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Introduction to Microscopy

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 A
Presentation
on

Introduction to Microscopy
In-partial submission of PhD course work of Advanced Instrument Analysis in chemical Engineering subject under
Chemical Engineering Department
by
Mr. Sameer Sheshrao Gajghate
16EDMER010
PhD Research Scholar

Mechanical Engineering Department

National Institute of Technology Agartala
1
2016-2017
 CONTENT
1. Introduction of Microscopy
2. Types of Microscope
3. Optical Microscope
4. Components of optical microscope
- Confocal optical Microscope
5. Electron microscopy
- Different types of Electron Microscopy
6. SEM
7. Components of SEM
8. Resolution of SEM
9. Application
10. Advantages & Disadvantages
11. TEM
12. Components of TEM
13. Advantages & Disadvantages 2
 Microscopy

• With the advent of high resolution microscopes modern microbiologists have access to
microscope

• That produces images with high clarity & magnification.

• The Leeuwenhoek’s single lens microscope has been transformed into a high resolution
multi-lens combination with magnification upto two thousands time.

• Further electron microscopes with magnification upto one lakh times

• Used to study fine structure of cells and sub- cellular components.

3
 Microscopy Categories
• Microscopes are categories
1. Light (optical) microscopes and 2. Electron microscopes
1.1. Light microscopy involves use of optical lenses and light radiations
• Optical microscopes further categorized as
1. Polarizing Microscope, 2. Reflected Light Microscope,
3. Bright field microscopy (Fig. 5b), 4. Dark field (Fig. 5a),
5. Phase contrast microscopy (Fig. 4.a), 6. Fluorescence microscopy (Fig. 4.(b-c)),
7. Confocal Microscope (Fig. 6)
- Stimulated Emission Depletion (STED) Microscopy
- Laser Scanning Confocal Microscope ,
- Multiphoton Microscope
- Three-Dimensional Optical Microscopy
• Electron microcopy is of two types
1. Transmission microscopy and
4
2. Scanning Electron microscopy
 Light (Optical) Microscope
• Light travels as wave in crests & troughs.
• The amplitude of the crests & troughs determine the brightness of the light.
• The number of time complete wave occur per unit time is called as frequency and the
distance between two consecutive crests is called wavelength (λ) of the light.
• Light microscope wavelength in the range 400-700 nm make up visible spectrum.
• While the UV region consists of wavelengths ranging from 100-385 nm.
• Visualizing any object directly by human eye involves incidence & reflection of light
in the visual range.
• Microscopes use day light or light emitted by incandescent bulb.
• Fluorescent & UV microscope employ UV radiations.

5
 Eyepiece

Revolving nosepiece Arm

Objectives

Stage

Stage clips Coarse adjustment knob

Diaphragm Fine adjustment knob

Light source

Base

Fig. 1. Parts of Optical microscope

6
 Functions of the Major Parts of a Optical Microscope [1-3]
• Light source and Condenser: project a parallel beam of light onto the sample for
illumination
- Condenser gathers light and concentrates it into a cone of light that illuminates the
specimen with uniform intensity over the entire view field [6].
• Sample stage with X-Y movement: sample is placed on the stage and different part of
the sample can be viewed due to the X-Y movement capability
• Fine/ Focusing knobs: since the distance between objective and eyepiece is fixed,
focusing is achieved by moving the sample relative to the objective lens
• Objectives: does the main part of magnification and resolves the fine details on the
samples (mo ~ 10 – 100)
• Eyepiece: forms a further magnified virtual image which can be observed directly with
eyes (me ~ 10)
• Beam splitter and camera: allow a permanent record of the real image from the
objective be made on film (for modern research microscope).
1. http://www.youtube.com/watch?annotation_id=annotation_100990&feature=iv&src_vid=L6d3zD2LtSI&v=ntPjuUMdXbg (I)
2. http://www.youtube.com/watch?v=VQtMHj3vaLg (II) Parts and Function of a Microscope (details)
7
3. http://micro.magnet.fsu.edu/primer/java/kohler/contrast/index.html
 Magnification
• To visualize any tiny object 0.1mm there is limit of unaided Human eye.
• To see micro organism much smaller than 0.1mm a system required convex lenses.
• When parallel rays pass through convex lens get converged at point called as Focal
length (f) of the lens.
• Hence, a lens with shorter focal length will have higher magnification power.
• In compound microscope it will be i.e 10X, f=16mm; 40X, f=4 mm; 100X, f= 1.8mm.
• Image produced by objective lens falls on the eyepiece lens serve as object.
• Image formed in the eyepiece is perceived by one eye.

