2Overview Systems of the TEM Illumination Condenser lens system Specimen manipulation stageImaging systemImage formationMagnificationRecording system
3Reading List Practical Electron Microscopy for Biologists. G. A. Meek. A copy of the Willams book is available as an online version through the Penn State catalog.The Bozzola there is a version two physical versions in the library.Practical Electron Microscopy for Biologists. G. A. Meek.
4The Transmission Electron Microscope Bizzola Electron Microscopy 1999
6Illumination System Thermionic Emission Source Electron source or gunProvides first coherent crossover of electron beamHigh voltage leading to filament- heated metal wire (cathode)Tungsten or Lab6 (lanthanum hexaboride)Work functionEnergy necessary to bring aboutelectron emissionElectrons would have no orderWehnelt CapElectrode that shapes and controls emissionNegative relative to filamentAnodePositive respective to filamentSystem is situated on top of the instrument column and consists of high voltage, and tungsten filament typically as the source of electrons. A certain amount of energy must put into the system to cause the electrons to leave Fermi level of the filament material (thermionic emission).Works in tandem with the Condenser system to collect and direct the illuminating electrons.All Metals contain positive ions and free electrons, must use temperature increase to increase kinetic energy and escape the Fermi level(measure of energy of least tightly held electrons), the Fermi level changes as a metal heats up Needing an amount of “work” to do this.When applying a fixed amount of negative high voltage kV, DC current runs through the filament to heat overcoming the work function of Tungsten which has an excellent yield of free electrons that are strongly attracted to the positive ions. just below its rather high melting point of 3,653 K. the filament will eventually melt and evaporate with diminishing filament life.LAB6 filaments have a lower work function than tungsten and operates more efficiently with an increase in brightnessPotential differences guide and shape what would otherwise be a cloud of electrons starting with the wehnelt cap which is a shield that is negative respective to filament repelling some electrons by otherwise guiding the accelerating electrons down to an apertured disc known as the annode with it’s positive chargeElectrostatic lenses have a positive and negatively charged surge to attract or repel so that electrons can be focused.Wikipedia
7Source Types Tungsten Lab6 Field Emission Gun Wire filament Lanthanum hexaboridecrystalField Emission GunTungsten tipThermionic (Schotkky) and ColdThe important properties of electron sources are their brightness, temporal coherency, energy spread, spatial coherency and stability. A smaller source size gives higher brightness value b and better spatial coherency, but less stability (harder to maintain, costly). (EMBook 77)
8Field Emission SourceAllows for emission of electrons from fine tip via charge differential of tip and anodes.BrighterMore coherent sourceAtomic diameter point source is future of FE gunsField emission guns operate on a much simpler principle than thermionic. There is a fine tipped usually Tungsten filament followed by two anodes. The first creates a positive charge with respect to the tip and allows the electrons to gain energy to escape the Fermi level and tunnel out of the point. No heat required just the charge difference is enough. The second anode accelerates the electrons to their first concentrated crossover point.100nm tip on the filamentSingle atom tips with brightness values on the order x times brighter and more coherent than current FEGs
9Coherent or Incoherent Waves have same wavelength and phase, in the ideal sense would be perfectly coherentIncoherentWaves have modulating phase relationships and wavelengthsTemporal coherencyWavelength differentialSpatial coherencySize of sourceField emission guns are said to be more coherent. Coherent vs. incoherence is classically defined asCoherent source of waves have same wavelength and phase, incoherent source would have different wavelengths and phase.In electron microscopy, spatial coherency is related to the size of the electron source.