Review Article TRANSMISSION ELECTRON MICROSCOPY-AN OVERVIEW

Abstract

Since its invention, electron microscope has been a valuable tool in the development of scientific theory and it contributed greatly to biology, medicine and material sciences. This wide spread use of electron microscopes is based on the fact that they permit the observation and characterization of materials on a nanometer (nm) to micrometer (µm) scale. The study of properties of small particles is one of the important lines of modern physical electronics because of their mesoscopic size. Transmission Electron Microscopy (TEM) has long been used in materials science as a powerful analytical tool. In transmission electron microscopy (TEM), a thin sample, less than 200 nm thick, is bombarded by a highly focused beam of single-energy electrons. The beam has enough energy for the electrons to be transmitted through the sample, and the transmitted electron signal is greatly magnified by a series of electromagnetic lenses. Transmission Electron Microscope (TEM) combined with precession 3D electron diffraction tomography technique has produced very promising results in the field of crystal structure determination and has the great advantage of requiring very small single crystals (from 25-500 nm) and very small quantity of material.

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International Research Journal for Inventions in Pharmaceutical Sciences, September 2013 Vol 1 Issue 2 1
International Research Journal for Inventions in
Pharmaceutical Sciences
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Review Article
TRANSMISSION ELECTRON MICROSCOPY- AN OVERVIEW
Rukari Tushar G*1, Alhat Babita R1
1VJSM’S Institute of Pharmacy Ale, Junnar, Pune 411 412 Maharashtra, India..
Email id: princetush@gmail.com
Article Received on: 21/07/13, Revised on: 01/08/2013, Approved for publication: 25/08/2013
Abstract
Since its invention, electron microscope has been a valuable tool in the development of scientific theory
and it contributed greatly to biology, medicine and material sciences. This wide spread use of electron
microscopes is based on the fact that they permit the observation and characterization of materials on a
nanometer (nm) to micrometer (µm) scale. The study of properties of small particles is one of the
important lines of modern physical electronics because of their mesoscopic size. Transmission Electron
Microscopy (TEM) has long been used in materials science as a powerful analytical tool. In transmission
electron microscopy (TEM), a thin sample, less than 200 nm thick, is bombarded by a highly focused
beam of single-energy electrons. The beam has enough energy for the electrons to be transmitted through
the sample, and the transmitted electron signal is greatly magnified by a series of electromagnetic lenses.
Transmission Electron Microscope (TEM) combined with precession 3D electron diffraction tomography
technique has produced very promising results in the field of crystal structure determination and has the
great advantage of requiring very small single crystals (from 25-500 nm) and very small quantity of
material.
Keywords: Electron microscope (EM), Transmission electron microscopy (TEM), 3D electron diffraction
tomography technique etc.
Introduction:
Characterization of crystal form is an
important issue in pharmaceutical materials
science.1,2 The existence of polymorphs, for
example, is considered to be a key concern,3–5
as is the stability of a chosen form to various
processing (and storage) conditions, for
example, milling and tableting.6
Address for correspondence:
Rukari Tushar G
E mail: princetush@gmail.com
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The recent emergence of pharmaceutical
cocrystals as alternatives to salts and
amorphous forms is recognized,7 but raises
questions during development of the
importance that will need to be attached to the
purity of the phases produced, for example,
components of the target cocrystal present as
‘‘impurity’’ phases or the possible generation
of small amounts of a different cocrystal
stoichiometry beyond the usual detection limits
of routine analytical methods such as powder
X-ray diffraction (PXRD). X-Ray powder
diffraction (XRPD) has already successfully
been used for a long time in the field of
pharmaceuticals for polymorph screening
(fingerprinting) and more recently for the
structural and microstructural analysis of active