The overall magnification is given as the product of the lenses and the distance over
which the image is projected:
M = (D *M1 * M2)/ 250 mm – (1)
Where, D = projection (tube) length (usually = 250 mm); M1, M2 = magnification of objective and ocular.
250 mm = minimum distance of distinct vision for 20/20 eyes. 8
 Resolution
• The ability to see two close objects as two distinct objects called resolution.
• The limit of resolution is the closest distance between two points at which the points
still can be distinguished as separate entities.
• Magnification should be coupled with good resolution to visualize small micro
organisms, else magnification alone will produce an inconclusive or blurred image.
• The resolution (Fig. 2.a-c) can be calculated as shown in Eqn. 2.
Maximum resolution: R = (0.061λ)/ N.A – (2)
Where, 0.61 is a geometrical term, based on the average 20-20 eye,
λ = wavelength of illumination, N.A. = Numerical Aperture
- The N.A. is a measure of the light gathering capabilities of an objective lens.
N.A. = n sin α – (3)
Where, n = index of refraction of medium,
α = < subtended by the lens 9
Continued…

(a) (b) (c)

Fig. 2(a, b, c). Numerical aperture & working distance of the 10X, 40X & 100X (oil immersion) objectives of a
compound microscope [4]

4. Philip D. Rack, on “Optical Microscopy”, Dept. of Materials Science and Engineering University of Tennessee & lecture was generated by Professor James Fitz-Gerald at the University of 10
Virginia.
 Continued….

Fig. 3. Ray diagram showing effect of immersion oil on cone of light (Numerical aperture) [5]

5. Gabriel Popescu, “Chapter 4. Principles of Optical Imaging” Electrical and Computer Engineering, University of Illinois at Urbana‐Champaign, Beckman Institute Quantitative 11
Laboratory, Light Imaging http://light.ece.uiuc.edu
 Types of Light (Optical) Microscope

(b)

(a) (c)
Fig. 4. (a)Working of phase contrast microscope, (b) Line diagram of working od a fluorescent microscope & (c) Fluorescent
treponent antibody absorption (FTA-ABS) for diagnosis of syphilis [6]. 12
6. Text of Microbiology websitehttps://www.ikbooks.com/home/samplechapter?filename=147_Sample-Chapter.pdf
Continued…..
Dark Field Microscopy Bright field Microscopy
• an opaque disc is placed underneath • “normal” wide-field illumination method
• the condenser lens • bright background • low contrast
• scattered light
• dark background
• high contrast (structural details)

Fig. 5a. Dark Field Microscopy [7] Fig. 5b. Phase contrast Microscopy [5]

7. http://www.geog.ucl.ac.uk/~jhope/lab/micro23.stm 13
 Confocal Microscope
• In the Confocal microscope (Fig. 6) all
structures out of focus are suppressed at
image formation.
• This is obtained by an arrangement of
diaphragms which, at optically conjugated
points of the path of rays, act as a point of
source and as a point detector respectively.
• Rays from out-of focus are suppressed by the
detection pinhole.
• The depth of the focal plane is, besides the
wavelength of light, determined in particular
by the N. A. of the objective used and the
diameter of the diaphragm.
Fig. 6. Components of Confocal microscope [4]
14
4. Philip D. Rack, on “Optical Microscopy”, Dept. of Materials Science and Engineering University of Tennessee & lecture was generated by Professor James Fitz-Gerald at the University of Virginia.
 Major improvements offered by a confocal microscope
• Over the performance of a conventional microscope may be summarized as follows:
1. Light rays from outside the focal plane will not be recorded.
2. Defocusing does not create blurring, but gradually cuts out parts of the object as they move
away from the focal plane.
3. Thus, these parts become darker and eventually disappear. This feature is called optical
sectioning.
3. True, three-dimensional data sets can be recorded.
4. Scanning the object in x/y-direction as well as in z-direction (along the optical axis) allows
viewing the objects from all sides.
5. Due to the small dimension of the illuminating light spot in the focal plane, stray light is
minimized.
6. By image processing, many slices can be superimposed, giving an extended focus image
which can only be achieved in conventional microscopy by reduction of the aperture and thus
sacrificing resolution.
15
 Applications of Optical Microscopy

1. Crystal morphology and symmetry

• Crystal fragments (characteristic shape)
• Classify isotropic and anisotropic substances
• Check possible symmetry (parallel extinction)
Fig. 7a. Crystal

2. Phase identification, purity and homogeneity

• Standard optical data (refractive indices and
optical axes) for comparison
• Phase analysis (impurities with separated
crystalline/amorphous phase)
• Single vs. twinned crystal
Fig. 7b. Irregular Crystal
16
Continued….

