Progress and applications of transmission electron microscopy: A review

Hira Khaleeq
Department of chemical engineering
Sichuan university, Chengdu, china

Abstract

Nanotechnology is the emerging field and has its application in every other field. Similarly, it has its
application in medicine and pharmacy. Many bionanomaterials have been prepared and in use like drug
delivery vehicles, tissue engineering products etc. Characterization of these materials is vital and is done
by transmission electron microscope (TEM). TEM is an important instrument and is used for structural
analysis for all kinds of nanomaterials. Conventional TEM has its limitations but with time researchers
work on it and produced techniques and methods that improve it. Modern TEM is assisted with many
spectroscopic techniques and other technology. This article will present a review of progress in TEM.

Keywords: Nanotechnology, bionanomaterials, TEM

1. Introduction
1.1 Nanomedicine
Nano-pharmaceutical is quite new and progressing field with applications of nanotechnology to
pharmaceutical science for production of drugs and biomedical materials at nano scale. [1] It is a
revolutionary field that has the capacity to reform the course of treatment of disease including its
diagnosis. [2] Nano drug delivery systems are novel and have many prominent benefits over conventional
drug delivery. Taking the drug on nano level improves various pharmacological properties of drug as
compared to traditional dosage form. NDDS have more bioavailability, potency, efficiency, safety profile
and targeted delivery of drug to specific site. [3] Nanoparticles have also found their applications in
diagnostic tests. Nanoparticle probes can be used for diagnosing cancers, tumors etc. Nanoparticles have
also the capacity to deliver drugs to specific cell sites or organs to fight malignant tumors with minimal
damage to adjoining normal tissues. [4] Particles in between size of 1nm to 100nm fall in nanoparticle
category. The morphology, size and surface chemistry of nanoparticles is very important when it comes to
their interaction with surrounding media. Physical and chemical properties influence the pharmacokinetic
properties of drug nanoparticles including their absorption, bioavailability, solubility, distribution and
elimination.

Various types of nanoparticles used in nanomedicine are there including

• Solid lipid nanoparticles

• Liposomes
• Nanostructured lipid carriers
 • Fullerenes
• Nanoshells
• Quantum dots
• Super paramagnetic nanoparticles [3]

Medicinal nanoparticles can be prepared by various techniques using various different materials. Drug
nanoparticles can be prepared by incorporating drug on polymers ideally biodegradable polymer such as
polyethylene glycol, chitosan, cellulose, chitin etc. Metallic nanoparticles such as gold, silver etc. have
also been prepared having medical applications. Nanoparticles can be prepared by different physical and
chemical methods for example ultra-sonication, solvent evaporation, micro-emulsion, thermal
decomposition, polyol method etc. [5]

Nanomedicine has showed promising results and is future approach to cure and diagnose diseases. Gold
nanoparticles (GNPs) have been prepared and evaluated for their medical properties. GNPs are loaded
with cetuximab and gemcitabine to act as drug carrier and showed positive results against pancreatic
cancer. Gold nanoshells have been used for hyperthermia therapy in mice tumor models and displayed
better results than the controlled group. GNPs have also been studied as radiosensitisers. [6] Lactoferrin
modified docetaxel nanoparticles have been prepared using solvent evaporation-emulsion method and
evaluated. Results showed elevated concentration of docetaxel in brain making SLNPs as ideal drug
carriers. [7] Ciprofloxacin loaded poly lactide co-glycolic acid nanoparticles were prepared using double
emulsion method and deposited on skin scaffolds and investigated. Results offer concrete evidence for
satisfying the requirements of local nano antibiotic delivery systems having biodegradability and
biocompatibility to use in tissue engineering, including mid ear tissues (e.g., tympanic membranes) which
are vulnerable to bacterial infections. [8] The most significant recent medical applications of
nanotechnology comprise of cancer treatments and some COVID-19 vaccines. [9]

