Transmission Electron Microscopy Reference Notes

8/2/2019 Transmission Electron Microscopy Reference Notes

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TRANSMISSION ELECTRON MICROSCOPY REFERENCE NOTES

by

Franklin BaileyElectron Microscopy Center

University of IdahoMoscow, Idaho, 1989 Franklin Bailey, 1989yRevised 1999

TABLE OF CONTENTSCOURSE OUTLINE ................................................................ ivRULES AND REGULATIONS ............................................. viCHAPTER IHistory of Electron Microscopy ......................................... 1CHAPTER IIOptical Studies ............................................................. 3CHAPTER IIIThe Electron Microscope . ...............................................

9CHAPTER IVFormvar Grid Preparation ................................................ 22CHAPTER VShadow Casting ............................................................ 24CHAPTER VILens Aberrations .......................................................... 26CHAPTER VIIThe JEOL 1200 EX II Transmission Electron Microscope ........ 32CHAPTER VIIIElectron Diffraction ....................................................

43CHAPTER IXPhotographic Theory and Technique ................................. 46INDEX .............................................................................. 52

TABLE OF ILLUSTRATIONSFig. 2.1 - Image Formation By An Ideal Lens ................................ 6Fig. 3.1 - Light vs Electron Microscopes ...................................... 8


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Fig. 3.2 - Biased vs Unbiased Gun .............................................. 9Fig. 3.3 - Simple Electromagnetic Lens ........................................ 13Fig. 3.4 - Lens Magnification Factors ........................................... 17Fig. 3.5 - Oil Diffusion Pump .................................................... 18Working Vacuum Systems ......................................................... 20Fig. 4.1 - Shadow Casting Setup ................................................. 24Longitudinal Chromatic Aberration .............................................. 26Lateral Chromatic Aberration .................................................. 27Coma ............................................................................... 27Spherical Aberration ................................................................ 28Astigmatism .......................................................................... 29Asymmetry ........................................................................... 29Curvature of Field .................................................................. 30Pin Cushion Distortion ............................................................. 30Barrel Distortion .................................................................... 31Rotational Distortion ............................................................... 31

COURSE OUTLINE; FOR FS&T 527

TRANSMISSION ELECTRON MICROSCOPYWeek 1:

Course IntroductionOutline of SemesterE. M. Terminology and General InformationHand Out Study GuidesPreparation of Co-polymer support films and "Holey Grids"

Week 2:Electron Microscopy Theory I (The Electromagnetic Lens)Introduction to Ray DiagramsShadow Casting

Week 3:Electron Microscopy Theory II (The Electrostatic Lens)Equipotential LinesPractical Exercise I (The Cold Startup, Filament Saturation and Alignment)

Week 4:

ExaminationElectron Microscopy Theory III (The Vacuum System)Practical Exercise II (Compensation for Astigmatism, Through Focus Series, EMPhotography)Handout Problem Set # 1

Week 5:Electron Microscopy Theory IV (Photographic Theory)


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Practical Exercise III (Preparation of Particulate Grids)Practical Exercise IV (Print Darkroom)Problem Set #1 Due

Week 6:Electron Microscopy Theory V (Resolution and Artifacts)Practical Exercise V (Preparation of Bacterial Grids)Through Focus and Stigmation Micrographs DueHandout Problem Set #2

Week 7:ExaminationParticulate Grid Micrographs DuePractical Exercise VI (Magnification Verification)

Week 8:Bacterial Micrographs DueElectron Microscopy Troubleshooting I (The Missing Beam)Practical Exercise VII (Filament Change and Alignment, Aperture check)Magnification Micrographs and Calculations dueWeek 9:Electron Microscopy Troubleshooting II (No Vacuum)Practical Exercise VIII (Purging Pumps and Replacing "O" rings)Problem Set #2 Due

Week 10:Electron Microscopy Theory VI (Electron Diffraction)Practical Exercise IX (Electron Diffraction Patterns)Handout Problem Set #3

Week 11:ExaminationElectron Microscopy Troubleshooting III (No Camera)Practical Exercise X (Camera Disassembly and Cleaning)

Week 12:Open (Finish your problem sets and catch up if necessary.)

Week 13:Problem Set #3 Due

Week 14:Open

Week 15:All Portfolios Due

Week 16:Final Exam

Requirements for Satisfactory Completion of Course(1) Final Examination (Score of 70% or better)

(2) Three Major Exams (Score of 70% or better)(3) Three Problem Sets (Score of 70% or better)(4) Turn in All Required Projects(5) Demonstration of Good Laboratory and Microscope Technique(6) Return of All Materials Loaned YouRULES AND REGULATIONS OF THE ELECTRON MICROSCOPELABORATORYNEVER ATTEMPT TO OPERATE ANY EQUIPMENT WITHOUT PRIOR


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INSTRUCTION BY QUALIFIED PERSONNEL IN THE LABORATORYANDWITHOUT CAREFULLY READING THE INSTRUCTION MANUALTHERE WILL BE NO EATING, DRINKING, OR SMOKING IN THEMICROSCOPE ROOM.THERE WILL BE ABSOLUTELY NO SMOKING IN THE ENTIRE HOLMRESEARCH CENTER.MEASUREMENTS :The metric system of measures is the primary standard used in electronmicroscopy.Beloware some units that will provehelpful in this course of study.1 inch = 2.54 centimeters (cm)1 cm = 10-2 m1 millimeter (mm) = 10-3 meters = 10-2 cm1 micron () = 10-6 meters = 10-4cm

1 nanometer (nm) = 10

-9

meters = 10

-7

cm

1 Angstrom Unit () = 10-10 meters = 10-8 cm

The following compilation should be used as a study guide only and should not be

considered a complete set of notes for the class.Annotate whenever necessary to obtain a

finished notebook.CHAPTER I

HISTORY OF ELECTRON MICROSCOPY


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Since the introduction of the first practical microscope by Anton van

Leeuwenhoek, the desire to resolve smaller and smaller bits of information has been ever

burning in scientists from all disciplines.Improvements on the microscope and the

preparation methods for microscopy continued to make it a powerful tool for

investigation in biology and medicine as will as other fields.Abbe', however, in 1873,showed that the smallest resolvable distance which could ever hope to be achieved was

about the wavelength of light, or about 2,000 (.2).Therefore the magnification of

anything greater than 1000X would not reveal any smaller objects to the human

eye.When the X?ray was discovered around the turn of the century and it was

demonstrated to have a very short wavelength, there was considerable hope for the

development of an X-ray microscope.This would obviously overcome the limitations of

the existing microscopes and further the development of the science of microscopy.There

was, however, one major barrier; the index of refraction of all substances for X?rays is

close to unity so that effective refracting lenses cannot be made. X?ray microscopy would

later play an important part in providing quantitative data on the composition of certain

specimens.When the electron was discovered in 1897 by J.J. Thompson; it was soon

characterized as a charged particle with a rest mass, but wasn't thought to possess any

wave properties.The testing that followed led to the development of the cathode-ray

oscilloscope, and verified the fact that high accelerating voltages and magnetic fields

could manipulate electrons.It wasn't until 1924, however, that Louis de Broglie advanced

the hypothesis that there was a wave characteristic associated with electrons; and showedthat the wavelength of an electron beam accelerated by 60,000 volts would be only .05

or1/100,000 that of visible light.This lead to the development of the theory of wave

mechanics by E. Schrodinger in 1926; and the experimental verification of this theory on

electron waves by Davisson and Germer in the United States and Thomson and Reid in

England lead to further interest in the use of the electron beam.After 15 years of work on

the effects of magnetic fields on electrons, H. Busch published a paper showing that

magnetic fields having axial symmetry acted as lenses for electron beams.This began the

new science of electron optics.


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E. Ruska made a significant contribution in magnetic lens design by surrounding

the wire coil windings with iron except for a small gap.This produced a lens with a

stronger and shorter field that also possessed a more efficient focusing action.Following

his research on electromagnetic lenses, in 1934 Ruska described the first electron

microscope expressly designed to be used for high resolution work.This was the firstinstrument used to surpass the resolution of the light microscope, as was shown by E.