3. Crystal defects – grain boundaries and

dislocations
• Defects always present, even in single
crystal,
Fig. 8a. Crystal structure
• Chemical etching may preferentially
occur at stress sites.

4. Refractive index determination

• Becke line method:
• Sample (n1) is immersed in a liquid (n2),
• Out of focus, light is seen to emerge from
region of higher n.

Fig. 8b. Formation of Becke lines 17

 Advantages and Disadvantages of Optical Microscope
1. Advantages
• Direct imaging with no need of sample pre-treatment, the only microscopy for real
color imaging.
• Fast, and adaptable to all kinds of sample systems, from gas, to liquid, and to solid
sample systems, in any shapes or geometries.
• Easy to be integrated with digital camera systems for data storage and analysis.

2. Disadvantages
• Low resolution, usually down to only sub-micron or a few hundreds of nanometers,
mainly due to the light diffraction limit.

18
 Electron Microscopy
• Electron microscopes are scientific instruments that use a beam of energetic to examine
objects on a very fine scale.
• Electron microscopes use beam of electron in place of light
• Object cannot be perceived by our eyes directly.
• The image produced by electron microscopes is perceived by CRT or X- ray plates.
• Electron microscopes were developed due to the limitations of light microscopes which
are limited by the physics of light.
• in the early 1930’s this theoretical limit had been reached and there was a scientific
desire to see the fines details of the interior structure of organic cells (nucleus,
mitochondria, etc.)
• This required 10,000x plus magnification which was not possible using current optical
microscopes.

19
 Summary of Electron Microscopes components
1. Electron optical column consist of:
- Electron source to produce electrons
- magnetic lenses to de-magnify the beam
- magnetic coils to control and modify the beam
- aperture to define the beam, prevent electron spray, etc.
2. Vaccum systems consists of:
- Chamber which “holds” vaccum, pumps to produce vaccum
- valves to control vaccum, gauges to moniter vaccum
3. Signal detection & display consists of:
- Detectors which collect the signal
- electronics which produce an image from the signal
20
 Different types of Electron Microscopy
1. Scanning Electron Microscope (SEM)
2. Transmission Electron Microscope (TEM)
3. Scanning Transmission Electron Microscope (STEM)
4. Modern Methods of Atomic level microscopy
-Field Ion Microscope
-Scanning Probe Microcopy
a) Atomic force microscopy (AFM), b) Ballistic Electron emission Microscopy (EFM),
c) electrochemical scanning tunneling microscope (ESTM), d) force modulation microscopy (FMM),
e)Kelvin Probe force microscopy (KPFM), f) Magnetic force microscopy (MFM),
g) Magnetic resonance force microscopy (MRFM), h) Near- field scanning optical microscopy (NSOM) or
Scanning Near- field scanning optical microscopy (SNOM), i) Piezo force nmicroscopy (PFM),
j) Photon scanning tunneling microscopy (PSTM), k)Scanning Atom probe (SAP),
l) Scanning electrochemical microscopy (SECM), m) Scanning capacitance microscopy (SCM),
n) Scanning gate microscopy (SGM), o) Scanning ion- conductance microscopy (SICM),
p) Spin Polarized scanning tunneling microscopy (SPSM), q) Scanning thermal microscopy (SThM),
r) Scanning tunneling microscopy (STM), s) Scanning Voltage microscopy (SVM),
t) scanning hall probe microscopy (SHPM).