\ Perfect spatial coherence would imply that the electrons were all emanating from the same point at the source. Small source the higher the spatial coherency as well as higher the brightness. More instances for the source waves to be out of phase.The more coherent the beam the better the quality of the phase contrast in the image formationThe ideal source would be a single atom of e.g. Tungsten and the electron beam would be tunneled out of that single atom source. They are producing scopes with this capability now.Temporal coherency is a way of describing how well the electrons are in step with one another. Ideal source would emit electrons of all same wavelength to be coherent, typically with a stable beam and voltage this value is not as limiting as spatial coherency.In the equation the coherence of lambda, is the velocity of electrons times h (planks constant) divided by the energy spread of the source, the lower the energy spread the better the coherence.So this also leads to a term used a lot and that is the point spread function which is measurement of the airy disc phenomena or degree of blurring in the point source the more coherent the better the psf, the lower value of psf will determine the resolving power of the imaging system. Better source = better information.When thinking about how the emission guns work (thermionic and field) the source for starters is smaller and more pointed in a FE gun therefor from the beginning there is less opportunity for waves of differing energies from exiting the source. W filaments are usually pointed, where as the FE guns are very finely pointed. (cost issue) (vacuum issue)Temporal coherence length= product of velocity and planks constant divided by the energy spreadSpatial coherence length= wavelength divide by 2 times the alpha (angle formed from source at at the specimen)
10Comparing the Electron Sources Brightness here is really a value of intensity. When we say the beam is brighter it’s actually a more intense source.To summarize: the important properties of electron sources are their brightness, temporal coherency, energy spread, spatial coherency and stability. A smaller source size gives higher b and better spatial coherency, but less stability. (EMBOOk 77)Energy spread the tail of the Fermi distribution that can overcome the work function results for distribution of the exit momenta p or energies (EMBook 87)
12Condenser Lenses C1 Crossover C1 is spot size Determines size of beam on specimenC2 is beam brightness knobVaries and magnifies C1Aperture is a physical aperture in range of sizesReduces spherical aberrationAssociated stigmator to correct astigmatismC1 CrossoverThe first condenser limits the first crossover coming from the gun. It’s known as C1 or also spot size on the physical controls of the scope controls the initial illumination amount hitting the specimen. It converges the beam to a focus at or near the plane of the specimen, changing the current changes the intensity of beam hitting the specimen and the overall brightness of the image. As magnification increases the illuminating spot must be reduced in size.C2 enlarges the C1 spot and magnifies it, also referred as Brightness in the physical controls.The condenser Aperture is a fixed hole of ranging size from uM. The reduces spherical aberration of the beam but also the overall amount of beam hitting the specimen. Classically the larger the aperture the more signal is generated and more contrast but loss of resolution due to aberration. The smaller the lens, the more resolution, but loss of brightness and contrast. Another tradeoff.Main control of spatial coherence correction: the smaller and more parallel the beam to the specimen, the better the coherence and better the phase contrast product. However when starting with a less bright source as in the Lab6 there is a tradeoff and I can’t get the appropriate electron counts to take an image
17Grid Types Supports used must be strong yet electron transparent Plastic (formvar)CarbonHoley carbonQuantifoilC-flatSpecimen supports Carbon, plastic (formvar) holey carbon, and quantifoil,
18Image Formation Four fundamental processes Scattering Absorption Gives rise to amplitude contrastContrast from absence of electronsDiffractionUsed to enhance contrast in cryoEM but with loss of resolutionInterferenceGives rise to phase contrastHalo or fringe around objectIn it’s most basic form what we are dealing with is radiation bearing no relevant information is made to interact with a specimen by passing through it. When the radiation reappears on the other side of the specimen, it carries information about the specimen which is then processed in such a way that the eye can see it and the brain can interpret it. (Meeks 83)There are four fundamental physical process that take part in the formation of the image part of these we talked about last week but I want to expand on it. Meeks (83)