Driest and H.O. Muller; in 1934.About the same time, Krause used the instrument to

introduce specimen techniques and obtained some of the first micrographs of thin

sections.Ruska and B. von Borries; improved upon Ruska's original microscope and

designed a practical electron microscope for the Siemens and Halske company in

1938.This was the first transmission electron microscope designed for general laboratory

use and it had the capability of resolving details of approximately 100 .This led to a

series of electron microscopes built by different researchers, each an improvement upon

the preceding one.The first electron microscope built on this continent was by A. Prebus

and J. Hillier in Toronto in 1939, and C. Hall built a similar one in the United States in

1941.The first commercial electron microscope in this country was introduced by RCA in

1940, and by 1945 the TEM's were capable of a resolution of 10 .Thus the development

of the basic instrument.Improvements continued throughout the subsequent years with

the evolution leading to the sophisticated instrumentation we enjoy today.The evolution

continues, however, not only with the progress in the development of hardware, but in the

improvement of techniques and supporting equipment.CHAPTER II

OPTICAL STUDIESFollowing are a few definitions concerning our analysis of optics.Optics - The study or consideration of radiationGeometric Optics - The study or consideration of radiation by means of straight line

representations ("rays") of the direction of the progressive motion of radiation, including

the concepts of perspective and the distribution of "brightness" and "shadow".Physiological Optics - The study or consideration of radiation upon the basis of

psychological response.For example, our mental response to the color we call "red" is aphenomenon entirely different from a description of the causative radiation in terms of

"frequency", "wave length", etc.Physical Optics - The study or consideration of radiation via use of "wave forms"

wherein a wave form represents a probability distribution of energy in space-time.Quantum Optics - The study or consideration of radiation via investigation or


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examination of the interaction of radiation with the atomic entities of matter.A "ray" is a linear (straight line) representation of the progressive motion of radiation,

i.e., the "path" of the radiation.A "pencil of radiation" is a "cone" of rays emanating from a single reference point source

of radiation.A "beam of radiation" is the summation of all "pencils" of radiation emanating from a

single broad source of radiation.Vergence - The changing of the direction of the progressive motion (path) of radiation. Convergence - The changing of the direction of the progressive motion of radiation such

that the radiation approaches, or tends to approach, a single point or location in geometric

space.Divergence - The changing of the direction of the progressive motion of radiation such

that the radiation extends, or tends to extend, from a single real, or apparent, point in

different directions.Collimation- A special case of vergence of radiation in which a "set" of "rays" is rendered

such that each ray of the set is parallel to each, and every, other ray of the set of rays.A "lens" is a device used for verging radiation. A "bundle of rays"is a pencil of rays from one end of an optical system to the other end

of such system, or from one point in geometric space to another point in geometric space,

through any and whatever materials or events that are, or become, active along the path

of the motion of the pencil of radiation.LENSES:Lenses, particularly lenses made of materials such as glass or quartz, are often classified

into "types" on the basis of external surface curvature or curvatures.Some representative

types are often labeled:1) Spherical2) Cylindrical3) Toroidal4) ParabolicalThe following representative kinds of single "spherical lenses" shall receive ourimmediate attention:1) Plano-convex

2) Double-convex3) Equi-convex4) Meniscus5) Plano-concave6) Double-concave7) Equi-concaveSuch lenses are often called positive if the lens action tends to converge radiation

andnegative if the lens action tends to diverge the radiation of concern.All "convex


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spherical lenses are usually "positive" and all "concave" spherical lenses are usually

"negative" lenses.A meniscus lens may be "positive or negative" in this sense depending

upon the respective radii of curvature of its external surfaces.The principal axis of a lens is a straight line through the geometric center of the lens and

perpendicular to the lens surfaces at points of contact with such surfaces.Each lens has two principal focal points.The locations of these principal foci in geometric

space depend upon lens shape, material or materials of which the lens is constructed, type

or types of radiation concerned, and the types of media on each side of the lens and

through which the radiation passes.The focal length of a lens is the linear, "optical" distance from either the geometric center

of the lens, or from another "reference point" within or associated with the lens, along the

principal axis of the lens to a principal focal point.The "reference point" selected depends

upon lens type considerations and the precision required for a given application.A focal point, in general, is a point, on or off the principal axis of the lens, to which, or

from which, incident "parallel" rays converge, diverge, tend to converge, or seem to

diverge after passing through the lens.In a ray diagram, these are abbreviated by the

symbol F1, F2, etc.All "focal points" are not "principal focal points."A focal plane is a two-dimensional geometric space that passes through, or contains, a

focal point.Such a "plane" may be considered perpendicular to any selected axis of the

lens, including but not necessarily always applicable, the principal axis of the lens.A principal focal plane is a focal plane through, or including, a principal focal point andis perpendicular to the principal axis of the lens concerned.Image Space is the three dimensional geometric space into which radiation emerges, or

would emerge, after passing through the lens, or through a selected reference plane

within, or associated with, the lens.In ray diagrams, image space is abbreviated with the

symbol si.

Object space is the three dimensional geometric space through which the radiation,

emanating from the object, passes, or would pass, in order to become incident upon(to

"strike") the lens or a selected reference plane, within or associated with, the lens.In ray

diagrams, object space is abbreviated with the symbol so.The principal focus or principal focal point of a lens is a point on the principal axis of a

lens toward which incident "parallel" rays( or rays from a source that is an infinite

distance from the lens) are converged, would converge, or from which such rays diverge

or seem to diverge.In ray diagrams, focal points are abbreviated with the symbol F1, F2,

etc.


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A principal point of a lens is the point of intersection of a principal plane with the

principal axis.A nodal point of a lens is a location on the principal axis of a lens such that radiation that

is aimed at, or passes through, a "first" nodal point, N1 to become incident upon the lens,

will emerge from the lens (into image space) without change in direction (parallel toincident ray) and appear to arise from a "second" nodal point, N2.The six major (primary) "cardinal points of a lens" are:1) Two "principal focal points" (F1 and F2)2) Two "principal points" (P1 and P2)3) Two "nodal points" (N1 and N2)The principal planes of a lens are two-dimensional geometric spaces, not necessarily

within the lens material or outline, perpendicular to the principal axis of the lens that can

be effectively utilized to describe and to determine the action of a lens.Lenses are often classified as "thick" or "thin."A thin lens is a lens for which the principal points and geometric center of the lens

coincide at the geometric center of the lens.The focal length is said to be large compared

with the length of the lens.

A thick lensis one in which all refraction does not take place in a single plane.Electron

lenses are of the thick type.

When the media on both sides of a lens (object space and image space) are the same, the

nodal points of a lens coincide with the principal points of the lens concerned.An object plane is a two dimensional geometric space that includes, or passes through,

the object, or the object location.An "object plane" may be perpendicular, or transverse at

any angle, relative to the principal axis of the lens or may be coincident, or parallel, to

such principal axis.An image plane, or "image focal plane", is a two dimensional geometric space that

includes, or passes through, a point (1) at which radiation arising from an object point is

converged by the lens, or (2) from which radiation, arising from an object point, seems to

be diverged by the lens.An ideal lens is a lens that will "verge" all incident rays arising from any single object

point in the same object plane to corresponding single points within a correspondingimage plane without relative geometric dislocation of points within the plane.Practically

speaking, an "ideal lens" does not exist.A lens may be described as "symmetrical" or "non-symmetrical" (asymmetrical) in the

handling of "rays" from a single object point.A symmetrical lens "verges" all incident

rays from a single object point to the same image point or location.A "non-symmetrical"


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lens may "verge" such rays to different image points in correspondence with incidence

within different geometric planes through, or within, the lens.IMAGE FORMATION BY A LENSA real image is an image that can be "caught" on a screen located at the image focal plane

position, i.e., it is an image formed by the actual convergence of "real rays" or of theprogressive motion extension of the radiation concerned in terms of rays. A virtual image is an image that cannot be "caught" on a screen located at the image focal

plane position, i.e., it is an image "formed" by the hypothetical extension of ray segments

to an apparent point of divergence or convergence either opposite to, or in the direction

of, the progressive motion of the radiation.INTRODUCTION TO RAY DIAGRAMS:

Throughout our discussion of optics we will be talking about lenses in terms,

which have been previously defined as being nonexistent.That is, we shall refer to the

lenses for which we draw diagrams as ideal lenses.Also, although we have already

determined that the electromagnetic lenses are of the thick kind, we shall be using the ray

diagrams, which are associated with thin lenses.This helps simplify the diagrams and,

hopefully, the understanding of how lenses work.To further our definition of the "ideal lens", we need to take note of the three

conditions of such a lens which were enunciated by Clerk Maxwell:1)Every ray of the pencil proceeding from a single image point of the object, must

afterpassing through the instrument, converge to, or diverge from, a single point of the

image.2)If the object is a plane surface, perpendicular to the axis of the instrument, the

image of any point of it must also lie in a plane perpendicular to the axis.3)The image of an object on this plane must be similar to the object, whether its

lineardimensions be altered or not.