21
8. P. R. Khangaonkar, 2010. “An Introduction to material characterization”, Penram Int. publishing pvt. Ltd. ISBN978-81-87972-80-8.
 Scanning Electron Microscope (SEM)
Introduction
It provides a valuable combination of high resolution imaging, elemental analysis, and
recently, crystallographic analysis.
It is used for inspecting topographies of specimens at very high magnifications using a
piece of equipment called the scanning electron microscope.
SEM magnifications can go to more than 300,000 X but most semiconductor
manufacturing applications require magnifications of less than 3,000 X only.
It is often used in the analysis of die/package cracks and fracture surfaces, bond failures,
and physical defects on the die or package surface.
Identifying crystalline compounds and determining crystallographic orientations of
microstructural features as small as 1 µm (recently developed capability--not currently
widely used, but likely to become so).

22
 Working of Scanning Electron Microscope
tungsten filament (electron source)
e- electrostatic lens
accelerating voltage anode

electromagnetic lenses
(condenser lenses)

electromagnetic lens (objective lens)

& deflector coils (raster scan)
SE detector (scintillator & PMT)

sample

Fig. 9. Simplified drawing of Scanning Electron Microscope [9]

23
9. Doug, Holly & Oleg, “SEM Microscope”.
 Working Principle
• The main components of a typical SEM are
electron column, scanning system, detector(s),
display, vacuum system and electronics controls
in figure.
• The electron column consists of an electron gun
and two or more Electromagnetic lenses
operating in vacuum.
• The electron gun generates free electrons &
accelerates these electrons to energies in the
range 1-40 keV in the SEM.
• Purpose of the electron lenses is to create a
small, focused electron probe on the Specimen.
• Most SEMs can generate an electron beam at the
specimen surface with spot Size less than 10 nm.
Fig. 10. Working principle of SEM [10] • Max. size of specimen can used upto 2.5 X
10^-7 nm. 24
10. M.T. Postek, K.S. Howard, A.H. Johnson and K.L. McMichael, Scanning Electron Microscopy: A Student’s Handbook, (Ladd Research Ind., Inc Williston, VT., 1980).
continued…..
• In order to produce images the electron beam is focused into a fine probe.
• Its scanned across the surface of the specimen with the help of scanning coils (Fig. 10).
• With a higher accelerating voltage the electron beam penetration is greater and
the interaction volume is larger.
• Therefore, the spatial resolution of micrographs created from those signals will be reduced.
• So there will be a brighter image because the no. of backscattered electrons (BSEs) will
increase but the resolution will be worse.
• For secondary electron (SE) imaging at typical voltages (say 15 keV), BSEs can enter the
secondary electron detector and degrade resolution because they come from deeper in the
sample.
• Complex interactions of the beam electrons with the atoms of the specimen produce wide
variety of radiation.
• In such case knowledge of electron optics, beam-specimen interactions, detection, and
visualization processes is necessary for successful utilization of the power of the SEM.25
 Components of SEM
1. Electron Column
• The electron column is where the electron beam is generated under vacuum (Fig. 11).
• Focused to a small diameter, and scanned across the surface of a specimen by
electromagnetic deflection coils.
• The lower portion of the column is called the specimen chamber.
• The secondary electron detector is located above the sample stage inside the specimen
chamber.
• Specimens are mounted and secured onto the stage which is controlled by a goniometer.
• The manual stage controls are found on specimen chamber and allow for x-y-z
movement, 360 rotation and 90 tilt (Fig. 11).

26
continued

Fig. 11. SEM Column [9]

27
continued
A. Electron gun:
• It located at the top of the column.
• Free electrons are generated by thermionic emission from a tungsten filament at ~2700K.
• The filament is inside the Wehnelt which controls the number of electrons leaving the
gun.
• Electrons are primarily accelerated toward an anode that is adjustable from 200V to 30
kV (1kV=1000V) as shown in Fig. 12.

28
Continued…..

Fig. 12a. Schematic of a generalized W-Hairpin electron source [13]

29
13. From Scanning Electron Microscopy and X-Ray Microanalysis, Joseph I. Goldstein et al. Plenum Press
 Major Electron Beam Parameters
• Four electron beam parameters define the probe
which determine resolution, contrast, and depth of
focus of SEM images:
Probe diameter – dp
Probe current – Ip
Probe convergence angle – αp
Accelerating Voltage – Vo
• These interdependent parameters must be balanced Electron optical brightness, β, is a
by the operator to optimize the probe conditions constant throughout the column, thus
depending on needs: is a very important electron source
Resolution parameter [13] -
Depth of Focus
Image Quality (S/N ratio)
Analytical Performance 30
 Secondary electron Yield

Fig 12c. Secondary electron Yield [10] 31

Continued….

