• Generation/maintainence of the EM beam
  • Without vacuum, gases would ionize and arcing would result
    • Small amount of oxygen in air would cause filament to burn out
  • MFP of molecule in air is 5nm vs. 6.5nm in EM

• Sputter coating
• Vacuum equipment
• FFE

• Two stages
• Low vacuum followed by high vacuum
• Low vacuum (roughing vacuum) is often created by mechanical/rotary vane pump
• High vacuum is often via diffusion pump or turbomolecular pump

• Gas molecules bumping into each other
• MFP is 65nm
• Roughing/mechanical pump

• Gas interacts with walls of chamber
• MFP greater than column diameter
• Diffusion pump

• Physical transfer of gas from a volume
• Oil-containing
• May or may not have moving parts

• Sequesters or entrains (gas molecules still present in chamber)
• Physical or chemical means
• Non-oil containing

• Found in nearly all EM equipment
• Works by positive displacement
• Use
  • Lower the pressure to a point that a high vacuum pump can take over (works as a roughing pump)
  • Remove gas from the outlet of a high vacuum pump that cannot efficiently discharge its gases (works as a backing pump)

• Construction
  • Rotor with a spring-loaded vane
• Function
  • As rotor turns, space between rotor and chamber wall varies 
• Pumping characteristics
• 0.1Pa and 100 to 1000L/min

• The most widely used vacuum pump
• Mechanical pump with no working parts
• Works by momentum transfer (=impact) rather than by diffusion

• Oil vapor created by heating oil
• Vapor rises
• Supersonic oil vapor knocks down gas molecules
• Gas accumulates before first annulus
• Withdrawn
• Condensation via water
• 1/2 hour warm up
• Reaches 10-4Pa at speeds of 100 to 1500 1/s

• Mechanical pump
• Provides a function similar to DP
• Advantages
  • No backward diffusion of oil vapors
  • No warm-up needed
• Characteristics
  • Attain 10-7 to 10-8Pa at rate slower than DP

• Structure
  • 5-15 stacks of spinning slotted rotors and stationary slotted plates (stators)
• Function
  • Gas molecules hit rotor and travel to next stage
  • Rotor spins at 50,000 rpm, stators encourage downward movement
  • Each stage acts as a compressor stage to further concentrate the gas
  • Gas is exhausted via a rotary vane pump

• Entrainment via a getter surface
  • Reactive gases bond to cooler getter surface
    • Gettering material is often a Ti alloy with 15% Mo
    • Often cooled with water or LN2
  • Renewed by subliming a coat of fresh gettering metal
    • Coats over previously trapped atoms

• Cylindrical chamber with sublimation filaments
• During regeneration of gettering surface, filament is heated to sublimation (requires initial vacuum of DP or TMP)
• Normal operating range for TSP is <10^-4Pa
• Used for ultrahigh vacuum gun chambers for field emission scopes

• Entrainment pump that ionizes gases
• Central anode of steel cylinders and cathode plates coated with gettering material (Ti)
• Incoming gas molecules are ionized by externally applied magnetic field
  • Ions are unaffected
  • Electrons are trapped in cylinders by magnetic field
• Ions fly directly to cathode, lose their charge and are entrained
• When ions hit, they sputter away some of the cathode material with is eventually used up
• Initial vacuum must be high (from DP or TMP)
• Used as gun chamber pumps on FE scopes

• Entrainment pump
• Efficient, quiet
• Condenses some gases on cold surface and traps others in an adsorptive material
• Can reach ultrahigh vacuum of 10^-10Pa (used to pump FE gun chamber)
• Common type is two-stage helium gas refridgerator-cooled cryoadsorption pump

• Furst stage is cryocondensation
• Consists of radiation shield kept at -190C that traps water vapor and thermally shields inner stages
• May be cooled by LN2 to reduce load on helium refridgerator

• Second stage is cryocondensation and adsorption
• At -250C; also cooled by liquid He
• Traps all gases by freezing or adsorbing them - activated charcoal, molecular sieves
• Needs to be brought to room temp once a week to pump away adsorbed and condensed gases

• Need to measure vacuum in a chamber or know vacuum level for operation of control valves and pumps in automated systems
• The gauges used are indirect readings
  • Measure some pressure-related property and transduce the property to electronic symbol