Taking these into consideration we can draw a representation of image formation by an

ideal lens.

Fig. 2.1If we assume that the locations of F1, F2, H1, and H2 are known, we can then follow some

simple rules for the graphic construction of ray diagrams.1) Given any point Po in object space, find its conjugate


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a) Draw a ray through Po and F1 to intersect h1.From the point of intersection

draw a line parallel to the axisb) Draw a ray through Po parallel to the axis to intersect h2.From the point of

intersection draw a line through F2 to intersect the second line in a).The point of

intersection is Pi.c) If the point is in image space, reverse the procedure.

2) Given any ray segment in object space, find its continuation in image space

a) Take the point P where the given segment or its extension cuts the first focal

plane.Draw a line through P, parallel to the axis, to h2 and from this point through

F2b) Extend the given segment to cut h1, and from this point of intersection draw a

segment parallel to the axis to intersect h2c) Draw a ray form this last intersection parallel to the ray through F2 to obtain the

required rayd) If the given ray is in image space, reverse the procedure

Lens equation for thin lenses

With the illustration to guide us, we can now understand the equation for the

geometry of a thin lens.The equation for the hypothetical condition of a perfect thin lens

is as follows:

Then, if f1 = f2 = f, which is true under certain conditions, the lens equation takes on a

simpler and more familiar form of:which is the form applicable to glass lenses in air.

CHAPTER IIITHE ELECTRON MICROSCOPE:

This course is primarily designed to be a study of the practical applications of

electron microscopy and not a mathematical study of the physical theories


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involved.Those wishing to investigate further may consult any of the many good

references on the subject.Some of those are: Zworykin et.al. 1945; Cosslett 1951; Hall

1953; Haine 1954, 1961; Siegel 1964; Grivet 1972; Hawkes 1972.

The electron microscope is quite parallel to the familiar light microscope in the

arrangement of the various elements, which compose the working columns.Figure 3.1allows you to observe thesimilarities between the two microscopes, while pointing out

themore obvious differences.It should be noted that the rendering is not to scale. Fig. 3.1

While both instruments were designed to assist the unaided eye in resolving objects,

which are too small to be seen, the light microscope is adequate only for those, which are

greater than a few tenths of a micrometer in diameter.The need to observe objects in

greater detail or at higher magnification led to the development of the electron

microscope.The limiting factor to the resolving power of a good light microscope is the

wavelength () of the illuminating source in object space.In theory, the shorter the

wavelength, the greater the resolving power of the system.In the formation of the electron

beam, the wavelength is directly proportional to the energy of the electrons in the

microscope.

This can be given with the following equation: = ~

Where is the wavelength of the electron in nanometers andEis the energy of the

electron in kV.As you can see, the calculations for a 100kV electron would have the ataround .04 which is less than half the diameter of an atom.In theory, then, it is possible

to easily resolve the atomic structure of any material.However, the electromagnetic lenses

of a transmission electron microscope are much less than perfect, and resolution is only

2-3 .Some resolution limits are as follows:

The human eye at optimum distance --------- 0.1 mmVisible light at optimum distance ------------- 0.2 The ultraviolet microscope -------------------- 0.1 The transmission electron microscope ---->2

Resolution will be discussed in greater detail in the section on the objective lens system. THE ELECTRON GUN SYSTEMFig. 3.2

Haine and Einstein (1952), in studying the model system of a biased electron gun,

discovered two major properties of that system.First, they labeled four important

parameters of a biased gun system.These being 1) the distance from the tip of the


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filament to the opening in the grid of the Wehnelt cylinder, 2) the temperature of the

filament, 3) the negative bias of the Wehnelt cylinder with respect to the cathode and 4)

the brightness of the beam.The biased electron gun is simply one, which stabilizes itself against gun emission

fluctuations, which arise as a result of the changes in the filament temperature.To bias agun, a strong resistance is inserted between the filament and the high voltage lead; and

the electron beam current generates a voltage across the resistance.This bias voltage is

applied to the Wehnelt cylinder so that any increase in the beam current causes an

increase in the bias voltage.This acts as a check on the system and in turn reduces the

beam current again.The second major property they discovered was the relationship between the

accelerating voltage on the gun and the brightness of the beam.As the accelerating

voltage increases, the brightness increases proportionately.This fact is directly related to

bias in that as the accelerating voltage increases, the bias increases, and therefore the

brightness increases.

Interrelating the four parameters of the biased gun and producing a final working

electron beam can summarize the electron gun system.First, the filament height has

probably the most influence on the bias of the gun.Bias increases as the filament height

decreases, therefore dictating an optimum height for the production of the brightest

beam.

An operating filament temperature should remain between 2700 and 2900 Kelvin

units.Below this range produces a beam, which is insufficient for high magnificationviewing; while above it causes rapid wear on the filament metal without an increase in

beam brightness.A stable filament temperature contributes greatly to the stability of the

gun bias, as was previously discussed.The negative bias on the Wehnelt cylinder acts to reduce the size of the electron

beam to a diameter smaller than that of the opening in the Wehnelt cylinder shield.This

condensation increases the brightness of the beam without a corresponding increase in the

accelerating voltage.

The final element in the electron gun assembly is the anode.This is usually a flat

plate, which has a hole in the center and is electrically at ground potential.The electrons

are accelerated from the filament and pass through the anode into the column of the

electron microscope.This entire unit forms the first "lens" of the electron microscope, and acts much

the same as a stacked double convex/plano concave optical lens system. This lens is

referred to as anelectrostatic lens.


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The above discussion of the electron gun was with the assumption that the

filament of choice was the tungsten hairpin type.However, there are other electron

sources (emitters), which should be considered because modern technology has allowed

them to be produced at a cost which makes their use practical to the average laboratory.LANTHANUM HEXABORIDE (LaB6) - a crystal emitter made of the boride of the rareearth lanthanum.The LaB6 crystal produces an increase in brightness of about ten fold

over the tungsten filament and produces a much smaller spot size because the effective

source size is smaller.The increase in brightness allows the operator to work at very low

accelerating voltages without loss of signal, preventing beam damage to fragile

specimens.Another advantage to the LaB6 emitter is the narrow energy spread

(chromacity) of the beam, with the resultant decrease in chromatic aberration. The LaB6 emitter requires a gun vacuum of around 10-6 to10-7 torr; too low for the

conventional pumping system.Therefore, most manufacturers utilize at least one ionpump in the gun area (we will discuss ion pumps in the vacuum system section).The very

low vacuum in the gun area extends the emitter life to many times that of the

conventional filament.The combined advantages of the LaB6 cathode results in a marked

increase in resolution without a corresponding increase in the signal to noise ratio.The

optimum operating temperature of the LaB6 is around 1500 Kelvin units, a considerably

cooler cathode than the hairpin tungsten emitter.FIELD EMISSION - A different theory from the thermionic sources (tungsten and LaB6)

allows the field emission emitter to produce a beam of electrons from a point source that

is much smaller than even the LaB6.This source is a single tungsten crystal,electrolytically etched to a micro?fine point, and whose axis is aligned with the optical

axis of the microscope.Thus the planes of the most intense emission are perpendicular to

the and crystallographic indices, andproduce a beam of less than 5 nm in

diameter.The field emission source is several orders of magnitude brighter than the LaB 6

emitter with a much better chromacity and a very great depth of field.This combination of

factors allows the resolution of the field emission microscope to approach that of the

conventional transmission electron microscope.Because of the need for near perfect stability in the gun area, the vacuumrequirements are much more critical for the field emission gun.Ions pumps are necessary

to lower the vacuum into the 10-9 to 10-10 torr range.The term "cold cathode" is often applied to the field emission emitter because of

the very low voltage applied to the tungsten crystal.Unlike thermionic emitters, which are

heated to a temperature, which allows electrons to "boil" from the tip, the electrons are

"drawn" from the tip of the field emission emitter as results of the current differential


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between the cathode and the dual anodes of the system.THERMAL FIELD CATHODE (TFE) - A different type of thermionic emitter, which

incorporates the tungsten crystal of the field emitter and the increased heating of the

thermionic systems.This tip cathode makes use of the Shottky effect and is therefore

referred to as the Shottky cathode emitter (SE).This tungsten crystal is coated with a verythin (< 1 atom) layer ofZrO, which reduces the work function of the escaping electrons

by almost one-half.