• Fig.12d. Edge effect on secondary electron yield [15]

32
15. Scanning Electron Microscopy: Physics of Image Formation and Microanalysis (Springer Series in Optical Sciences) by Ludwig Reimer and P.W. Hawkes (Hardcover - Oct 16, 1998)
Continued…
B. Condenser Lenses:
• After the beam passes the anode it is
influenced by two condenser lenses that cause
the beam to converge and pass through a focal
point (Fig. 13).
• The electron beam is essentially focused down
to 1000 times its original size.
• It is responsible for determining the intensity
of the electron beam when it strikes the
specimen [10].
(1/u)+ (1/v)= (1/f)
M=v/u Fig. 13. Condenser lens [13]

33
 Continued…..
C. Apertures:
• Depending on microscope one or more apertures
may be found.
• The function of apertures is to reduce and exclude
extraneous electrons in the lenses.
• The final lens aperture located below the scanning
coils determines the dia. or spot size of the beam at Aperture diffraction causes a fundamental
the specimen. limit to the achievable probe size
• It will in partly determine the resolution and depth
of field.
• Decreasing the spot size will allow for an increase
in resolution and depth of field with a loss of
brightness [10].

Fig. 14. Optimum aperture angle determined by combined effect of

spherical aberration and aperture diffraction [13].
34
 Continued…..
D. Scanning System:
• Images are formed by rastering the electron beam
across the specimen using deflection coils inside
the objective lens.
• The stigmator or astigmatism corrector is located
in the objective lens.
• Uses of a magnetic field in order to reduce
aberrations of the electron beam.
• Beam should have a circular cross section when it
strikes the specimen [10,11].
• Contrast from predominately angular dependence
of secondary electron yield and edge effects.
Fig. 15a. Everhart-Thornley SE detector [13]

35
11. I.M. Watt, The Principles and Practice of Electron Microscopy, (Cambridge Univ. Press. Cambridge, England, 1985).
Continued…..

• Fig. 15a. Energy dispersive X-rays detector: Si(Li) [16]

16. A Guide to X-Ray Microanalysis, Oxford Microanalytical Instruments 36

Continued……

E) Specimen Chamber:

• At the lower portion of the column the specimen stage and controls are located.

• The secondary electrons from the specimen are attracted to the detector by a
positive charge.

37
 Continued…..
2. Vacuum System
• To provide a controlled electron beam requires column be under vacuum at a pressure of at
least 5x10^-5 Torr.
• A high vacuum pressure is required for a variety of reasons.
• First, the current that passes through the filament reach temperatures around 2700K [12].
• A filament will oxidize and burn out in the presence of air at atmospheric pressure.
• Secondly, the column optics to operate properly requires a fairly clean, dust-free
environment.
• Third, air particles and dust inside the column can interfere and block the electrons [10].
• In order to provide adequate vacuum pressure inside the column, a vacuum system
consisting of two or more pumps is typically present.

12. C.E. Lyman, D.E. Newbury, J.I. Goldstein, D.B. Williams, A.D. Romig, J.T. Armstrong, P. Echlin, C.E. Fiori, D.C. Joy, E. Lifshin and Klaus-Ruediger Peters, Scanning Electron 38
Microscopy, X-Ray Microanalysis and Analytical Electron Microscopy: A Laboratory Workbook, (Plenum Press. New York, N.Y., 1990).
Continued….

3. Electron Beam-Specimen Interactions

• To achieve higher resolutions, an electron source is
required instead of light as the illumination source.
• which allows for resolutions of about 25 Angstroms.
• The use of electrons not only gives better resolution
but, due to the nature electron beam specimen
interactions there are a variety of signals
• that can be used to provide information regarding
characteristics at and near the surface of a specimen.

Fig. 16. Signals that result from electron beam-

specimen interaction [14].
14. C.E. Lyman, D.E. Newbury, J.I. Goldstein, D.B. Williams, A.D. Romig, J.T. Armstrong, P. Echlin, C.E. Fiori, D.C. Joy, E. Lifshin and Klaus-Ruediger Peters, Scanning Electron 39
Microscopy, X-Ray Microanalysis and Analytical Electron Microscopy: A Laboratory Workbook, (Plenum Press.New York, N.Y., 1990).
Electron Beam/ specimen interaction

• Fig. 12b. Electron Beam interaction [13]

40
 Some Common errors & images of SEM

1. Image Disturbances

2. Effect of Accelerating Voltage

3. Effect of Working Distance

4. Effect of Spot Size

Fig. 17. Reactive ion etching of Al/Si(001)

(a) (b)
Fig. 18. Secondary a) Electron image & b) Backscattered Electron image 41
Continued….