Rukari Tushar g et al. Transmission Electron Microscopy- An Overview
International Research Journal for Inventions in Pharmaceutical Sciences, July 2013 Vol 1 Issue 2 2
pharmaceutical ingredients (API) and
excipients. Large enough single crystals are in
fact either very difficult to growth or do not
represent well the properties of the
polycrystalline pharmaceutical compounds.
Furthermore quantitative phase analyses can
only be performed with powder materials.
Although XRPD is well established, there are
often limitations in its successful application,
in particular when facing complex structures
whose XRPD patterns are characterized by
many overlapping peaks and the determination
of the cell parameters and the extraction of the
diffracted intensity is very difficult and often
unsuccessful. In such cases the use of
synchrotron radiation and advanced
experimental methods need to be explored (e.g.
texture method8, anisotropic thermal expansion
9) but the determination of the crystal structure
is never guaranteed and far from being routine.
Transmission electron microscopy
(TEM) has long been used in materials
science as a powerful analytical tool.9–14
Transmission electron microscopy (TEM)
is a microscopy technique whereby a beam of
electrons is transmitted through an ultra-thin
specimen, interacting with the specimen as it
passes through. An image is formed from the
interaction of the electrons transmitted
through the specimen; the image is magnified
and focused onto an imaging device, such as a
fluorescent screen, on a layer of photographic
film, or to be detected by a sensor such as a
CCD camera (Figure 1 & Figure 2). 16
Figure 1: The first practical TEM
Figure 2- Layout of optical components in a
basic TEM
Although TEM has been less widely
applied to the study of organic materials, primarily
due to perceived difficulties with sample
preparation and beam damage, recent studies of
pharmaceutical compounds have shown not only
that these difficulties can be overcome, but also
that TEM analysis can provide a range of useful
information about samples that cannot be obtained
by other, more routinely used, analytical methods.
17,18
INSTRUMENTATION:
Source formation:
From the top down, the TEM consists of
an emission source, which may be a tungsten
filament, or a lanthanum hexaboride (LaB6)
source. For tungsten, this will be of the form of
either a hairpin-style filament, or a small spike-
shaped filament (Figure 3 & Figure 4). LaB6
sources utilize small single crystals. By connecting
this gun to a high voltage source (typically ~100–
300 kV) the gun will, given sufficient current,
begin to emit electrons either by thermionic or
field electron emission into the vacuum. This
extraction is usually aided by the use of a Wehnelt
cylinder. Once extracted, the upper lenses of the
TEM allow for the formation of the electron probe
to the desired size and location for later interaction
with the sample. Manipulation of the electron
beam is performed using two physical effects. The
interaction of electrons with a magnetic field will
cause electrons to move according to the right hand
rule, thus allowing for electromagnets to
manipulate the electron beam. The use of magnetic

Rukari Tushar g et al. Transmission Electron Microscopy- An Overview
International Research Journal for Inventions in Pharmaceutical Sciences, July 2013 Vol 1 Issue 2 3
fields allows for the formation of a magnetic lens
of variable focusing power, the lens shape
originating due to the distribution of magnetic flux.
Additionally, electrostatic fields can cause the
electrons to be deflected through a constant angle.
Coupling of two deflections in opposing directions
with a small intermediate gap allows for the
formation of a shift in the beam path, this being
used in TEM for beam shifting.16
Figure 3- Single crystal LaB6 filament.
Figure 4- Hairpin style tungsten filament
Optics:
The lenses of a TEM allow for beam convergence,
with the angle of convergence as a variable
parameter, giving the TEM the ability to change
magnification simply by modifying the amount of
current that flows through the coil, quadrupole or
hexapole lenses. The quadrupole lens is an
arrangement of electromagnetic coils at the vertices
of the square, enabling the generation of a lensing
magnetic fields, the hexapole configuration simply
enhances the lens symmetry by using six, rather
than four coils.
Typically a TEM consists of three stages of
lensing. The stages are the condensor lenses, the
objective lenses, and the projector lenses. The
condensor lenses are responsible for primary beam
formation, whilst the objective lenses focus the
beam that comes through the sample itself (in
STEM scanning mode, there are also objective
lenses above the sample to make the incident
electron beam convergent). The projector lenses
are used to expand the beam onto the phosphor
screen or other imaging device, such as film. The
magnification of the TEM is due to the ratio of the
distances between the specimen and the objective
lens' image plane. 16
Display
Imaging systems in a TEM consist of a phosphor
screen, which may be made of fine (10–100 µm)
particulate zinc sulphide, for direct observation by
the operator. Optionally, an image recording
system such as film based or doped Yttrium-
Aluminum Garnet (YAG) screen coupled CCDs.
Typically these devices can be removed or inserted
into the beam path by the operator as required.16
Vacuum system:
To increase the mean free path of the electron gas
interaction, a standard TEM is evacuated to low
pressures, typically on the order of 10−4 Pa. The
need for this is twofold: first the allowance for the
voltage difference between the cathode and the
ground without generating an arc, and secondly to
reduce the collision frequency of electrons with gas
atoms to negligible levels—this effect is
characterized by the mean free path. TEM
components such as specimen holders and film
cartridges must be routinely inserted or replaced
requiring a system with the ability to re-evacuate
on a regular basis. As such, TEMs are equipped
with multiple pumping systems and airlocks and
are not permanently vacuum sealed. Sections of the
TEM may be isolated by the use of pressure-
limiting apertures, to allow for different vacuum
levels in specific areas, such as a higher vacuum of
10−4 to 10−7 Pa or higher in the electron gun in
high-resolution or field-emission TEMs. High-
voltage TEMs require ultra-high vacuums on the
range of 10−7 to 10−9 Pa to prevent generation of an
electrical arc, particularly at the TEM cathode.16