The following table shows a comparison of the different types of emitters.W LaB6 W(FE) W-ZrO(SE)

Work function(eV) 4.6 2.7 4.5 2.8Current density

(A.cm-2) 1.3(2800K) 25 104 - 106 500

WorkingTemperature (K) 2800 1400-2000 300 1800

Brightness (A.cm-2.sr-1) 5.10

4 -5.105 3.105 5.107 - 2.109 108Crossover diameter

(m) 20 - 50 10 - 20 0.005 - 0.01 0.015Lifetime (H) 25 150 - 200 >1000 >5000

Working Pressure(Pa) 10

-2 - 10-3 10-3 - 10-4 10-7 - 10-8 < 10-6From Armin Delong, Institute of Scientific Instruments of the Academy of Sciences of

the Czech Republic, BrnoThe emission of electrons from a source, in the numbers necessary to produce a

usable beam, is impeded by a potential barrier between the material from which the

electron originate and the necessary vacuum environment within the gun area.Thus, there

are two parameters which restrict the emission of electrons:1) The temperature to which

the emitter must be raised in order to free a sufficient number of electrons into the

vacuum, and 2) the height of the potential barrier of the emitter material.This barrier is

referred to as the work function ( ) of the emitter, and can vary within a range of 1.9

5 eV depending upon the emitter material.

THE ELECTROMAGNETIC LENSThis system introduces the electromagnetic lens into our discussion of electron

microscopy.A simple electromagnetic lens consists of several thousand turns of copper

wire around a soft iron core, while a hole through the center allows for the passage of the

electron beam.By varying the current through the coils, the electromagnetic lens will


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impart upon the electron beam the properties of light rays passing through optical

lenses.Because the current is moving in a circular motion through the coils of the lens,

the electrons are formed into a spiral cone rotating about the lens axis as they pass

through the magnetic flux.This property has no affect on the focusing behavior of the

lens, however.All manipulation of the electron beam is done hereafter by the variouselectromagnetic lenses and their associated apertures.The physical aperture placed in C 2

is used to limit the angular aperture of illumination when the lens is defocused.There are

usually three adjustable apertures in a typical set having openings of 100, 200, and 500

nm.These can be selected and aligned by the user from outside the column.Although the

magnetic flux of the lenses is controlled by the current passing through the coils, the iron

core retains some of the properties of permanent magnetism after all current is

removed.This property is known as remanence and can only be counteracted by reversing

the current of the lens until it is resaturated in the opposite direction.Upon increasing lens

current in the initial direction, the saturation curve will not follow the original curve but

will vary.This phenomenon is called hysteresis and can cause an error of 10% in

computing magnification.This should be taken into consideration when calibrating

magnification of the lens systems.DEPTH OF FIELD AND DEPTH OF FOCUS OF A LENS

The depth of field of a lens is the maximum distance of separation of two object

planes, parallel but not equidistant from the lens, that permits apparent sharpness of focus

in the image of the object concerned; i.e., it is the thickness of the object that may be in

focus at any one lens image plane at any single time.

The depth of field for the optical microscope is less than its resolution limit;

consequently, full advantage of the resolution limit of the lens cannot be realized except

in a very thin object.However, focusing on different planes within the object is possible

and this is an advantage provided "out of focus" adjacent planes (at different depths

within the object) do not produce blurred overlapping images of sufficient opacity to

obscure detail of plane in focus.All planes within object penetration by electron radiation may be focused sharply

as a single image with maximum resolution provided thickness of the object does not

exceed the depth of field.It is therefore concluded that the depth of field of an electron

microscope is much larger than the depth of field for an optical microscope.The depth of focus of a lens is the maximum distance of separation of two image

planes, parallel but not equidistant from the lens, that permits images of an object to

appear in focus; i.e., images of a single object plane will appear to be in focus throughout

the depth of focus.


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Depth of focus of an optical microscope is only 10-20 cm at best while depth of

focus of an electron microscope is for all practical purposes infinite.However, if an object

exposed to an optical microscope is thicker than the depth of field, the practical depth of

focus without interference from other image planes of object positions within the thick

object is essentially equal to the depth of field.Such is not the case in electronmicroscopes until an object thickness is reached (1-2 ) that would not transmit electron

radiation of speed commonly used in electron microscopes.Therefore, the photographic

plate of an electron microscope may be located at any convenient distance below the

observation screen upon which the image is focused without introducing an "out of

focus" image.THE CONDENSER LENS SYSTEM

A simple condenser system is composed of a single, weak lenswhich projects the

concentrated beam onto the specimen plane without undue heat buildup.The single

condenser system, however, cannot concentrate the beam to a brightness required for

high magnification work.Thus the double condenser system is used in all of the modern

electron microscopes.This system uses two stacked condenser lenses; an upper strong

lens and a lower weak lens.The strong lens shortens the focal length allowing the beam to

be more concentrated when it enters the field of the weak second lens.The second lens

then performs the same purpose as a single condenser system, but with a brighter beam

focused on the specimen.The lens gap of the first lens is smaller than the lens gap of the

second , weaker lens, thus allowing the magnetic flux to have greater controlover the

electron beam.This type of lens is often referred to as a polepiece lens, although thisnarrow gap does not constitute a true polepiece.THE OBJECTIVE LENS SYSTEM

This is the most important element of the electron opticsystem.Any defect in the

operation of the objective system will only be magnified by the remaining optics.Not

only must theoptics work perfectly, but the many accessories, which accompany the

objective lens, must also function flawlessly.The lens itself must have a very short focal

length which dictates that it be a powerful magnet with a very small lens gap.This

combination should result in a system that would give truly high magnification and

resultant resolution.However, the size of the lens gap is limited by the amount of "extras"

required by thepractical electron microscope. These include specimen holder, objective

aperture, specimen traverse unit, specimen cooling device, lens cooling coils, etc.As with

lens defects, any problem induced by the accessory equipment will also be compounded

further down the column.The specimen traverse unit must be able to move the specimen over the whole of


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its area without any movement along the Z axis. This allows the microscopist to view the

entire area of the grid without moving it from the plane of focus.The objective apertureis used to enhance the contrast of the resulting image of the

specimen.By limiting the illumination of the specimen image, the electron dense areas

appear darker while the electron opaque areas are changed very little.The objectiveaperture is usually in the 20-50nm range.