(a) (b)
Fig. 17. a) ZnO Nano wires & b) Carbon Nano-tubes
42
 Advantages & Disadvantages
Advantages
• High resolution & Depth of focus (1 X 10^-6 nm)
• Elemental analysis attachments
• Almost all kinds of samples, conducting and non-conducting (stain coating needed)
• Based on surface interaction --- no requirement of electron-transparent sample.
• Imaging at all directions through x-y-z (3D) rotation of sample.
Disadvantages
 Cost
 More knobs
 Vacuum
 Low resolution, usually above a few tens of nanometers.
 Usually required surface stain-coating with metals for electron conducting.
43
 Transmission electron microscopy (TEM),

Introduction

• In it a beam of highly focused electrons are directed toward a thinned sample (<200 nm).

• Normally no scanning required --- helps the high resolution, compared to SEM.

• These highly energetic incident electrons interact with the atoms in the sample producing
characteristic radiation

• and particles providing information for materials characterization.

• Information is obtained from both deflected and non-deflected transmitted electrons,

backscattered and secondary electrons, and emitted photons.

44
 Sampling on a TEM?

• Lower resolution/large area techniques should be first performed to get a ‘broad picture’
about the sample.
• This includes XRD and SEM. We can even start with optical microscopy.
• Phase related information should be obtained via XRD.
• Chemical information via EDX in SEM should also be obtained (any chemical
inhomogeneity should be noted).
• On ‘usual’ samples conventional TEM should be performed before trying out HRTEM.

45
 Operating conditions of TEM

• The wavelength of the electrons in a 10 kV SEM is 12.3 x 10−12 m (12.3 pm)

• In a 200 kV TEM the wavelength is 2.5 pm

• Wavelength of X-rays usually used in XRD is in the order of 100 pm (Cu kα: λ=154 pm).

Instrument Operating Voltage λX-ray or λElectron

X-rays - 1.54 Å
SEM 10 kV 0.12 Å
TEM 200 kV 0.02 Å

46
 The TEM sample & the projected image
• The sample is a thin 3mm disc. The central portion of the disc is thinned down further to make
it electron transparent (< 1000 Å in thickness).
• The process of sample preparation usually leaves a hole(s) in the ‘middle’ with electron
transparent region next it.
• The thin regions in the sample can bend.
• The standard image seen on the screen or captured in the camera is a projected image
integrated through the thickness.

47
 Introduction to TEM
The first transmission electron microscope was invented in 1933 by Max Knoll and Ernst
Ruska at the Technical College in Berlin.

Fig. 18. A transmission Electron Microscope is anologous to a slide projector as indicated by Philips above [17].
48
17. A transmission Electron Microscope is anologous to a slide projector as indicated by Philips
Instrument components

Electron gun
Condenser system (lenses & apertures for
controlling illumination on specimen)
Specimen chamber assembly
Objective lens system
A. image - forming lens - limits resolution;
B. Aperture - controls imaging conditions
Projector lens system (magnifies image or
diffraction pattern onto final screen)

49
Working Principles
Working
1.In a conventional transmission electron
microscope, a thin specimen is irradiated with
an electron beam of uniform current density.
2.Electrons are emitted from the electron gun and
illuminate the specimen through a two or three
stage condenser lens system.
3.Objective lens provides the formation of either
image or diffraction pattern of the specimen.
4.The electron intensity distribution behind the
specimen is magnified with a three or four stage
lens system and viewed on a fluorescent screen.

50
Continued….

Fig. 19. Line diagram of TEM 51

 Examples

Matrix - '-Ni2AlTi SiO2 precipitate dislocations

particle in Si in superalloy
Precipitates - twinned L12 type '-Ni3Al
52
 TEM Facility
• TEM provides information of:
- Structural
- Chemical
Modes
- Imaging
- Diffraction
1. In Tem we can switch from an image (Real Space) to diffraction (reciprocal space) by the
switch of a button.
2. We can get spectroscopic information and use it for forming an image or diffraction
patterns.

