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TRANSMISSION ELECTRON MICROSCOPY

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DOI: 10.13140/RG.2.2.21280.71681

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TRANSMISSION ELECTRON MICROSCOPY
Sehasree Mohanta
Indian Association For The Cultivation Of Science

(Dated: 8th June 2021)

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 (m)
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 trans-
mission 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 Micro-
scope (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.
Keyword: Electron microscope (EM), Transmission electron microscopy (TEM), 3D
electron diffraction tomography technique etc. Secondary publications and information
retrieval purposes.

CONTENTS produced, for instance , components of the tar-

get cocrystal present as “impurity” phases or the
I. INTRODUCTION:- 1
possible generation of small amounts of a special
II. INSTRUMENTATION:- 2 cocrystal stoichiometry beyond the standard de-
A. Electrons: 2
B. Source formation: 3 tection limits of routine analytical methods like
C. Optics: 3 powder X-ray diffraction (PXRD). X-Ray pow-
D. Display: 4
E. Vacuum System: 4 der diffraction (XRPD) has already successfully
F. Specimen stage: 4 been used for an extended time within the field
G. Electron gun: 5
H. Electron lens: 6
of pharmaceuticals for polymorph screening (fin-
I. Apertures: 6 gerprinting) and more recently for the structural
J. Sample preparation: 7 and microstructural analysis of active pharma-
III. APPLICATIONS OF TRANSMISSION ELECTRON ceutical ingredients (API) and excipients. Large
MICROSCOPY:- 7
enough single crystals are in fact either very dif-
IV. CHALLENGES IN TRANSMISSION ELECTRON ficult to growth or don’t represent well the prop-
MICROSCOPY:- 7
erties of the polycrystalline pharmaceutical com-
V. CONCLUSION AND OUTLOOK:- 7 pounds. Furthermore quantitative phase analy-
VI. ACKNOWLEDGEMENT:- 8
ses can only be performed with powder materi-
als. Although XRPD is well established, there
VII. CITATION:- 8
are often limitations in its successful applica-
References 8 tion, in particular when facing complex struc-
tures whose XRPD patterns are characterized
I. INTRODUCTION:-
by many overlapping peaks and therefore the de-
termination of the cell parameters and therefore
The recent emergence of pharmaceutical the extraction of the diffracted intensity is ex-
cocrystals as alternatives to salts and amorphous tremely difficult and sometimes unsuccessful. In
forms is recognized,7 but raises questions dur- such cases the utilization of synchrotron radia-
ing development of the importance which will tion and advanced experimental methods got to
got to be attached to the purity of the phases be explored (e.g. texture method8, anisotropic
 2

FIG. 1 Layout of optical components in a basic

TEM,@copyright
wikipedia.com

FIG. 2 The first practical TEM,@copyright

The first practical TEM, originally installed at IG
Farben-Werke and now on display at the Deutsches Museum
thermal expansion 9) but the determination of in Munich, Germany
the crystal structure is never guaranteed and
much from being routine. II. INSTRUMENTATION:-
Transmission microscopy (TEM) has long been
A. Electrons:
utilized in materials science as a strong analyti-
cal tool.9-14 Transmission microscopy (TEM) is
Theoretically, the utmost resolution, d, that
a microscopy technique whereby a beam of elec-
one can obtain with a light-weight microscope
trons is transmitted through an ultra-thin spec-
has been limited by the wavelength of the pho-
imen, interacting with the specimen because it
tons (λ) that are getting used to probe the sam-
passes through. a picture is made from the inter-
ple and therefore the numerical aperture NA of
action of the electrons transmitted through the
the system. (12)
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 λ
d= (1)
detected by a sensor like a CCD camera (as it is 2nsinα
shown in the Figure1 and 2
where n is that the index of refraction of the
medium during which the lens is functioning and
(16) (13) Although TEM has been less widely λ is that the maximum half-angle of the cone of
applied to the study of organic materials, pri- sunshine which will enter the lens (see numeri-
marily due to perceived difficulties with sample cal aperture). Early twentieth century scientists
preparation and beam damage, recent studies of theorized ways of getting round the limitations
pharmaceutical compounds have shown not only of the relatively large wavelength of light (wave-
that these difficulties are often overcome, but lengths of 400–700 nanometers) by using elec-
also that TEM analysis can provide a variety of trons. Like all matter, electrons have both wave
useful information about samples that can’t be and particle properties (as theorized by Louis-
obtained by other, more routinely used, analyt- Victor de Broglie), and their wave-like proper-
ical methods. (2) ties mean that a beam of electrons are often fo-
 3