• Used for low vacuum (roughing vacuum)
  • Laminar flow range or 100Pa - 10^-1Pa
• Operational principle
  • Heat loss from a hot wire is transferred to surrounding gas and the resistance of wire changes
  • Ammeter detects current difference which can be read as vacuum

• Two resistor wires
  • Reference wire (standard condition, fixed resistance) and measurement wire (under vacuum, resistance changes)
• As vacuum increases, temp of measurement wire increases
• Resistance increases and current decreases
• Resistance is detected by Wheatstone bridge circuit which applies more I to maintain R

• Aka Penning gauge
• Used for high vacuum
  • Molecular flow range 10^0-10^-6 Pa
• Operational principle
  • Ionized gas molecules create a current proportional to the vacuum

• Voltage of several thousand volts is maintained between cathodes and anodes
• High voltage ionizes gas molecules
• Positive ions travel to negative cathode and lose their charge, causeing a current to be generated
• Current is calibrated to measure vacuum
• System is made more efficient when e- formed during initial ionization head toward + anode and ionize additional gas molecules as result of an externally applied mag. field that lengthens their time of flight

• Used for low vacuum (roughing vacuum) - 100Pa -10^-1 Pa
• Theory and operation
  • Heat lost from a hot wire is transferred to a gas
  • As vacuum increases wire gets hotter
  • Temperature of the wire is measured by thermocouple or voltmeter attached to thermocouple
  • Temperature or current is correlated to vacuum level

• Aka Bayard-Alpert of ionization gauge
• Used for high and ultrahigh vacuum - 10^-4 to 10^-10 Pa
• Operational principle
  • e- ionize gas molecules which, when collected, create a current inversely proportional to the vacuum

• Hot filament cathode generates e- by thermionic emission
• 200V accelerates e- towards + anode (Grid=wire coil)
• e- ionize gas molecules
• Ions attracted to a central wire/ion collector
• Ions collide with collector and lose charge, create current

• First stage: rotary rough-pump pumps column/chamber down to vacuum DP can be used
• Second stage: Valve (roughing) between rotary pump and column/chamber is closed and valve (main/plate) isolating DP is opened, backing valve opened so rotary pump backs DP by removing gas compressed in bottom stage of DP

• May not need uniform vacuum in the system
• FE Microscope
  • Gun chamber kept at ultrahigh vacuum, rest of system kept at high vacuum
  • Accomplished by small diameter of EM column
• ESEM
  • Specimen chamber kept at low vacuum
  • series of apertures separate chamber from column so that upper parts of column above apertures can be kept at a vacuum thousands of times better than specimen chamber

• Advantages
  • Last a long time
  • Can pump a lot of gas
• Disadvantages
  • Back-streaming of oil vapor
  • One-half hour warm-up and cool-down

• Simplest system
  • Pumps from ambient to working
  • At working is backed by rotary pump
• Advantages
  • Clean, fast, don't need valves of 2-stage system
• Disadvantages
  • Cost, foreign material drawn into rotor, lubrication of rotor-shaft bearing

Lenses bend rays of light or electrons so that the rays are deflected in a predictable fasion

• Light lenses cause a slowing down of the velocity of light as it passes through the material of the lens
• Electron lenses cause a spiraling of the electrons as they pass through the bore of the lens

• Glass lenses of magnifying glasses, camera lenses, and LM can be convergent (positive) or divergent (negative)
• Magnetic lenses of the EM are convergent (positive)

- Distance from the lens to the focal point

- A strong lens has a short focal length

- A weak lens has a long focal length

• Ability to distinguish fine detail
• Minimum separation at which two objects can be seen as separate entities and not blurred together as one
• Not the same as magnification

• X life size
• Maximum magnification = r.p(eye)/r.p(scope)
• Empty magnification

• Diffraction limits resolution in an aberration-free optical system
• Object size and wavelength must be of similar magnitude for diffraction to occur
• We can quantitate the magnitude of the diffraction effect using the Abbe equation .6lambda/nsinalpha

• Bending of light by a barrier
• Part of wavelength that clears barrier continues in straight line (primary wavefront, undiffracted)
• Part of wavelength that hits edge of barrier goes around the corner (secondary wavefront, diffracted)

• When wavelength hits the edge of barrier and goes around the corner - diffracted
• Is a new spherical wavefront
  • The circular pattern effect is diffration
  • Explained by Huygen's principle
    • Each point on a wavefront may be regarded as a new source of waves
• Primary wavefront interferes with secondary