19Specimen Beam Interactions: Scattering Elastic (Rutherford scattering)Electron collides with or passes close to a nucleus of atom, no loss of energy of initiating electronchanges direction without losing velocity orenergyInelasticElectron collides with cloud electrons,measurable loss of energy of initiating electronAs sample thickness increases more electronsare backscatteredAssumed to occur only once in TEM (either the e- scatters or it doesn’t)Sometimes referred to as the direct transmitted beam if no scattering occurredCan either forward or back scatterScatter measured in spatial deviation manifests as contrast, scatter of angular deviation manifests as diffraction patternsBefore we get to the Imaging system it’s necessary to pause and examine what happens now that the beam has hit the sample. Electron scattering is a term that is involved inElectrons have a particle and a wave function. In their particle form they can interact with the nuclei of the specimen atoms or with the electrons in the cloud surrounding the nucleiMatter is virtually empty space the majority of incident electrons will pass straight through a very thin specimen. (electron transparency)When the beam passes through a thin specimen the atoms of the specimen will interact with the incident fast electrons in one or both of two ways elastically or inelasticallyElastic: if a fast electron collides with a nucleus it will be deflected at a large angle but will have practically no energy loss (bounce a ball bearing a dense steel plate and it will bounce back to the same height dropped)If that same electron is on a plate rolling and collides with another one they will share their velocities obeying the laws of conservation of momentum. This is an Inelastic interaction. The high velocity imaging electron has a change in direction but also a change in velocity.Multiscattered electrons are harder to predict, again another reason for very thin samples for TEM.The spatial distribution of scattering can be observed as contrast in images, the angular distribution can be viewed in patterns known as diffraction patterns.There are more electrons in specimen than there are nuclei therefore inelastic scattering is the most important effect in formation of image.
20Scattering and Coherence Electrons remain in phase with one another after passing through sampleIncoherent electrons are those that have no consistent phase relationship upon passing through sampleElasticUsually coherentInelasticAlmost always incoherentWave nature of electronsAngular dependency• Most of the electrons are scattered overlarge angles• Go either through the OL aperture (phasecontrast) or are blocked (aperture contrast)
21Accelerating Voltages How does this all relate in choice of your electron accelerating voltage or kV?The faster the speed of the electrons the better resolution obtainedBut at the sacrifice of contrastSlower electron speeds have more opportunity for inelastic scattering, inelastic scattering produces energy (heat) therefore lower kV has more specimen damageI wanted to put this slide in to talk about the choice of kV and it’s relation to the scattering concept. Typically noted that the faster the kV the better resolution that is obtained. But at a cost of contrast formation.
22Mass ThicknessTypical thick sections are at 100nm while high resolution is limited to 10’s of nm.The maximum useful specimen thickness depends on the type of electron– specimen interaction used to form the image and on the mode of operation.For high-resolution imaging (≤1 nm) in the bright-field mode, phase-contrast effects are important.
23Objective Lens and Aperture So we covered the illumination system and the second section the specimen manipulation system, now we start on the third part the imaging system which begins with the Objective Lens.
24Objective Lens Most important lens Forms initial image further magnified by other lensesResponsible for focusBlocking of more peripherally deflected electrons with Objective apertureThe larger the aperture used the more phase contrastImportant for cryo EM and higher resolutionThe smaller the aperture the more aperture contrastAssociated stigmator to correct any astigmationThe single most important lens in the TEM. Responsible for the the initial image and diffraction patters that are sent down to the projector lenses to the CCD. Any defects in the lens will be further magnified by the imaging g system. Must be machined as symmetrical as possible and free of astigmatism.Terminology: While the aperture is the hole in the disk, the metal surrounding the aperture is called the diaphragmMost of the electrons are scattered over large angles• Go either through the OL aperture (phasecontrast) or are blocked (aperture contrast)
25Objective ApertureYou can say in this ray diagram the peripheral electrons are blocked reducing phase interference to increase contrast, but at the cost of resolution.Bozzola 176
27Intermediate and Projections Lenses Similar in construction to objective lensMajor function is to assist in the magnification of the image from the objective lensIntermediate aperture is for viewing the diffraction patterns, the IM and Proj lenses further magnify the image from the objective lens and project to recording system.