cused and diffracted very similar to light can. is performed using two physical effects. The in-
The wavelength of electrons is said to their K.E. teraction of electrons with a magnetic flux will
via the Broglie equation, which says that the cause electrons to maneuver consistent with the
wavelength is inversely proportional to the mo- proper hand rule, thus allowing electromagnets
mentum. Taking under consideration relativistic to manipulate the beam .
effects (as during a TEM an electron’s velocity (7)
may be a substantial fraction of the speed of sun-
shine , c) the wavelength is

h
λ= q
E
2mE(1+( ))
2mc2

(2)
where, h is Planck’s constant , m0 is that the
mass of an electron and E is that the K.E. of
the accelerated electron. Electrons are usually
generated in an microscope by a process referred
to as thermal emission from a filament, usually
tungsten, within the same manner as a light-
weight bulb, or alternatively by field electron
emission. The electrons are then accelerated by
an electrical potential (measured in volts) and fo- FIG. 3 Single crystal LaB6 filament.@copyright
cused by electrostatic and electromagnetic lenses researchgate.net
onto the sample. The transmitted beam con-
tains information about electron density, phase The use of magnetic fields allows for the for-
and periodicity; this beam is employed to make mation of a magnetic lens of variable focusing
a picture . power, the lens shape originating thanks to the
distribution of magnetic flux. Additionally, elec-
trostatic fields can cause the electrons to be de-
B. Source formation: flected through a continuing angle. Coupling of
two deflections in opposing directions with alit-
From the highest down, the TEM consists of
tle intermediate gap allows for the formation of
an emission source, which can be a tungsten fila-
a shift within the beam path, this being used in
ment, or a lanthanum hexaboride (LaB6) source.
TEM for beam shifting. (9) (4)
For tungsten, this may be of the shape of ei-
ther a hairpin-style filament, or alittle spike-
shaped filament as shown within the Figure 3 C. Optics:
and 4. LaB6 sources utilize small single crys-
tals. By connecting this gun to a high volt- The lenses of a TEM leave beam convergence,
age source (typically 100- 300 kV) the gun will, with the angle of convergence as a variable pa-
given sufficient current, begin to emit electrons rameter, giving the TEM the power to vary mag-
either by thermionic or field electron emission nification just by modifying the quantity of cur-
into the vacuum. This extraction is typically rent that flows through the coil, quadrupole or
aided by the utilization of a Wehnelt cylinder. hexapole lenses. The quadrupole lens is an ar-
Once extracted, the upper lenses of the TEM rangement of electromagnetic coils at the ver-
leave the formation of the electron probe to the tices of the square, enabling the generation of
specified size and site for later interaction with a lensing magnetic fields, the hexapole config-
the sample. Manipulation of the electron beam uration simply enhances the lens symmetry by
 4

Yttrium- Aluminum Garnet (YAG) screen cou-

pled CCDs. Typically these devices are often
removed or inserted into the beam path by the
operator as needed . (11)

E. Vacuum System:

To increase the mean free path of the electron

gas interaction, a typical TEM is evacuated to
low pressures, typically on the order of 10−4 Pa.
The need for this is often twofold: first the al-
lowance for the voltage difference between the
cathode and therefore the ground without gener-
ating an arc, and secondly to reduce the collision
frequency of electrons with gas atoms to negli-
gible levels—this effect is characterized by the
mean free path. TEM components like specimen
holders and film cartridges must be routinely in-
serted or replaced requiring a system with the
power to re-evacuate on a daily basis. As such,
TEMs are equipped with multiple pumping sys-
tems and airlocks and are not permanently vac-
FIG. 4 Hairpin style tungsten filament.@copyright uum sealed. Sections of the TEM could also
researchgate.net be isolated by the utilization of pressurelimiting
apertures, to permit for various vacuum levels
using six, rather than four coils. Typically a in specific areas, like a better vacuum of 10−4 to
TEM consists of three stages of lensing. The 10−7 Pa or higher within the electrode in high-
stages are the condensor lenses, the objective resolution or field-emission TEMs. High voltage
lenses, and therefore the projector lenses. The TEMs require ultra-high vacuums on the range
condensor lenses are liable for primary beam for- of 10−7 to 10−9 Pa to stop generation of an elec-
mation, whilst the target lenses focus the beam trical arc, particularly at the TEM cathode.
that comes through the sample itself (in STEM
scanning mode, there also are objective lenses Φ
J = AT 2 e(− KT )
above the sample to form the incident electron
beam convergent). The projector lenses are wont
to expand the beam onto the phosphor screen or F. Specimen stage:
other imaging device, like film. The magnifica-
tion of the TEM is thanks to the ratio of the dis- TEM specimen stage designs include airlocks
tances between the specimen and therefore the to allow for insertion of the specimen holder into
objective lens’ image plane. (3) (2) (6) the vacuum with minimal increase in pressure
in other areas of the microscope. The specimen
D. Display: holders are adapted to carry a typical size of
grid upon which the sample is placed or a typical
Imaging systems during a TEM contains a size of selfsupporting specimen. Standard TEM
phosphor screen, which can be made from fine grid sizes are a 3.05 mm diameter ring, with a
(10 − 100µm) particulate zinc sulphide, for di- thickness and mesh size starting from a couple
rect observation by the operator. Optionally, a of to 100µm. The sample is placed onto the in-
picture recording system like film based or doped ner meshed area having diameter of roughly 2.5
 5

