higher –vecharge than the charge than the filament…….filament…….and thus and thus focuses thefocuses theelectronselectrons



ElectronsElectrons areareattracted to theattracted to thepositively chargedpositively charged‘‘anode plate’ anode plate’ andandpass through anpass through anaperture within itaperture within it

Cross-Cross-SectionSectionthrough through

thetheColumn of Column of

a TEMa TEM(side (side view)view)

The TEM ‘column’TEM ‘column’ stands vertically with

a cathode guncathode gun assembly at the top

housing the tungsten filament

Electrons are boiled off the tip of this

filament by ‘thermionic emission’‘thermionic emission’ when a high voltagehigh voltage

is applied





Beneath this is an anode plateanode plate, to which

electrons are attracted and an

aperture allows their passage down the

TEM columnTEM column





The TEM columnTEM column is maintained under a

high vacuumhigh vacuum as electrons have insufficient energy to pass through gas and

water molecules


This vacuum is achievedusually via oil diffusionpumps, backed up byrotary pumps




A series of lead-shrouded and water-cooled

electromagnetic electromagnetic lenseslenses make up the

bulk of theTEM column





Condenser lensesCondenser lenses condense and focus

the electrons onto the area of the specimen

being examined





AnAn objective lens objective lens surrounding the surrounding the

specimen insertion specimen insertion area primarily area primarily

focusesfocusesand initially magnifiesand initially magnifies

the imagethe image





IntermediateIntermediate and projector lenses

magnify and project the focused image

onto the fluorescent fluorescent screenscreen (converts

electrons to photons) at the base of the

column or to a CCD cameracamera beneath that


Transmission EMsTransmission EMsElectromagnetic lens defects

are similar to those of optical lenses…

…and these detract from achievement of the maximumtheoretical resolution. They are:

1. Spherical aberration:

Electrons passing through the lens periphery are refracted more than those passing along the lens axisand therefore do not have the same focal point.

Apertures are used in the TEM to limit the peripheral electrons and minimise this aberration



2. Chromatic aberration: electrons of different energiesconverge at different focal points and this is essentiallyequivalent to chromatic aberration in light microscopy

This can be minimised by:• increasing the accelerating voltage• an improved vacuum• use of the thinnest possible specimen



3. Astigmatism: occurs when the field within the electromagnetic lens is not perfectly symmetrical. Can be due to imperfect boring of the lens polepiecesor contamination of the column, specimen or apertures

.....TEMs have astigmatism controls to correct for this

Preparation of biological Preparation of biological samples for TEMsamples for TEM

• FixationFixation: : ‘Greater care’ needed for samples ‘Greater care’ needed for samples prepared for TEM, owing to the improved prepared for TEM, owing to the improved resolution and the higher magnifications resolution and the higher magnifications possiblepossible

• Samples have to be dry* Samples have to be dry* (as the TEM operates under a high vacuum): therefore samples are dehydrated therefore samples are dehydrated ((** the exception to this are frozen-hydrated samples viewed by the exception to this are frozen-hydrated samples viewed by cryo-TEM)cryo-TEM)

• Samples have to be ultrathinSamples have to be ultrathin: this is because : this is because of the limited energy of the electron beam of the limited energy of the electron beam Therefore: Therefore: • special resins designed to allow cutting of ultrathin special resins designed to allow cutting of ultrathin

(c.50-100nm) sections are used to infiltrate and (c.50-100nm) sections are used to infiltrate and ‘embed’ samples‘embed’ samples

• Or Or small particulates small particulates may be viewed (after air-drying)may be viewed (after air-drying)

• FixationFixation: : Normally a double-fixation in buffered Normally a double-fixation in buffered glutaraldehyde glutaraldehyde (c.2-5%; cross-links proteins) (c.2-5%; cross-links proteins) and and subsequently osmium tetroxide subsequently osmium tetroxide (1%; imparts (1%; imparts electron-density to lipidic components)electron-density to lipidic components)