• Primary wavefront interferes with secondary
• Part of advancing wavefront passes through (undiffracted or zero order)
• Edges of slit create two secondary spherical wavelet patterns close to edges of slit/pinhole
• Diffracted wave interferes with undiffracted wavefront
• Interference takes three basic forms: constructive, destructive, and partial

• Spectral bands are produced by diffraction and constructive and desctructive interference at a slit
• Undiffracted/zero order, first order, second order, third order, and so on
• Fringes/bands caused by diffraction at small openings and sharp edges are called Fresnel fringes
  • French mathematical physicist Augustin Fresnel (1788-1827)
  • Produced when e- beam strikes an opaque edge
  • Can be used as a focusing aid in TEM (look at small holes in a section)

• Diffraction at a small aperture or a slit has the effect of broadening the source
  • Degrades resolution
  • Is the reason we don't use small aperture

• 19th centurary British astronomer
• Infinitely small point of light cannot be imaged as a perfect point due to an aperture
• Diffraction at the aperture gives rise to fringes that surround the image
• Pattern is called an Airy Disc

• Aperture, wavelength, and refractive index of medium between lens and object
• 84% of incident energy found in central peak

• Radius related to resolution
• 1896 - examine resolution
• Two disc resolved when separation between centers is same as radius of first dark ring
  • Need 19% intensity drop between two central peaks
  • R.P. = lambda/nsinalpha

• Examine resolution in the LM
• Large aperture lens collects more diffracted light and give more information
• Introduced numerical aperture
  • NA = nsina where n is refractive index of the medium and a is half the lens acceptance angle
• The Abbe equation
  • R.P. = .6lambda/nsina
  • Wavelength and aperture angle set limites to resolution
  • R.P. is resolution of two minute pinholes in a metal film by the objective lens

• Bending of light at interface
• Index of refraction, n
  • Speed of light in vacuum/speed of light in medium
  • A measure of optical density
    • Speed of light in glass is less than in air: glass is optically more dense

• Lambda = 0.5um (green light)
• Highest NA: n=1.74 for methylene iopdide, 1.78 if S is dissolved in it, sina=0.87
• Lambda<UV is a problem
  • Eyes insensitive to shorter wavelengths
  • Waves shorter than UV cannot yet be bent
  • Energy assocaited with radiation is harmful

• Should have a resolution 100,000 times better than LM
• Why isn't it better? Because of spherical aberration
• Spherical aberration is reduced by using small aperture
  • BUT, small aperture gives diffraction
  • So, we compromise on aperture size

• Lens surfaces are spherical: zones father from the axis have a different refractive power
  • Axial rays are brought to a focus point further from the lens than medial or peripheral rays
  • Ds=ks*f*a^3
• Correction: combine positive and negative lens

• Results from dispersion of lens - refractive index varies according to lambda
• Light from a n axial point will not come to a common focus
  • Blue rays will be focused to a point closer to the lens
• In EM, e- have different velocities and therefore different wavelengths
  • e- with greater velocity are acted on by the lens for shorter time and are deflected less: focused further along the lens axis
• Dc=kc*f*a*deltaV/V

• Lenses are unable to produce a single point image of an off-axis point object
• Linear images at different focus result

• Instead of image lying in a plane, it falls upon the surface of a sphere

• Magnification varies from center to periphery
• Pincusion - less magnification at the center
• Barrel - greater magnification at the center

• Used in two main ways
• Provides magnified object image
  • Permits visualization of specimen structure
• An analytical tool
  • Measure brightness
  • Measure length, width, area
  • Count
  • Determine optical properties such as refractive index or reflectance

• Energy used to form image is in visible light portion of electromagnetic spectrum
• Human eyes respond to wavelengths from 730-760 nm (red/infrared) to 360-380 nm (violet)
• Greatest sensitivity in green region at about 550 nm

• Bends rays of light so that they are deflected from their original path in a predictable way
• Transparent glass causes light to slow down because the refraction between light in air (or oil) and the glass

• SinI/sinR=n^2/n^1
• I=incident, R=refracted, n^1 is the refractive index of the less dense medium

• An optical system with two refracting surfaces
• Two main types: Converging, diverging

• Used in geometrical optics to study:
  • Paths followed by rays of light or e- through lenses
  • Constructions used to find the relative positions and sizes of objects and their images
• By convention, rays travel from left to right