53
 TEM components
1. Electron gun
• At the top of the column, the electron gun delivers high-energy electrons to the instrument.
• Thermionic guns (tungsten or LaB6) are the most common types.
• The appropriate electron energy depends on the nature of the specimen and the kind of
information required.
• Higher electron energies allow thicker samples to be analysed and, due to their smaller
wavelengths, increase the resolution possible;
• however, it is rare now to see TEMs which operate at energies greater than 200 keV.
• The introduction of field emission guns and improvements in lens design have largely
made higher-energy microscopes unnecessary for high-resolution.
• Additionally, higher energy electrons cause increasing amounts of damage to samples.
Biological samples in particular require lower operating voltages.

54
Continued….
2. Condenser lens system
3. Specimen chamber
4. Objective and intermediate lenses
5. Vacuum System
a. Roughing Pump (Rotary Pump)
b. Diffusion Pump
c. Turbo-molecular Pump/Turbo Pump
d. Ion Pump/Ion Getter Pump/Sputter Ion Pump
e. Cryogenic/Adsorption Pumps
f. Vacuum Tube Gauge (Pirani Gauge)
g. Ion Discharge Gauge (Penning Gauge)

55
 Image Contrast in the TEM
• TEM images are simply magnified images of the electron intensity on the bottom surface of
the specimen and contrast arises only if the intensity varies significantly from one region to
another.
1. Absorption contrast
• Examination of samples which are thicker, denser, or with higher atomic number allow fewer
electrons to pass through
• It applies to both amorphous and crystalline specimens and is used extensively by biologists
who call it mass-thickness.
• In crystalline samples, this contrast mechanism is usually swamped by others.
2. Diffraction Contrast
• Diffraction contrast is simply a function of the diffraction conditions.
• It is the method most commonly used to study crystal defects like dislocations, stacking
faults, precipitates, etc.
• It is the mechanism which explains extinction (bend) contours and thickness fringes.
56
Continued….

57
Continued….

58
Continued……

59
Continued….
• We see something if: (i) Light (visible part of the spectrum) enter our eyes, (ii) the light has
sufficient intensity, (iii) There is sufficient contrast in the image.
• Contrast is a dimensionless number as defined below. It is to be noted that contrast is not the
difference in intensity between the light (I1) and dark (I2) regions, but the difference divided
by the (say) higher of the two intensities (I2).
• A contrast value of 5-10% can be picked discerned by our eyes.
• We have strong or weak contrast* (but not bright or dark contrast).

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Continued….
Contrast are of two types
1. Amplitude Contrast 2. Phase contrast
1.1 Diffraction contrast (Coherent elastic scattering )
- Bright Field & Dark Field Images
1.2 Absorption contrast (In-coherent elastic scattering )
- Mass-thickness contrast → small effect in a thin specimen
2. Phase contrast are of
• Thickness Fringes
• Bend Contours
• Fresnel Fringes
• Moiré Patterns - Beats is the time analogue of Moiré patterns
• Phase contrast
• Lattice Fringes 61
Continued….

62
Continued….

63
Continued….

64
65
Continued….
Phase contrast and high-resolution imaging
• Unlike absorption and diffraction contrast
mechanisms, which rely on the amplitude of
scattered waves, phase contrast results whenever
electrons of a different phase pass through the
objective aperture.
• If spots along a systematic row are allowed
through, a lattice image is formed.
10 nm
• Such images can be used to show the extent of
crystallisation of a grain-boundary film or the
habit plane of planar defects.
• If more diffracted beams are allowed to
contribute, then a structure image can be formed.

66
 Resolution
• Resolution of human eyes ~ (0.1 – 0.2) mm.
• Highest useful magnification is governed by the resolution.
• Raleigh criterion is used for the definition of resolution.
• Optical Microscope; δ= 0.61λ/μsinβ (Green light λ=550nm, δ=300nm, 1000 atomic
diameter)
• δ= λ/2, δ → Smallest distance that can be resolved, λ → Wavelength of radiation
β → Semi-angle of collection & μ → Refractive index of viewing medium
• TEM
λ ~ 1.22/√E (Eλ100keV electrons → λ =4 pm )
• Aspects determining the resolution -
1.Wavelength of the radiation
2. Aberration of the lenses in the imaging system
3. Diffraction limit of the imaging system
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 Requirement of interpretable images
1. very thin (about 5nm) specimens. If they are too thick, inelastic scattering degrades the
phase contrast information.
2. to be at a zone axis so that many beams are available and the crystallographic information
is interpretable.
3. Only those diffracted beams which correspond to distances within the point resolution of
the HREM will contribute to the image.
4. precise alignment of the electron beam down the optic axis. Also, any defects must lie
along the beam direction.
5. coherent illumination (i.e., LaB6 filament or FEG).
6. a specific value of objective defocus. In order to establish this value, the quantitative
defocus associated with each “click” of the objective focus control must be calibrated.