• DehydrateDehydrate: in an ethanol series: in an ethanol series

• Resin embeddingResin embedding: infiltration with epoxy resin : infiltration with epoxy resin for a few days and heat-polymerisedfor a few days and heat-polymerised

• Thin sectioningThin sectioning: must be ‘ultrathin’ to allow : must be ‘ultrathin’ to allow the electron beam to transmit through the the electron beam to transmit through the sectionsection

Preparation of biological Preparation of biological samples for TEMsamples for TEM

• Sections of c. 60-100nm are cut on an Sections of c. 60-100nm are cut on an ultramicrotome and collected on TEM ultramicrotome and collected on TEM support ‘grids’support ‘grids’

<<< <<< 3mm >>>3mm >>>

Thin sectioning/’Ultramicrotomy’ Thin sectioning/’Ultramicrotomy’

Contrast in thin sections examined in the TEM is Contrast in thin sections examined in the TEM is facilitated by the use of facilitated by the use of heavy metalsheavy metals that can that can occlude and absorb electrons. These are routinely: occlude and absorb electrons. These are routinely:

• Osmium tetroxideOsmium tetroxide: when used as a secondary : when used as a secondary

fixative imparts electron-density to the lipidic fixative imparts electron-density to the lipidic component, especially membranescomponent, especially membranes

• UranylUranyl acetate acetate and and leadlead citrate citrate: are used as ‘post-: are used as ‘post-stains’. The former binds nucleic acids and proteins stains’. The former binds nucleic acids and proteins and the latter subsequently enhances the contrastand the latter subsequently enhances the contrast

Staining to Achieve Contrast Staining to Achieve Contrast

‘‘Negative staining’ of Negative staining’ of particulate samplesparticulate samples

• A very simple but effective method to examine A very simple but effective method to examine particulate samplesparticulate samples at high resolution at high resolution

• A drop of the sample is aliquotted onto a coated TEM A drop of the sample is aliquotted onto a coated TEM support gridsupport grid

• A drop of A drop of heavy metal stainheavy metal stain (e.g. uranyl acetate) is (e.g. uranyl acetate) is then dropped onto the sample and allowed to dry then dropped onto the sample and allowed to dry down around itdown around it

• When viewed under the TEM the sample appears to be When viewed under the TEM the sample appears to be negatively-stainednegatively-stained as the heavy metal creates an as the heavy metal creates an electron-dense backgroundelectron-dense background

Negative Stainingbacteriophage

Life Sciences TEM: Hitachi-7100Life Sciences TEM: Hitachi-7100(<125kV; resolution = 0.204nm)

Electron gun regionElectron gun region

TEM columnTEM column

Specimen airlockSpecimen airlock

High voltage High voltage supply cablesupply cable

Viewing screen areaViewing screen areaplus binocularsplus binoculars

Hitachi-7100 TEMHitachi-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 formationTEM 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)


Elastically scatteredElastically scattered electrons (by the nucleus of an atom)contribute mostly toimage contrast contrast


Inelastically scatteredInelastically scattered electrons are concentrated withinsmaller scattering angles


UnscatteredUnscattered (or transmitted) electrons will pass throughthe specimen to form theelectron-lucent regions ofthe image


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 aperturesin the objective lens, but at the expense of some lowering

of the resolution


Or CCD camera

Objective lens apertureObjective lens aperture

Condenser lens apertureCondenser lens aperture

TEM imagingTEM imaging

CCD CCD cameracamera

Gatan Ultrascan 1000 CCD CameraGatan Ultrascan 1000 CCD Camera


Specifications:

• CCD active area 28.7mm X 28.7mm• 2048 X 2048 pixels (14m 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


Camera workstation alongside TEM

Camera controller(with low noise electronicsand high speed read-out at4 megapixels per second)

TEM imagingTEM imaging

TEM imaging to reveal ‘ultrastructure’TEM imaging to reveal ‘ultrastructure’ of cells & particulatesof cells & particulates

Useful LinksUseful 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):

VideosVideos

Structure and Function of the TEM

The TEM: part 1

The TEM: part 2

electron microscopy: lecture 1: introduction to the transmission electron microscope (tem)

Documents