Rukari Tushar g et al. Transmission Electron Microscopy- An Overview
International Research Journal for Inventions in Pharmaceutical Sciences, July 2013 Vol 1 Issue 2 4
Specimen stage:
TEM specimen stage designs include airlocks to
allow for insertion of the specimen holder into the
vacuum with minimal increase in pressure in other
areas of the microscope. The specimen holders are
adapted to hold a standard size of grid upon which
the sample is placed or a standard size of self-
supporting specimen. Standard TEM grid sizes are
a 3.05 mm diameter ring, with a thickness and
mesh size ranging from a few to 100 µm. The
sample is placed onto the inner meshed area having
diameter of approximately 2.5 mm usual grid
materials are copper, molybdenum, gold or
platinum. This grid is placed into the sample
holder, which is paired with the specimen stage. A
wide variety of designs of stages and holders exist,
depending upon the type of experiment being
performed. In addition to 3.05 mm grids, 2.3 mm
grids are sometimes, if rarely, used. These grids
were particularly used in the mineral sciences
where a large degree of tilt can be required and
where specimen material may be extremely rare.
Electron transparent specimens have a thickness
around 100 nm, but this value depends on the
accelerating voltage.16
Electron gun
The electron gun is formed from several
components: the filament, a biasing circuit, a
Wehnelt cap, and an extraction anode. By
connecting the filament to the negative component
power supply, electrons can be "pumped" from the
electron gun to the anode plate, and TEM column,
thus completing the circuit. The gun is designed to
create a beam of electrons exiting from the
assembly at some given angle, known as the gun
divergence semi angle, α. By constructing the
Wehnelt cylinder such that it has a higher negative
charge than the filament itself, electrons that exit
the filament in a diverging manner are, under
proper operation, forced into a converging pattern
the minimum size of which is the gun crossover
diameter. The thermionic emission current density
J, can be related to the work function of the
emitting material and is a Boltzmann distribution
given below, where A is a Richarson constant, Φ is
the work function, T is the temperature of the
material and K is Boltzmann constant.
This equation shows that in order to achieve
sufficient current density it is necessary to heat the
emitter, taking care not to cause damage by
application of excessive heat, for this reason
materials with either a high melting point, such as
tungsten, or those with a low work function (LaB6)
are required for the gun filament (Figure 5). 16
Figure 5- Cross sectional diagram of an electron
gun assembly, illustrating electron extraction
Electron lens:
Electron lenses are designed to act in a manner
emulating that of an optical lens, by focusing
parallel rays at some constant focal length. Lenses
may operate electrostatically or magnetically. The
majority of electron lenses for TEM utilize
electromagnetic coils to generate a convex lens.
For these lenses the field produced for the lens
must be radially symmetric, as deviation from the
radial symmetry of the magnetic lens causes
aberrations such as astigmatism, and worsens
spherical and chromatic aberration. Electron lenses
are manufactured from iron, iron-cobalt or nickel
cobalt alloys, such as Permalloy. These are
selected for their magnetic properties, such as
magnetic saturation, hysteresis and permeability.
The components include the yoke, the magnetic
coil, the poles, the polepiece, and the external
control circuitry. The pole piece must be
manufactured in a very symmetrical manner, as
this provides the boundary conditions for the
magnetic field that forms the lens. Imperfections in
the manufacture of the polepiece can induce severe
distortions in the magnetic field symmetry, which
induce distortions that will ultimately limit the
lenses' ability to reproduce the object plane. The
exact dimensions of the gap, pole piece internal