• Incident light rays converge after refraction
• Focal length is positive
  • Called positive, convex, condensing or magnifying lens
• Always thicker at its center than the edge
• Produces a real image or a virtual image

• An image that can be seen on a surface such as a screen or photographic film
• The rays intersect and physically reunite
• The image is magnified and inverted

• An image that cannot be received on a surface such as a screen or film but can be converted into a real image by an optical system such as the eye, a microscope, or other converging lens system

• By placing an object between the lens and focal point

• Incident light rays diverge after refraction
• Focal length is negative
  • Called a negative, concave or diminishing lens
• Always thinner at center than at edges
• When used alone can only form virtual image

• Meniscus
• Plano-convex
• Double-convex

• Meniscus
• Plano-concave
• Double-concave

• One or more lenses
  • Ordinary vs. Coddington vs. Hastings triplet
• forms virtual image
• Has practical limit to magnification
• Leeuwenhoek was able to get magnifications of 200 times and resolution of 1.5um so bacteria could be seen
• Haven't been able to do better than this to date

• LM uses a series of converging and diverging lenses to produce an image
• Lenses are present in the illuminator and substage diaphragm that focus the light on teh specimen
• Lenses are present in the objectives and oculars that are used to form the image

• Objective lens - constructed of converging and diverging elements arranged in groups
• Ocular - two converging plano-convex lenses

• 1. The objective produces a magnified real image and the ocular produces virtual image
• 2. The real image formed by the objective serves as an object for the ocular
  • An image formed by one lens can serve as the object for a second lens

• First image forming system is the objective
• Forms intermediate image: magnified real image
• Not intercepted but is viewed magnified through a magnifier called the eyepiece

• Eyepiece magnifies intermediate image and forms virtual image
• the lens of our eye (or a lens in a camera attachment of the microscope) converts the virtual image into a real image

• Quantitation of object and image distances, magnification
• Derived from geometrical comparisons of similar triangles
• 1/f=1/a+1/b
• b/a = magnification (ratio of object and image distances)
• f=focal length
• Objective lens: real image, short f (gives real, magnified first image)
• Ocular: virtual, longer f (gives virtual image, used to produce second image)

• In the substage
• Focuses light on specimen
• Abbe
• Aplanatic
• Aplanatic-achromatic
  • Corrected for red and blue chromatic aberration, spherical aberration, and field curvature

• In the base
• Make up the iluminator
  • Collector lens, field diaphragm, lamp

• In standard objective, rays from back aperture of lens come to focus and form an image
• For infinity corrected objective, rays from back aperture are focused to infinity and do not form an image
  • The tube lens (Telan lens) receives objective rays and forms real intermediate image

• Most common viewing mode
• Ridect light passes through condeser and objective aperture and illuminates the background against which the image is seen
• Image of the specimen becomes visible by way of absorption contrast
  • Specimen reduces amplitude of light passing through it (all light or certain wavelengths of light)

• Many objects absorb naturally but specimens like cells and tissues are transparent and therfore do not change the amplitude of light
• Specimen must be made absorbent with dyes (stains)

• Aka darkground microscopy
• Direct light is prevented from passing through the objective aperture by using oblique illumination
• Specimen features appear bright against a dark background
• Undeviated light misses the objective
  • No zero order contribution to image
• Image is formed from light scattered by specimen features
  • Weakly diffracted light enters objective
• Usually used for small specimens such as bacteria

• Aka reflected light microscopy or epiillumination microscopy
• Illumination falls on the object from the same side as that from which the object is observed
• Microscope is equipped with epi-illuminator
• Used for flruorescence microscopy (biological specmens), geological and metallurgical specimens, examination of semiconductor devices

• Image formed by object fluorescence
• Mechanism
  • Phton absorded, e- boosted to higher energy (excitation)
  • E- returns to ground state and emits quantum of light (deexcitation)
  • With energy lost as heat, emitted light has less energy and longer wavelength

What is light source is used to excite fluorescence in fluorescence microscopy?