Since this is the first lens system which the electron beam will traverse after it

encounters the specimen, here is where true resolution is determined.Using Abbe's

equation, we can compute the theoreticalresolution of any objective lens.This, of course,

does not consider any of the imperfections that can interfere with perfectpractical

resolution.The Abbe' equation is:Limit of Resolution =where 0.612 is constant; is the wavelength in object space; no is the Loschmidt number(the number of molecules per unit volume at 0 C and 1 atm); sine o is the half angle of

the objective lens.The value ofdiffers with the acceleration voltage of the gun; and the

thickness of the specimen is another variable, which must be taken into consideration

when computing resolution.The following table will illustrate this for you, and can be

used as an unrefined "rule of thumb" to assist in the calculation of resolution.RESOLUTION LIMIT vs. SPECIMEN THICKNESS

S) for point at surface of object (M) for point halfway through object Accelerating Potential Limit Object ThicknessResolution

(kV)(cm)()

100 1 x 10-6 8.4 (S)8.4 (M)

100 2 x 10-6 8.6 (S)8.7 (M)

100 4 x 10-6 9.0 (S)9.5 (M)

100 8 x 10-6 11.0 (S)14.0 (M)

100 1 x 10-5 12.4 (S)15.5 (M)

100 2 x 10-5 17.0 (S)


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33.0 (M)100 4 x 10-5 31.5 (S)

85.0 (M)100 8 x 10-5 59.4 (S)

240.0 (M)100 1 x 10-4 75.0 (S)

330.0 (M)50 1 x 10-6 9.0 (S)

9.0 (M)50 2 x 10-6 9.6 (S)

9.8 (M)

50 4 x 10-6 13.0 (S)14.0 (M)

50 8 x 10-6 18.6 (S)24.0 (M)

50 1 x 10-5 22.5 (S)29.5 (M)

50 2 x 10-5 42.0 (S)69.0 (M)

50 4 x 10-5 85.0 (S)180.0 (M)

50 8 x 10-5 175.0 (S)500.0 (M)

THE INTERMEDIATE/PROJECTOR LENS SYSTEMThis system consists of simple electromagnetic lenses that have the singular

responsibility of the magnification of the specimen image.The low magnification image

from the objective lens is further magnified by the intermediate or 1st projector lens

whose image is greatly magnified by the final or 2ndprojector lens.The formula for this

series is simply M0 * M1 * M2 = MT.M0 = the objective, M1 = the intermediate, and M2 =

the projector magnifications.If the objective lens image is 25X, the intermediate image

60X, and the projector 100X, the final magnification would then be 150,000X.This is a

typical series in high magnification microscopy.As you can see, no single lens possesses


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great magnification capabilities; but together they compound the image to a very

highmagnification.The intermediate aperture is a field-limiting aperture only. It is primarily used

during electron diffraction.Its purpose is to limit the area of the specimen image involved

in forming the diffraction pattern.For instance, aperture openings of 400, 200, and 50 diameters correspond to a diffracting specimen area of 8, 4, and 1 diameters

respectively(at reduced magnification 20, 10, and 2.5 diameter).Fig. 3.4THE VIEWING SYSTEM

The radiation produced by the electron beam is not of a wave length that can be

seen by the unaided human eye.Therefore, the final image must be projected in a form

that is useable to the observer.Thus, the viewing screen is coated with a substance that

fluoresces when excited by the bombardment of electrons. This substance usuallyfluoresces a green light because the dark adapted human eye is most sensitive to

green.This aids in the focusing of the image.The viewing system is usually composed of two phosphor coated screens; a large

screen which allows the viewing of the entire part of the specimen through which the

beam has passed, and a smaller focusing screen which is tilted to facilitate the optical

magnification of a small area by the binocular focusing aid.The large screen has zones

etched into the phosphor coating to indicate the approximate areas that will be included in

the micrograph, depending upon the type of film used.THE VACUUM SYSTEM

Any foreign matter inside the column of an electron microscope can deflect the

electron beam and impede the brightness of the image.This material is also carbonized as

the beam strikes it and causes a contamination buildup on the internal surfaces of the

column.Therefore, an efficient highvacuum system is imperative for the operation of the

electron microscope.

Vacuum systems can be complicated and must work in a sequential valving cyclefor accurate operation.The action will be explained in more detail during the following

discourse.The typical vacuum system actually consists of two assemblages connected and

working together.The low vacuum side is composed of one or two mechanical

forepumps, which are capable of vacuums down to the 10-2 Torr range.A Torr denotes


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1mm of mercury.One mechanical pump does nothing except "back" the high vacuum side

while the second mechanical pump "roughs out" the electron microscope system, taking

it from atmosphere to around 10-2 Torr.From here the high vacuum side takes over and

the second type of pump comes into play.This is a three stage oil diffusion pump that is

responsible for lowering the vacuum into the 10-6 Torr range.The oil diffusion pump hasno moving parts save the switching valve at its mouth.The instrument works by heating a

special high viscosity oil to its boiling point and forcing the vapors up through a series of

baffles.The oil vapor is then cooled by a water jacket surrounding the pump and

subsequently forced back down around the inner pump walls. When this occurs, a very

high vacuum is drawn on the upper area of the pump and everything within this area is

drawn down with the oil.The diffusion pump is "backed" by one of the forepumps to

cleanse the contaminants from the oil.(Fig 3.5)Fig. 3.5

OIL DIFFUSION PUMPIt must be remembered never to allow the oil diffusion pump to remain on for any

time without the cooling water or the backing of the forepump.The exclusion of either of

these will result in the "cracking" of the diffusion pump oil.Cracking is the term that

means the diffusion pump oil no longer has the ability to function as a condensable

liquid; and the pump must be removed, cleaned and the oil replaced.Two other high vacuum pumps that you may encounter are the turbomolecular

pump and the ion getter pump.The turbomolecular pump was designed to replace (or

accomplish the same purpose as) the oil diffusion pump.It works much like a turbine,

having stacked, multi?finnedrotors that turn at ultra?high speeds.The turbomolecular

pump can achieve a vacuum of 10-5 torr from atmosphere in a very short period of

time.The makers of these pumps claim no oil backstreaming into the specimen

chamber.There is, however, evidence that some bearing grease contamination does

occur.A major disadvantage of the turbomolecular pump is the weakness of the

bearings.The vanes spin so rapidly that the bearings soon become non-functional and the

pump ceases to work.Several companies advertise pumps with magnetic bearings that are

essentially friction free.The ion getter pump is used in conjunction with the oil diffusion pump or the

turbomolecular pump.It is capable of vacuums down into the 10-12 torr range.These pumps

are usually found on instruments with differentially pumped columns; meaning the gun

area is pumped to a greater vacuum than the chamber area because of the presence of

either a LaB6 emitter or a tungsten single crystal emitter..


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WORKING VACUUM SYSTEMS

INTERESTING FACTS ABOUT VACUUMCourtesy of Sloan Instruments

Santa Barbara, CA

PRESSURE(torr) 760 .01 10-5 10-9 10-12HOW ATTAINED Atmosphere

MechanicalPump DiffusionPump

IonPump BakedIon

PumpMOLECULES/m3 2.5 x 1019 3.2 x 1014 3.2 x 1011 3.2 x 10732,000DISTANCE BETWEENMOLECULES 3.3 nm 38.1 nm .3 cmCOLLISION/SECOND 7 x109 90,000 90 9 x 10-3 9 x 10-6MEAN FREE PATH

BETWEEN COLLISIONS670 nm 0.5 cm 5.1 m 51 km 51,000

kmPATH (English Units) 25 in 3/16 in 5.5 yds 30 mi 30,000

miMOLECULES/SEC/cm2

STRIKING SURFACE 2.9 x 1023 3.8 x 1018 3.8 x 1015 3.8 x 10113.8 x 108

TIME FOR ONE MONO-LAYER TO FORM 3 x 10

-9 sec .23msec .23 sec 38 min 27 daysMONOLAYERS/SEC 3.3 x 108 4400 4.4 .004 4 x 10-7COMPARATIVE NOTE:If molecules were enlarged 220 million times, their .35 nm diameter (O2 & N2) diameterwould appear to be the size of baseballs.On this scale, an inch becomes 3,500 miles and the following relationships would exist:

PRESSURE(torr) 760 .01 10-5 10 10-12BASEBALLSEPARATION 1 yard mile 50 milesPATH OF BASEBALLBETWEENCOLLISIONS

44feet 690 miles 6.9 x 10

5miles 6.9 x 10

9 miles6.9 x 1012miles

PATH EXAMPLES N.Y. toChicago 3 trips tomoon 37 trips to Sun& back 1.2 lightyears

BASEBALL HAILTHICKNESS BUILDUP 1100 ft./sec 4 ft./sec 100in/sec 3.9 x 10

-

3 in/sec

Calculation of velocity of molecules (G)G 3RT/M = 15794T/Mcm/secNote: O2 and N2 travel 340 m/sec at 25 C

Where:M = Molecular weightP = Pressure in mmT = Absolute temperature

Calculation of mean free path (L)L = 8.524 N/PT/M cm G = root mean square velocityR = Gas constant

N = Viscosity in c.g.s.Calculation of mass of gas striking a surface: Useful conversion factors:


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M = 58.32 x 10-3 PM/T gm/cm2/sec 1 = 10-8 cm = 10-4m1in = 25.4 nm1 torr = 1 mm Hg (to 6 decimals)

CHAPTER IVFORMVAR GRID PREPARATION

Particulate specimens are usually placed on mounting support films to facilitate

their observation with the electron microscope.To be practical for use as mounting

supports, the films must meet the following requirements:1)The film must be strong enough to support the specimen.2)The film must exhibit no resolvable structure that is smaller than the specimendetailbeinginvestigated.3)The film must be essentially transparent to the electron beam.The support film is usually a resinous polymer or co-polymer which adheres to

the specimen grid and is supported by the cross bars of the grid.The choice of film type is

often a function ofspecimen type, according to the solvent/film interaction. A polyvinyl formal resin called Formvar is the most popular of the support films,

and the one used in this lab.Formvar is a finely divided powder, white to faint tan in

color, which is soluble in 1,2-dichloroethane, and dissolves to a colorlessliquid.Care must

be taken when working with 1,2-dichloroethane since it is an extremely volatile liquid

and highly flammable.The concentration most used in the lab is 0.8 % by weight, and is

made by adding 0.2989 grams of formvar powder to 29.8 ml of 1,2-dichloroethane.The

solution should be stored in a tightly stoppered bottle because of the volatility of the

solvent.Another popular co-polymer for thin membrane formation is nitrocellulose

dissolved in amyl acetate.Be careful when using this solution; the amyl acetate doesn't

evaporate as rapidly as the 1,2-dichloroethane, and you might mistake this as the film

being too thin.Wait at least three to five minutes for the film to form.FILM FORMATION:

There are basically two methods of forming a support filmfor electron

microscopy.I have no preference as to the technique you use as long as the support films

meet the criteria mentioned above. The first method is the simplest but not necessarily the

best.

Method I : Fill a large vessel with triple distilled water and position it under a

light source in order to produce a good reflection from the surface of the water.Make sure

the surface is very clean.To do this, drop a few drops of the formvar solution on the

surface and allow it to dry.When it has dried, sweep the resulting film from the surface

with a wooden applicator stick.Any contaminant, which was on the surface of the water,


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should have been embedded in the formvar film and removed.When the water is still, put

two more drops on the surface and watch as they dry.Colors should appear on thefilm as

it dries.When it has dried, place 300 mesh grids onto the surface (shiny side down) in the

area where the color appears a very faint gray.This is the area of the film, which is in the

thickness range of 60-100 , and which best fulfills the support film requirements.Gridsshould be placed on the film with the width of a grid space between them. When all the

grids are on the film, a clean glass slide is positioned over thegrids, pushes them under

the water, and sweeps 180 until they are again above the surface of the water; this time

the grids are sandwiched between the formvar film and the glass slide.Stand the slide up

onto a paper towel or filter paper andallow it to dry thoroughly.When dry, the formvar

coated grids are removed from the glass slide by etching around them with the point of

your forceps and carefully picking them up.They may then be transferred to a grid box to

await your pleasure.

Method II :A clean glass slide is dipped into a formvar solution and allowed to

dry by standing it on its end on a paper towel or filter paper.When dry, the sides and end

of the slide are etched with a razor blade.The slide is carefully introduced (end first) into

a vessel of triple distilled water; making sure the thin formvar films separate from the

sides of the slide and float off onto the water.When rectangles of thin film are floating on

the water surface, place grids on them as in the first method, and pick them up in a

similar fashion.PREPARATION OF "HOLEY" FORMVAR GRIDS:

Approximately 10 parts of formvar solution to 1 part distilled water is placed in asmall vial and emulsified by vigorous shaking by hand or with the vortex mixer.Continue

as in Method I, and finish with a thin carbon coating.The extremely small water droplets

prevent the co-polymer from forming a continuous film, and holes form where the

droplets were.

CHAPTER VSHADOW CASTING

The two dimensional image rendered by the transmission electron microscope

often fails to reveal details of specimen surfaces, or structures which project parallel to

the plane of the electron beam.Before the introduction of the scanning electron

microscope, the only method to produce a three dimensional electron beam image was

shadow casting.This process involves the deposition of an electron dense substance upon

the specimen at such an angle that only a part of the specimen is coated.This leaves the

area to the "leeward" side of the specimen uncoated producing a "shadow" of the

specimen. (Fig.4.1).


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Fig.4.1The height of the specimen can be determined by measuringthe length of the shadow (l)

and the angle of the deposition ()using the formula:l = h/tan

The substance deposited is normally a precious metal, carbon, or a combination of

the two evaporated, in the molecular state, from electrodes; and the instrument involvedis called a vacuum evaporator.

Often the evaporated film will appear to be exceptionally granular when observed

with the electron microscope.There are two factors which directly affect the size of the

final particles; the vacuum within the bell jar during the evaporation process and the type

of substance evaporated.The "hardness" of the vacuum is of utmost importance in the shadow casting

process because the final deposition of the film is dependent upon the number of foreign

molecules within the evaporating area.The one exception to this is with the evaporation

of carbon, in which the forepump vacuum will suffice.The thin metal film is obviously formed on the specimen by condensation after

vaporization.It is therefore assumed that the metals with the higher vaporization

temperature will condense more quickly after vaporization, and form finer particle

sizes.Also, the concurrent evaporation of two or more elements will result in smaller

aggregate size by increasing the distance over which any atom must diffuse in order to

secure its place within a crystal lattice.The particle size of a film of evaporated gold will

therefore be larger than that of evaporated platinum or that of a 60/40 alloy of

gold/palladium.The "grain" size of evaporated tungsten is exceedingly fine, butdeposition time is very long and temperature is extremely high.

Another factor to consider is the amount of metal used in the evaporation

process.Too little metal will result in a poor or even non?existent shadow, where too

much will obscure the detail of the specimen which you were hoping to enhance in the

first place.A film of 20?40 will usually give quality results to most specimens.In

determining the amount of metal to use for a certain thickness of film, we must make two

assumptions that may or may not be always true.First, we must assume spherical

geometry of the evaporation process; that is, the same amount of metal will be given off

in all directions from the point of vaporization.The second assumption is that all of the

metal evaporates from the source.This is not always the case, as many metals alloy with

the tungsten filament during the high evaporating temperatures and do not

evaporate.Because of these two factors, as much as two times the metal may be

needed.This will take trial and error to determine as well as a little patients.This not

withstanding, the equation for calculating the correct amount of metal is as follows:


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Where: M= mass of material to be evaporated (g)

t= thickness of the evaporated film (nm)= shadowing angler= source to specimen distance (cm)d= density of material being evaporated (g cm-3)

By using the weight of the metal being used instead of the length of the metal wire, the

researcher isn't limited to using wire only, nor does the researcher worry about the

diameter of the wire being used.CHAPTER VILENS ABERRATIONS

Chromatic AberrationChromatic aberration is the blurring of images of objectpoints both on and off the

principal axis of the lens, due to different wavelengths of radiation upon the object, or

different wavelengths induced into the radiation via passageway through the object.Threefactors that usually cause chromatic aberration are:

1. High voltage fluctuation2. Distribution of velocities from filament3. Change in velocity after passing through specimenThere are two general types of chromatic aberration (although both occur

essentially at the same time).These are:

1.Longitudinal chromatic aberration - different wavelengths converge at differentdistances from the lens position in image space, the shorter wavelengths converge closest

to the lens and the longer wavelengths farther out.The area equidistant from convergent

points would be the area of "best focus" although it would still be blurred.

2.Lateral chromatic aberration - caused by the double convergence of the

extended rays on the periphery of the image space.This causes the short wavelengths to

"pool" toward the center of the image; and the longer wavelengths move to the outside.

ComaA form of lateral spherical aberration which is a blurring effect introduced into

the image of a non-axial object point because of width of aperture and/or a result of the

oblique incidence of rays upon a lens.It derives its name from the "comet-like"

appearance of the "blur" (occurs only for object points off the principal axis of the lens).