FIG. 6 TEM sample support mesh ”grid”, with ultramicro-

tomy sections.@copyright
wikipedia.com

FIG. 5 The electron source of the TEM is at the top, where

the lensing system (4,7 and 8) focuses the beam on the spec- FIG. 7 A diagram of a single axis tilt sample holder for inser-
imen and then projects it onto the viewing screen (10). The tion into a TEM goniometer. Tilting of the holder is achieved
beam control is on the right (13 and 14).@copyright by rotation of the entire goniometer.@copyright
wikipedia.com wikipedia.com

G. Electron gun:

The electrode is made from several compo-

mm usual grid materials are copper, molybde- nents: the filament, a biasing circuit, a Wehnelt
num, gold or platinum. This grid is placed into cap, and an extraction anode. By connecting
the sample holder, which is paired with the spec- the filament to the negative component power
imen stage. supply, electrons are often ”pumped” from the
electron gun to the anode plate, and TEM col-
A wide sort of designs of stages and holders umn, thus completing the circuit. The gun is
exist, depending upon the sort of experiment be- meant to create a beam of electrons exiting from
ing performed. additionally to three .05 mm the assembly at some given angle, referred to as
grids, 2.3 mm grids are sometimes, if rarely, the gun divergence semi angle, α. By construct-
used. These grids were particularly utilized in ing the Wehnelt cylinder such it’s a better neg-
the mineral sciences where an outsized degree of ative charge than the filament itself, electrons
tilt are often required and where specimen mate- that exit the filament during a diverging man-
rial could also be extremely rare. Electron trans- ner are, under proper operation, forced into a
parent specimens have a thickness around 100 converging pattern the minimum size of which
nm, but this value depends on the accelerating is that the gun crossover diameter. The thermal
voltage. (14) emission current density J, are often associated
 6

with the work function of the emitting material polepiece can induce severe distortions within
and may be a Boltzmann distribution given be- the magnetic flux symmetry, which induce dis-
low, where A may be a Richarson constant, φ is tortions which will ultimately limit the lenses’
the work function, T is that the temperature of ability to breed the thing plane. The exact di-
the material and K is Boltzmann constant. mensions of the gap, pole piece internal diame-
ter and taper, also because the overall design of
the lens is usually performed by finite element
analysis of the magnetic flux , whilst consider-
ing the thermal and electrical constraints of the
planning as shown in Figure 6.The coils which
produce the magnetic field are located within the
lens yoke. The coils can contain a variable cur-
rent, but typically utilize high voltages, and thus
require significant insulation so as to stop short
circuiting the lens components. Thermal distrib-
utors are placed to ensure the extraction of the
warmth generated by the energy lost to resis-
tance of the coil windings. The windings could
FIG. 8 Cross sectional diagram of an electron gun assembly, also be water-cooled, employing a chilled water
illustrating electron extraction.@copyright supply so as to facilitate the removal of the high
researchgate.net
thermal duty. (15)
(5)

H. Electron lens:

Electron lenses are designed to act during a

manner emulating that of an camera lens , by
focusing parallel rays at some constant focal dis-
tance . Lenses may operate electrostatically or
magnetically. The majority of electron lenses for
TEM utilize electromagnetic coils to get a con-
verging lens . For these lenses the sector pro-
duced for the lens must be radially symmetric, as
deviation from the radial symmetry of the mag-
netic lens causes aberrations like astigmatism,
and worsens spherical and aberration . Electron FIG. 9 Diagram of a TEM split polepiece design
lenses are manufactured from iron, iron-cobalt lens.@copyright
or nickel cobalt alloys, like Permalloy. These reasearchgate.net

are selected for his or her magnetic properties,

such as magnetic saturation, hysteresis and per-
meability. The components include the yoke, I. Apertures:
the magnetic coil, the poles, the polepiece, and
therefore the external control circuitry. The pole Apertures are annular metallic plates, through
piece must be manufactured during a very sym- which electrons that are further than a fixed
metrical manner, as this provides the boundary distance from the optic axis may be excluded.
conditions for the magnetic field that forms the These consist of a small metallic disc that is suf-
lens. Imperfections in the manufacture of the ficiently thick to prevent electrons from passing
 7