• Ultraviolet light is used to excite fluorescence and reemission is in the visible light range
• Light source and filter system required
  • Mercury lamp - high pressure
    • Gives 30% light at wavelength of 365 nm

Exciter filter

• Transmits short wavelength light to exicte fluorescence in the specimen

Barrier filter

• Protect the eyes from damaging UV light

Heat filter

• Located in the lamp housing and prevents excess heat build up

• Necessary in incident light excitation
• Reflects UV light onto specimen and at the same time allows transmission of fluorescence from the specimen to the viewer

• Unstained materials can't be visualized by brightfield microscopy
• Phase contrast microscopy converts optical path differences that we can't visualize into changes in amplitude that we can see

• Phase plate - developed by Zernike
• Annular diaphragm

• 1. Diffracted light is retarded 1/4 wavelength by specimen
  • The specimen diffracts light
    • This light is delayed by 1/4 wavelength
  •  Diffracted light goes through thick part of phase plate
• 2. Background light is advanced 1/4 wavelength by phase ring
  • This light is coming from condenser annulus
  • This light passes directly through specimen without contacting any details of structure
  • This light goes through a phase ring
    • Phase ring is etched to reduce path, producing phase advancement of the backgroung light
• 3. Desctructive interference can occur between specimen and background light
  • Optical path difference between background and specimen rays is lambda/2

• To prevent high intensity from swamping signal from thick part of plate, the ring is darkened with metallic coating
• Have positive phase contrast and negative phase contrast
  • Positive phase contrast is the most common

• Image formation based on ordered molecular arrangements/uneven density
• Ordered structures appear white on a dark background
• Examples
  • Mitotic spindles, actin and myosin, condensed DNA, crystals in biological specimens
  • Mineral ID
    • Used analytically to determine differences in optical path length that can be used to calculate refractive index

• Produced by crystals or precisely aligned molecules - more dense in one direction that the other

• The two rays are retarded or refracted to differing degrees because of the uneven density
  • Ordinary and extraordinary ray (O and E)
• Will split beam of light into two polarized beams a right angles

• Have the power to absorb one set of the rays produced
  • The orginary ray is absorbed and the extraordinary is transmitted
  • We make use of this property in polarizers, analyzers, and Nicol/Wollaston prisms 

• PLM is a compound LM fitted with a polarizer, analyzer, and compensator
• Compensator is used for quantitative measurement of birefringence
• Polarizers and analyzers are often nicol prisms
  • Light rays emerge vibrating in a single plane
• the polarizer lets light rays vibrating in a certain plane pass through which in turn excites light rays in the crystal which can be absorbed or transmitted by the analyzer
• The crystal appears light on a dark background

• Aka Nomarski differential interference contrast
• Produces amplitude contrast from a transparent specimen
• The image produced has a distinctive relief-like, shadowed appearance that appears 3D
• Parts
  • Polarizer, analyzer, Wollaston (Nomarski/DIC prisms) split and recombine light beam

• Light from the light source is polarized by a polarizing filter in the substage (polarized light)
• Light emerging from the polarizer is split into two components polarized at right angle to each other
• Specimen is sampled by pairs of closely spaced rays generated by lower DIC prism
• When ray pairs traverse region with a refractive index or thickness difference, an optical path difference is introduced between the two rays
• Optical path difference is translated into amplitude difference (path differences of lambda/10 to full wavelegth permit interference, thus imaging transparent detail
• Light beams recombined by upper Nomarski prism
• Analyser above prism passes certain amount of light depending on its orientation
• Out of phase light interferes at image plane to create image

• Aka stereomicroscope
• Used for erect, magnified image that shows depth - has considerable depth of field
• Binocular head with common objective lens
• Much of the light travels through objective at angle so objective must be of high quality
• Disadvantage is that NA is limited by double beam path to about 0.1

• Introduced by August Koehler in 1893
• Added collector lens for lamp
  • Used collector lens to focus lamp image on the fron aperture of the condenser
  • the field stop was then focused on the specimen with the condenser control
• Provided bright, even illumination and parallel unfocused light through the objective plane
  • Gives wide cone o0f light for optimum resolution and reduces effect of dust from condenser

• 1. Focus lamp on front aperture of condenser
• 2. Focus the specimen
• 3. Focus condenser to see field stop diaphragm (kohler illumination)
• Adjust condenser diaphragm using eyepiece telescope

• Plan
• Achromat
  • Chromatic aberration limited to two wavelengths: blue and red
• Apochromat
  • Chromatic aberration is minimized for three wavelengths (blue, green, red)

• Are indicated on lens barrel
• Indicated coverslip thickness (this is the standard number 1.5 coverslip)
• Working distance, magnification, application, numerical aperture/immersion medium
• Microscope slides should be 1.1mm thick