Rukari Tushar g et al. Transmission Electron Microscopy- An Overview
International Research Journal for Inventions in Pharmaceutical Sciences, July 2013 Vol 1 Issue 2 5
diameter and taper, as well as the overall design of
the lens is often performed by finite element
analysis of the magnetic field, whilst considering
the thermal and electrical constraints of the design
(Figure 6).The coils which produce the magnetic
field are located within the lens yoke. The coils can
contain a variable current, but typically utilize high
voltages, and therefore require significant
insulation in order to prevent short circuiting the
lens components. Thermal distributors are placed
to ensure the extraction of the heat generated by
the energy lost to resistance of the coil windings.
The windings may be water-cooled, using a chilled
water supply in order to facilitate the removal of
the high thermal duty. 16
Figure 6- Diagram of a TEM split polepiece
design lens.
Apertures:
Apertures are annular metallic plates, through
which electrons that are further than a fixed
distance from the optic axis may be excluded.
These consist of a small metallic disc that is
sufficiently thick to prevent electrons from passing
through the disc, whilst permitting axial electrons.
This permission of central electrons in a TEM
causes two effects simultaneously: firstly,
apertures decrease the beam intensity as electrons
are filtered from the beam, which may be desired
in the case of beam sensitive samples. Secondly,
this filtering removes electrons that are scattered to
high angles, which may be due to unwanted
processes such as spherical or chromatic
aberration, or due to diffraction from interaction
within the sample. Apertures are either a fixed
aperture within the column, such as at the
condenser lens, or are a movable aperture, which
can be inserted or withdrawn from the beam path,
or moved in the plane perpendicular to the beam
path. Aperture assemblies are mechanical devices
which allow for the selection of different aperture
sizes, which may be used by the operator to trade
off intensity and the filtering effect of the aperture.
Aperture assemblies are often equipped with
micrometers to move the aperture, required during
optical calibration.16
Sample preparation:
Preparation of TEM specimens is specific
to the material under analysis and the desired
information to obtain from the specimen. As such,
many generic techniques have been used for the
preparation of the required thin sections. Materials
that have dimensions small enough to be electron
transparent, such as powders or nanotubes, can be
quickly prepared by the deposition of a dilute
sample containing the specimen onto support grids
or films. In the biological sciences in order to
withstand the instrument vacuum and facilitate
handling, biological specimens can be fixated
using either a negative staining material such as
uranyl acetate or by plastic embedding. Alternately
samples may be held at liquid nitrogen
temperatures after embedding in vitreous ice.16
APPLICATIONS OF TRANSMISSION
ELECTRON MICROSCOPY:
1. The theoretical prediction of coexistence
of fcc-like and multiple twined AuCu
nanoparticles.19
2. TEM studies of functional biomolecules
and bio structures, where charge
interactions are known to play a dominant
role.20
3. Identification of defects in samples of
theophylline, solid-phase identification and
patent infringement, and has the potential
to provide a greater understanding of
defects, and related reactivity, in
pharmaceutical crystals.18
4. TEM itself is most useful in analyzing
images exhibiting high, uniform contrast of
isolated particles. 22
5. TEM images explain the geometric
properties of gold nanoparticles.23