27/36

Spherical aberration is a blurring of image points derived from object points

(occurs for object points on or off the principle axis of the lens) due to differing

convergent (or divergent) action by different sites within the lens, or by a difference in

lens strength transmitted through the lens.This is normally caused by an aperture defect

and is one of the major factors limiting resolution due to the radical changes in the focal

point of the lens.It occurs in both positive and negative forms; the positive image

converging in front of the geometric plane while the negative image converges behind the

geometric plane.

Astigmatism:There are two kinds of astigmatism:1. Image of object points in transverse object planes become located in transverse

image planes separated by a finite distance (from object points off the principal axis

only).

2.(Asymmetry) Image of object points (axial & non-axial) in mutually

perpendicular (or almost so) object planes become located in mutually perpendicular

image planes separated by a finite distance.

Curvature of FieldAssociated with astigmatism.Rays from object points in the same object plane do

not converge on "chief rays" after passing through lens, at the same distance from the

paraxial focal plane.A "chief ray" is a ray that passes through the lens without changing

direction (i.e. it strikes the lens perpendicular to the portion of the lens surface concerned.

Distortion: Images of object points in the same object plane are formed in the same focal

plane (via lens) but lateral magnification varies throughout the plane. A. Pin Cushion Distortion - Magnification increases with distance from principal

axis of lens system.Increase in magnification is greater in radial direction than in

circumferential direction.

B. Barrel Distortion - Magnification decreases with distance from principal axis

of the lens system.The decrease is greater in the radial direction than in circumferential


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direction.

C. Rotational Distortion - Actually a form of chromatic aberration that occurs

only in electromagnetic lenses.It is caused by the rotation of the rays of illumination from

the principle axis.

CHAPTER VIIIELECTRON DIFFRACTION

Some investigators need to understand more than just the morphology or size

characteristics of their specimen.This is especially true for those in certain physical

science fields such as geochemistry, metallurgy, crystallography, etc., where structural

information is necessary to compile all of the data necessary for the study.Electron

diffraction is a method by which a "record" of the specimen's structure can be obtained

and photographed, to be used by the investigator for crystal identification and alignment

purposes.Before we discuss the method by which a diffraction pattern is obtained, let's first

look at the diffraction pattern and the variations that we can obtain. ELECTRON DIFFRACTION PATTERNS

There are basically four types of diffraction patterns depending upon the

structural characteristics of the sample.AMORPHOUS PATTERN - Representative of a specimen whose constituent

atoms and molecules are arranged in a random manner with no consistent repeating

structure.The pattern consists of diffuse scattering of electrons, arranged in broad,

concentric circles,around a bright central area as seen in fig 9.1.

Since the diffraction pattern is a representation of the scattering of the electrons as

the main electron beam passes through the specimen, the appearance of the amorphous

pattern depends upon the thickness and density of the material being diffracted.SINGLE CRYSTAL PATTERN -This pattern is representative of the periodic

structure of the specimen, and is imaged as multiple "spots" or points of light around the

central spot.The pattern of spots is dependent upon the alignment of the crystal in respect

to the electron beam.This fact emphasizes the importance of a tilt stage on the electron

microscope when studying single crystal morphology.If the crystal is oriented as in fig.

9.2, the resulting pattern is a square array of spots.

If this single crystal were arrayed in a cubic form instead of a thin crystal form, the


29/36

resulting diffraction pattern would basically be the same as long as the plane of the

crystal remained perpendicular to the electron beam.However, if the cube were to be

rotated and the beam struck the crystal at the point of one of its corners, the resulting

pattern would form an array of spots in a hexagon arrangement around a central spot as

seem in fig. 9.3.

POLYCRYSTALLINE PATTERN - This pattern is indicative of a sample that consist of

a large number of small individual areas which all have the same atomic array, but are

arranged in different orientations.The pattern is actually a large number of single crystal

patterns that are all slightly rotated with respect to each other and therefore form very

thin, concentric rings in the positions where the spots were in the separate single crystal

pattern.PREFERRED ORIENTATION PATTERN - This type of pattern is prevalent

when the grains of a specimen tend to align in a specific orientation as in metal that has

been drawn through a die to form a thin wire.This type of specimen is said to contain

preferred orientation and the resultant diffraction pattern is composed of very close

concentric rings, but with certain portions of the rings at a different brightness than

others.This represents the majority of the grains aligned in certain orientations.Of course, the patterns you will see on the electron microscope will not always be

so clear and definitive.As mentioned earlier, the angle at which the beam strikes the

specimen plays a major role in the resultant diffraction pattern.Often, many trials are

required to achieve good results.

CHAPTER IXPHOTOGRAPHIC THEORY AND TECHNIQUE

Although the electron microscopic image contains a wealth ofinformation, very little use

can be made of it unless it isrecorded for study and interpretation. Thus the camera

becomes a very important part of the electron microscope.Cameras for theelectron

microscope come in many different formats, the mostpopular being the 3 X 4 inch

size.The physics and chemistry involved in the entire photographic process can fill and

has filled volumes.I will present a highly simplified explanation of what happens to a

sheet of film from exposure to print, taking each step in sequence and considering the

factors involved.The Photographic Emulsion:The typical composition of a film is a photosensitive layerin the form of a hard gel, which

is resting on a support substancesuch as glass or a cellulose ester.This photosensitive


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layer isreferred to as the photographic emulsion.The emulsion is achemical suspension

normally composed of the following :1. Gelatin 1.7 g.2. AgBr1.0 g.3. AgI0.03 g.4. AgCl0.02 g.5. KBr0.004 g.6. Water0.3 g.The silver halide grains are the emulsion components thatimpart the

photosensitivity to the gel.They form a discontinuous surface over the film substrate at a

concentrationof approximately 109 grains per square centimeter in a typical photo

emulsion.The silver halide grains vary in size dependingupon the "speed" of the film.The

high speed emulsions carry a grain size of 0.5 to 2.0 in diameter while the low speed

emulsions carry a grain size of 0.02 to 0.5 .The finer the grain size the higher the

resolution of the film.Each of the silver halides is sensitive to a different wavelength of

light (color) and, therefore, necessary to the emulsion in order to impart the full range of

gray levels to the negative or paper.Most photographic emulsions also contain complex

organic molecules to extend the range of grays.

Latent Image FormationA photographic emulsion subjected to radiation fromany source is said to be

"exposed".When this occurs some of the silver salt grains in the emulsion are changed

chemically in a way that renders them "developable" by a chemical system.Thischange inthe grains is called the latent image.Developingaction starts at random, but discrete,

points on the silver grains, known as sensitivity centers.A sensitivity center is considered

to be composed of finely divided (colloidal) silver as a result of the reaction:2AgBr 2Ag

+ Br2.When radiation interacts with the negative bromine ion, an electron isreleased and

taken up by the positive silver ion.This "loose" electron fills the sensitivity center and

initiates latent image formation.Photoprocessing

Development is the process of changing a latent image into avisible image by

reducing the silver ions to metallic silver.The developing (reducing) agent supplies

electrons to the latent image centers allowing more neutralsilver ions to be deposited onto

the centers.These silver ions are the dark areas of the photographic emulsion that

eventuallyform the visual image.The active ingredient in the developingsolution is

hydroquinone, which supplies two electrons per molecule to the photographic

emulsion.Usually, organic developers cannot adequately develop a silver ion by


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themselves, and must have an activator present.This is an alkali, most usually sodium

hydroxide; and the developing process takes place as represented in the following

manner:The reaction between the hydroquinone and sodium hydroxide forms the sodium salt of

the developing agent which dissociates in the water solution.

The two extra electrons from the negative ion can be donated to the silver ions formed

from the silver halide allowing: Ag++e -AgThe normal setup for the negative processing darkroom is asfollows:1.The developing agent (D-19, D-76, Microdol-X, etc.)