68
Continued…

• As the image obtained is a function of a number of variables, these must be defined and
calibrated in order to interpret the image.
1. Sample thickness
2. Objective lens defocus
3. Microscope parameters like kV, Cs, Cc, etc.
4. Number and type of beams included in the objective aperture.
5. Beam tilt

69
 Advantages and disadvantages of TEM

Advantages

• High resolution, as small as 0.2 nm.

• Direct imaging of crystalline lattice.

• Delineate the defects inside the sample.

• No metallic stain-coating needed, thus convenient for structural imaging of organic materials,

• Electron diffraction technique: phase identification, structure and symmetry determination,

lattice parameter measurement, disorder and defect identification.

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Continued….
Disadvantages
• Low sampling volume and rather slow process of obtaining information.
• High capital and running cost.
• Special training required for the operation of the equipment.
• Difficult sample preparation. Possibility of electron beam damage.
• Samples which are not stable in vacuum are difficult to study.
• Magnetic samples require special care.
• Non-conducting samples require gold or carbon coating.
• Difficulty in the interpretation of images. In usual mode of operation information is
integrated along the beam direction.

71
 References
1.http://www.youtube.com/watch?annotation_id=annotation_100990&feature=iv&src_vid=L6d3zD2LtSI&v=ntPjuUMdXbg (I)
2. http://www.youtube.com/watch?v=VQtMHj3vaLg (II) Parts and Function of a Microscope (details)
3. http://micro.magnet.fsu.edu/primer/java/kohler/contrast/index.html
4. Philip D. Rack, on “Optical Microscopy”, Dept. of Materials Science and Engineering University of Tennessee & lecture was generated
by Professor James Fitz-Gerald at the University of Virginia.
5. Gabriel Popescu, “Chapter 4. Principles of Optical Imaging” Electrical and Computer Engineering, University of Illinois at
Urbana‐Champaign, Beckman Institute Quantitative Laboratory, Light Imaging http://light.ece.uiuc.edu
6. Text of Microbiology websitehttps://www.ikbooks.com/home/samplechapter?filename=147_Sample-Chapter.pdf
7. http://www.geog.ucl.ac.uk/~jhope/lab/micro23.stm
8. P. R. Khangaonkar, 2010. “An Introduction to material characterization”, Penram Int. publishing pvt. Ltd. ISBN978-81-87972-80-8.
9. Doug, Holly & Oleg, “SEM Microscope”.
10. M.T. Postek, K.S. Howard, A.H. Johnson and K.L. McMichael, Scanning Electron Microscopy: A Student’s Handbook, (Ladd Research
Ind., Inc Williston, VT., 1980).
11. I.M. Watt, The Principles and Practice of Electron Microscopy, (Cambridge Univ. Press. Cambridge, England, 1985).
12. C.E. Lyman, D.E. Newbury, J.I. Goldstein, D.B. Williams, A.D. Romig, J.T. Armstrong, P. Echlin, C.E. Fiori, D.C. Joy, E. Lifshin and
Klaus-Ruediger Peters, Scanning Electron Microscopy, X-Ray Microanalysis and Analytical Electron Microscopy: A Laboratory
Workbook, (Plenum Press. New York, N.Y., 1990).
13. From Scanning Electron Microscopy and X-Ray Microanalysis, Joseph I. Goldstein et al. Plenum Press
14. All the remaining figures are taken from google.com website
15. Scanning Electron Microscopy: Physics of Image Formation and Microanalysis (Springer Series in Optical Sciences) by Ludwig Reimer
and P.W. Hawkes (Hardcover - Oct 16, 1998)
16. A Guide to X-Ray Microanalysis, Oxford Microanalytical Instruments
17. A transmission Electron Microscope is anologous to a slide projector as indicated by Philips
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 Thank you……

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