Rukari Tushar g et al. Transmission Electron Microscopy- An Overview
International Research Journal for Inventions in Pharmaceutical Sciences, July 2013 Vol 1 Issue 2 6
6. Evaporation rate of volatile nanoparticles
during electron beam exposure in TEM
analysis indicated a composite nature of
volatile nanoparticles emitted from internal
combustion engines. 24
CHALLENGES IN TRANSMISSION
ELECTRON MICROSCOPY:
There are two major reasons for the
underdevelopment of TEM in this field. The first
relates to sample preparation; because of the strong
interaction of the electron beam with the sample 25
it is required that the specimens be very thin
(˂500nm even for light, organic compounds).26The
second deterrent is the inherent susceptibility of an
organic material to electron beam damage. For
certain types of analysis, such as a full defect
characterization, this will certainly limit the
application of TEM.
CONCLUSION:
This overview conclude that, apart from
challenges, Transmission Electron Microscopy
analysis could be advantageous in the
pharmaceutically important areas of solid-phase
identification and patent infringement, and has the
potential to provide a greater understanding of
defects, and related reactivity, in pharmaceutical
crystals. Transmission Electron Microscopy should
be helpful for exploring the geometry of
nanoparticles and also to know crystal habit,
polymorphism etc.
REFERENCES:
1. Brittain HG. Physical characterization of
pharmaceutical solids. New York: Marcel
Dekker; 1995.
2. Vippagunta SR, Brittain HG, Grant DJ.
Crystalline solids, Adv Drug Deliv Rev.
2001;48:3.
3. Haleblian J, McCrone W. Pharmaceutical
applications of polymorphism, J Pharm
Sci. 1969;58:911.
4. Singhal D, Curatolo W. Drug
polymorphism and dosage form design: A
practical perspective, Adv Drug Deliv Rev,
2004; 56: 335.
5. Bauer J, Spanton S, Henry R, Quick J,
Dziki W, Porter W, Morris J. Ritonavir:
An extraordinary example of
conformational polymorphism, Pharm Res.
2001;18:859.
6. Effects of pharmaceutical processing on
drug polymorphs and solvates: Brittain
HG, Fiese EF. In: Brittain HG, editor.
Polymorphism in pharmaceutical sciences.
New York: Marcel Dekker; 1995.p. 331–
78.
7. Fleischman SG, Kuduva SS, McMahon
JA, Moulton B, Bailey Walsh RD,
Rodriguez-Hornedo N, Zaworotko MJ.
Crystal engineering of the composition of
pharmaceutical phases: Multiple-
component crystalline solids involving
carbamazepine, Cryst Growth Des. 2003;
3:909.
8. Nicolopoulos S, Gemmi M, Gozzo F.
crystal structure analysis of
pharmaceuticals with electron diffraction.
Cited 2013 June 24: Available from:
www.icdd.com/ppxrd/12/abstracts/P11.pdf
9. Brunelli M, Wright JP, Vaughan GBM,
Mora AJ & Fitch AN. Angew. Chem,
2003; 115:2075.
10. Hirsch PB, Howie A, Nicholson RB,
Pashley DW, Whelan MJ. Electron
microscopy of thin crystals. London:
Butterworths; 1965.
11. McLaren AC. Transmission electron
microscopy of minerals and rocks. 2nd
edition. Cambridge: Cambridge University
Press; 1991.
12. Thomas JM, Midgley PA. High-resolution
transmission electron microscopy: The
ultimate nanoanalytical technique.
Cambridge United Kingdom; Chem
Commun; 2004. p. 1253–67.
13. Midgley PA, Dunin-Borkowski RE.
Electron tomography and holography in
materials science. Nat Mater; 2009. p.271–
80.
14. Armigliato A. Analysis of crystal defects
by transmission electron microscopy.
Mater Chem; 1979. p. 453–71.
15. Hirsch P, Cockayne D, Spence J, Whelan
M. 50 years of TEM of dislocations. Past,
present and future. Philos Mag ; 2006. p.
4519–28.
16. Transmission electron microscopy. Cited
2013 July 09 Available from:

Rukari Tushar g et al. Transmission Electron Microscopy- An Overview
International Research Journal for Inventions in Pharmaceutical Sciences, July 2013 Vol 1 Issue 2 7
http://en.wikipedia.org/wiki/Transmission_
electron_microscopy
17. Li ZG, Handow RL, Foris CM, Li H, Ma
p, Vickery RD et al, Microscopy and
Microanalysis. 2002; 8(2): 134.
18. Eddleston MD, Bithell EG, Jones W.
Journal of Pharmaceutical Sciences, 2010;
99(9):4072.
19. Ascencio JA, Liu HB, Pal U, Medina A,
Wang ZL. Transmission Electron
Microscopy and Theoretical Analysis of
AuCu Nanoparticles: Atomic Distribution
and Dynamic Behavior. Microscopy
Research and Technique, 2006; 69:522.
20. White ER, Mecklenburg M, Shevitski
Brian, Singer SB, and Regan BC. Charged
Nanoparticle Dynamics in Water Induced
by Scanning Transmission Electron
Microscopy, Cited 2013 July 09 Available
from: http://pubs.acs.org/Langmuir
21. Woehrle GH, Hutchison JE, Ozkar S,
Finke Richard G, Analysis of Nanoparticle
Transmission Electron Microscopy Data
Using a Public- Domain Image-Processing
Program, Image, Turk J Chem, 2006; 30:1.
22. Kaushik V, Lahiri T, Singha S, Dasgupta
AK, Mishra H, Kumar U. Exploring
geometric properties of gold nanoparticles
using TEM images to explain their
chaperone like activity for citrate synthase.
Bioinformation, 2011;7(7): 320.
23. Mathis U, Kaegi R, Mohr M, Zenobi R,
TEM analysis of volatile nanoparticles
from particle trap equipped diesel and
direct-injection spark-ignition vehicles.
Atmospheric Environment, 2004; 38:4347.
24. Reimer L, Kohl H. Transmission electron
microscopy: Physics of image formation.
5th edition. New York: Springer; 2008.
Jones W, Thomas JM. Applications of
electron microscopy to organic solid-state
chemistry. Progress Solid State Chem,
1979;12:101.
Cite This Article as:
Rukari Tushar G. Transmission Electron Microscopy-An Overview IRJIPS 2013: 1(2);1-7.
Source of Support: - Nil, Conflict of Interest: Nil
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