through the disc, whilst permitting axial elec- III. APPLICATIONS OF TRANSMISSION ELECTRON
trons. This permission of central electrons in a MICROSCOPY:-
TEM causes two effects simultaneously: firstly,
apertures decrease the beam intensity as elec- 1. The theoretical prediction of coexistence of
trons are filtered from the beam, which may fcc-like and multiple twined AuCu nanoparticles.
be desired in the case of beam sensitive sam- 2.TEM studies of functional biomolecules and
ples. Secondly, this filtering removes electrons bio structures, where charge interactions are
that are scattered to high angles, which may be known to play a dominant role. (8)
due to unwanted processes such as spherical or 3. Identification of defects in samples of theo-
chromatic aberration, or due to diffraction from phylline, solid-phase identification and patent in-
interaction within the sample. Apertures are ei- fringement, and has the potential to provide a
ther a fixed aperture within the column, such greater understanding of defects, and related re-
as at the condenser lens, or are a movable aper- activity, in pharmaceutical crystals.
ture, which can be inserted or withdrawn from 4. TEM itself is most useful in analyzing im-
the beam path, or moved in the plane perpen- ages exhibiting high, uniform contrast of isolated
dicular to the beam path. Aperture assemblies particles.
are mechanical devices which allow for the se- 5. TEM images explain the geometric proper-
lection of different aperture sizes, which may be ties of gold nanoparticles.
used by the operator to trade off intensity and 6. Evaporation rate of volatile nanoparticles
the filtering effect of the aperture. Aperture as- during electron beam exposure in TEM analysis
semblies are often equipped with micrometers to indicated a composite nature of volatile nanopar-
move the aperture, required during optical cali- ticles emitted from internal combustion engines.
bration. (1)
IV. CHALLENGES IN TRANSMISSION ELECTRON
MICROSCOPY:-

There are two major reasons for the underde-

velopment of TEM during this field. The first
relates to sample preparation; due to the strong
J. Sample preparation: interaction of the beam with the sample 25 it is
required that the specimens be very thin (500
Preparation of TEM specimens is specific to nm even for light, organic compounds).26 The
the material under analysis and the desired in- second deterrent is that the inherent susceptibil-
formation to obtain from the specimen. As such, ity of an organic material to beam damage. For
many generic techniques have been used for the certain sorts of analysis, like a full defect char-
preparation of the required thin sections. Ma- acterization, this may certainly limit the appli-
terials that have dimensions small enough to be cation of TEM.
electron transparent, such as powders or nan-
otubes, can be quickly prepared by the deposi- V. CONCLUSION AND OUTLOOK:-
tion of a dilute sample containing the specimen
onto support grids or films. In the biological This overview conclude that, apart from chal-
sciences in order to withstand the instrument lenges, Transmission Electron Microscopy anal-
vacuum and facilitate handling, biological speci- ysis could be advantageous in the pharmaceu-
mens can be fixated using either a negative stain- tically important areas of solid-phase identifica-
ing material such as uranyl acetate or by plastic tion and patent infringement, and has the po-
embedding. Alternately samples may be held at tential to provide a greater understanding of
liquid nitrogen temperatures after embedding in defects, and related reactivity, in pharmaceuti-
vitreous ice. (10) cal crystals. Transmission Electron Microscopy
 8

should be helpful for exploring the geometry of [7] Lucille A Giannuzzi and Frederick A Stevie. A review
nanoparticles and also to know crystal habit, of focused ion beam milling techniques for tem specimen
preparation. Micron, 30(3):197–204, 1999.
polymorphism etc. [8] Jiro Hirokawa and Makoto Ando. Single-layer feed
waveguide consisting of posts for plane tem wave excita-
tion in parallel plates. IEEE Transactions on Antennas
VI. ACKNOWLEDGEMENT:- and Propagation, 46(5):625–630, 1998.
[9] Kazuo Ishizuka. Contrast transfer of crystal images in
Author would like to acknowledge Prof. Mintu tem. Ultramicroscopy, 5(1-3):55–65, 1980.
[10] Judy S Kim, Thomas LaGrange, Bryan W Reed, Mitra L
Mondol for critical reading of the manuscript Taheri, Michael R Armstrong, Wayne E King, Nigel D
and valuable comments. Browning, and Geoffrey H Campbell. Imaging of tran-
sient structures using nanosecond in situ tem. Science,
321(5895):1472–1475, 2008.
VII. CITATION:- [11] M Kuznetsov, F Hakimi, R Sprague, and A Mooradian.
High-power (¿ 0.5-w cw) diode-pumped vertical-external-
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