2.The stopping bath (usually running water)3.The fixing/hardening agentThe stopping bath is used to "stop'' the action of the developingagent on the

photographic emulsion.This can be in the form of a mild oxidizing agent such as a dilute

acetic acid solution; or,in the negative darkroom, simple immersion in a running water

bath.This action simply dilutes the reducing agent to the point of inactivity. The fixing agent is simply a solvent for the unexposed andundeveloped silver

halide grains.This is usually sodium thiosulfate, pentahydrate that is commonly known as

sodium hyposulfite or simply "hypo". A hardening agent is included in the fix bath totoughen the gelatin against abrasion.Chemicalsfrom the stop bath or hypo should never

be allowed to mix withthe developing bath.The photographic print processing darkroom setup is verysimilar to the negative

setup except that water is almost neverused to stop the action of the developer.Because of

the rapiddeveloping time of prints, an oxidizing agent must be used as astop bath in order

to obtain the correct contrast and overalldensity of the print.Most photographers do not

include a hardener in the hypo of the print darkroom as the dried paperemulsion is quite

tough and the inclusion of a hardener tends to cause the paper to curl excessively.After the developing process is completed, the film or papermust be rinsed in cool

running water for about twenty or thirtyminutes to clear any residual photochemicals

from the emulsion.The film is placed on hangers and allowed to dry, while the papermay

be treated in several ways depending on its type.The resin coated papers are blotted and

allowed to air dry, or are run through a warm air dryer; while the non resin papers must


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either be dried on a ferrotype plate or on a matte dryer.Preparation of Stock Chemicals(For Your Information)FILMFormula I - A modification of Kodak D-76 Developing Solution for use when a

resolution of 1000 is adequate.Develop film 4-6 minutes with agitation at 22-25C.Use solution without dilution.Capacity: 20 sheets of film per liter.Dissolve all

ingredients in order and manner listed.1.Measure 1500 ml of distilled water in a mixing vessel and warm the water to

approximately 50C.2.Dissolve 4.0 to 4.4 grams of p-monoethyl aminophenol sulfate (Elon, Metol, or Pictol)

in the water.

Agitate until visual inspection indicates solid is almost completely dissolved.

3.Dissolve completely 200 to 202.4 grams of anhydrous sodium sulfite in the solution

from 2.4.Dissolve completely 10 to 10.2 grams of hydroquinone in the solution from 3. 5.Dissolve completely 10 to 10.4 grams of sodium tetraborate (Borax) in the solution

form 4.6.Pour solution from 5. into a clean 2000 ml. erlynmeyer flask and carefully add more

distilled water to make a total of 2000 ml.7.Pour this solution into a clean amber jug and seal tightly.

Formula II - Special Fine Grain Developing Solution for use when a resolution of less

than 1000 is required.Develop film 15-18 minutes with agitation at 22-25 C.Capacity:20 sheets of film per liter.1.Boil 3600 ml of distilled water.Let cool to 60 C.2.Dissolve each of the following ingredients to maximum extent in separate small

portions of the freshly boiled water from 1.Try to restrict the amounts of water used to

only that required for complete solution in the case of all ingredients except O-phenylene

diamine and p-monoethyl phenol sulfate.These two ingredients will not dissolve

completely until the two "solutions" have been mixed.(a)o-phenylene diamine 42 grams.Do not use p-phenylenediamine as a

substitute.(b) p-monomethylaminophenol sulfate 42 grams. (Elon, Metol, or Pictol)(c) Sodium sulfite, anhydrous, 330 grams.(d) Potassium metabisulfite (crystal) 36 grams.

3.Mix solutions of (a) and (b) until solution is clear.Add additional boiled water if

necessary.


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4.Add the sodium sulfite solution to solution from 3.Agitate until solution is clear.Add

additional boiled water if necessary.

5.Add the potassium metabisulfite solution to the solution from 4.Agitate until solution is

relatively clear. Add any additional boiled water that remains of the original 3600 ml and

agitate.Filter the solution.6.Do not further dilute the solution from 5.Store as described in the previous formula. 7.Use this developer only in conjunction with a 3.5% acetic acid stop bath.Rinse film

three minutes in stop bath after removing from developer.PAPERFormula III - A modification of Kodak D-72 Stock Developing Solution for use when

prints are made from medium to low contrast film negatives. Develop 90 seconds to two

minutes, with agitation, at 22-25 C.Dilute 1 part stock solution with two parts distilled

water before use.Capacity: 10 8x10" sheets per liter of diluted stock. 1. Follow the procedures described in the preparation of the film developing

solutions, but use the following ingredients and dissolve in order listed.(a) Distilled water (warm, 50 C.); 1,000 ml(b) p-monomethylaminophenol sulfate; 6 grams(c) Sodium sulfite, anhydrous; 120 grams(d) Hydroquinone; 36 grams(e) Sodium carbonate, monohydrated; 160 grams(f) Potassium bromide; 4 grams(g) Distilled water to make 2000 ml

2. Store as previously indicated.3. Dilute one part solution to two parts distilled water before use.Formula IV - Stop bath1.Prepare a 3300 ml stock solution of 28% acetic acid by adding 3 parts (900 ml) of

glacial acetic acid to 8 parts (2400 ml) of distilled water.Caution:Add the acid slowly to

the water with agitation.Do not add the water to the acid.2.Add 125 parts of 28% acetic acid to 1000 parts of distilled water to make a volume of

solutionconvenient for use.Formula V - Fixer Solution1.Prepare a fixer stock solution by dissolving 600 grams of sodium thiosulfate in 2000 ml

of distilled water (60 C.).Cool solution to room temperature before storing in a clean

bottle.2.Prepare a hardener stock solution in accordance with the following formula.Dissolve all


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ingredients in order listed.(a) Distilled water (50 C)-1800 ml(b) Sodium sulfite, desiccated - 225 grams(c) 28% acetic acid - 705 ml(d) Boric acid crystals - 114 grams(e) Potassium Aluminum Sulfate - 225 grams(f) Cold distilled water to make - 3000 ml(g) Store in clean vessel

INDEXAbbe', 1Abbe's equation, 15angle of the deposition, 24anode, 10Astigmatism, 29Asymmetry, 29beam of radiation, 3biased electron gun, 9biased gun system, 9bundle of rays, 3Busch, 1Chromatic Aberration, 26Cold startup, 41Collimation, 3Coma, 27Compensation for Astigmatism, 42CONDENSER LENS, 14Convergence, 3Cracking, 19Curvature of Field, 30Davisson, 1de Broglie, 1depth of field, 14depth of focus, 14Distortion, 30Divergence, 3Driest, 2Einstein, 9electromagnetic lens, 12Electron Diffraction Mode, 42electron microscope, 8electrostatic lens, 10equation for thin lenses, 7filament heating knob, 41filament temperature, 10FILM, 49fixing agent, 48


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focal length, 4focal plane, 4focal point, 4focal points, 4Formvar, 22Geometric Optics, 3

Germer, 1Hall, 1Haine, 9Halske, 2high voltage button, 41high voltage selector, 41Hillier, 2hydroquinone, 47ideal lens, 5image plane, 5Image Space, 4

intermediate aperture, 17INTERMEDIATE/PROJECTOR LENS SYSTEM, 17ion getter pump, 19Krause, 2Latent Image Formation, 46Lateral chromatic aberration, 27lens, 3Lenses, 3Longitudinal chromatic aberration, 26Loschmidt number, 15magnetic flux, 12Maxwell, 6MEASUREMENTS, vimechanical pump, 18metric system, viMuller, 2negative, 4nodal point, 5object plane, 5Object space, 4objective aperture, 15OBJECTIVE LENS, 15oil diffusion pump, 18Optics, 3PAPER, 50pencil of radiation, 3Photographic Emulsion, 46Photoprocessing, 47Physical Optics, 3


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Physiological Optics, 3polepiece, 14positive, 4Prebus, 2Preparation of Stock Chemicals, 49principal axis of a lens, 4

Quantum Optics, 3ray, 3ray diagrams, 6RCA, 2real image, 5Reid, 1remanence, 12Requirements for Satisfactory Completion of Course:, vresolution limits, 9resolving power, 8Ruska, 2

Schrodinger, 1shadow casting, 24Siemens, 2silver halide, 45Spherical aberration, 28stopping bath, 48THE ELECTRON GUN SYSTEM, 9thick lens, 5thin lens, 5Thompson, 1Thomson, 1turbomolecular pump, 19vacuum evaporator, 24VACUUM SYSTEM, 18van Leeuwenhoek, 1Vergence, 3VIEWING SYSTEM, 17virtual image, 6von Borries, 2Wehnelt cylinder, 9X-ray microscope, 1

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Transmission Electron Microscopy Reference Notes

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