Characterization and evaluation of nanomedicine is very important. The shape and structure of nanodrug
helps it to reach the target site, maintain its level in body and produce effect even at low concentration. [1]
Many different techniques are in use for analysis of nanodrugs including Fourier transform infrared
spectroscopy, ultraviolet spectrophotometry, gas chromatography mass spectrometry, high-performance
liquid chromatography, scanning electron microscope. Atomic force microscopy, thermogravimetry,
Raman spectroscopy and transmission electron microscopy etc. [10][11][12][13][14]

1.2 Transmission electron microscopy (TEM)

Transmission electron microscopy is the most popular technique for characterization of nanomaterials in
electron microscopy. It provides the chemical information and imageries of the nanoparticles at
spatiotemporal resolution at atomic level. [15]

1.2.1 Principle
TEM utilizes the same principle as scanning electron microscopy that is using electrons as an excitation
source. An electron beam is transmitted through the thin specimen to produce an image.[16]

1.2.2 Structure
TEM mainly comprise of three systems
1. An electron gun: that yield electron beam and a condenser system : that focused the beam
on specimen
2. An image producer: to create image. Comprise of lenses, stage to mount specimen
3. And image recorder: that change the beam of electron into an image discernible to human
eye. A fluorescent screen and a digital camera are parts of it.
Additionally, a vacuum system containing pumps, linked gauges and power supplier also
exist.[17]

Figure 1: structure of TEM

 2. TEM; Progress and Applications

Figure 2: TEM classification

Transmission electron microscopy is swiftly evolving field. Its scope, extent to collect data and different
TEM techniques are escalating rapidly. A contemporary TEM offers a sophisticated characterization
instrument that has a capability to collect diffraction patterns of nanosized particles and pictures to the
atomic level. On combining with tomography, it can create images in three dimensions making it easier to
know the shape and structure of nanoparticles. In addition to this, TEM when combined with
spectroscopy techniques, not only helps to understand and examine constituents of materials but also give
info abouts bonds, optical and electronic structure to atomic resolution in both two and three dimensions.
Lastly, a wide range of experiments can now be done in situ in the TEM with concurrent characterization,
presenting valued insight in various processes.[18]

2.1 TEM as structural tool

Transmission electron microscope is an imperative instrument for structural evaluation of zeolite
nanomaterials. TEM can be used for characterizing zeolite structures, their growth patterns, porous
structure determination and analyzing their metal sites. Usually X-ray diffraction is used to study
structure of zeolites but TEM is used to evaluate structures that are very small in size or very complex to
be evaluated by X-ray diffractometer. To directly get the image of sample, scanning transmission electron
microscope (STEM) or high-resolution electron microscope (HRTEM) is applied. Electron
crystallography and tomography have been employed to study the microporous and mesoporous crystal
structure respectively when combined with TEM. In addition to structural identification, TEM also used
to give information about the size and shape of nano-zeolites material. Nano scaled zeolites have shown
better catalytic activity.[19]

Magnetic nanoparticles are of great importance and their significance are increasing day by day. They are
multipurpose and can perform variety of functions due to their magnetic nature e-g usage in magnetic
resonance imaging and magnetic hyperthermia. Scientist have prepared magnetic zeolites nanoparticles
by using iron to be used as biosensors in marking breast cancer cells. TEM evaluation showed cell
internalization of magnetic nano zeolites by breast tumor tissues. Cytotoxic analysis exhibits that nano-
zeolites are not toxic even at higher concentrations. Lastly the ability of magnetic zeolite nanoparticles to
act as a probe was confirmed invitro.[20]

Nanoparticles of magnetic zeolite nature has been prepared by scientist by using ferrous oxide that show
size of 4-6 nanometer by TEM analysis. These zeolites can have multiple applications such as can be used
as biosensors, for water management plants, energy etc. [21]

The one of the most influential tools for illustrating nano materials is High‐resolution transmission
electron microscopy (HRTEM) and it is crucial for nanotechnology. HRTEM is used for evaluation
dynamic shape shifting of nanocrystals, to observe surface reactions; to study electrical, mechanical and
field emission characteristics of nanowires and nanotubes; aberration‐free angstrom‐resolution imaging of
light elements (such as oxygen and lithium); high‐angle annular‐dark‐field scanning transmission electron
microscopy (STEM); taking images of atom groups at atomic level; electron holography of magnetic
materials; and high‐spatial resolution electron energy‐loss spectroscopy (EELS) for analyzing
nanomaterials for their electronic and chemical properties. [22]

2.2 TEM as quantitative imaging tool

The standard technique for characterizing structure and composition of nano materials at atomic level is
High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) imaging. The
quantitative analysis of materials is very challenging. During the last decade scientist have tried to
develop tactics to quantify the data obtained from STEM. The key aim is to abstract specific and correct
numbers for unidentified structure parameters that includes position of atoms, concentration of chemicals,
and atomic numbers. To extract quantitative data from STEM images, analytical stats approaches are
required. To achieve this, statistical parameter approximation theory has revealed dependable outcomes.
In this context, imageries are measured as data planes and structure analysis is done from it via parametric
model. Images obtained from HAADF-STEM at atomic level are expressed through parametric model.
The position of atoms, altitude and breadth of Gaussian peaks are included as this model’s parameters and
these are elucidated by means of the least square estimator. The atom columns position is calculated to the
accuracy of picometers. Furthermore, minor variances in average atomic number, that are not visually
notable, can be measured via HAADF-STEM image. Additionally, this theory permits one to quantify the
changes in composition at interfaces, and to calculate atoms accurately unto single atom susceptibility and
similarly to rebuild atomic assemblies three dimensionally(3D). StatSTEM is a software developed by
EMAT lab of University of Antwerp. It’s an easy and accessible software that helps to quantify images
from STEM.

In HAADF-STEM the intensity of image is leveled to sample thickness and atomic number. In
homogenous materials the cross section that scatters, i-e to quantify the cohesive intensities at atomic
column position, only rely on width and thus can be of usage for counting the atoms in each column by
utilizing statistical approach or by comparing it to simulator reference. While in heterogenous materials
the scattering cross section is contributed by all the elements present in it in a different way that
complicates the quantification of atoms. Moreover, even a tiny change in column of atoms effects the
scattering cross section. To overcome this and to obtain information atomic lensing model was presented
which helps in configure, to know constituents and thickening of material from scattering cross section.
The number of gold and silver atoms were calculated by means of this model on HAADF-STEM pictures
of silver coated gold nanorods. [18]

2.3 Investigation of Beam Sensitive Materials in a TEM

TEM permits the structural elucidation of materials in multidimensions of different materials at an atomic
scale. But the electron beam can cause the damage to the specimen structure due to its high energy. Many
materials that are sensitive to electron damage include zeolites, many biomaterials, organic-inorganic
hybrid materials etc. to overcome this problem modifications with TEM technique are required. Electron
beam damage can be minimized bye using low energy electron beam, by changing the suitable imaging
mode e-g materials directly related to dose of illumination, TEM mode can be utilized and with inverse
dose relationship i-e damage occur due to slow diffusion limited mass loss or precipitation, STEM mode
can be used. Controlling the electron beam has also been used to minimize damage of sample. Also,
strategies related to changes in sample and stage has also been used e-g freezing of specimen, coating the
sample with insensitive material, single particle tomography etc. changing the detector to suitable one
also decreases the sample damage. [23]

2.4 Atomic resolution tomography

Electron tomography has been used for studying the nanomaterials structure in multidimensions. Latest
progress in this field take the structure elucidation to atomic level. Though most of the studies showed
remarkable structure determinations, it also has some deviations ins structures specially of crystalline
materials. Determination of unknown structure to atomic scale and to know position of each atom electron
etc. of crystal structure is still difficult. To alleviate this difficulty High-angle annular dark-field scanning
transmission electron microscopy (HAADF-STEM) can be used along with electron energy loss
spectroscopy (EELS) or energy dispersive X-ray spectroscopy (EDS). Recently structures have been
determined on nano scale but still to be determined at atomic scale. Hence achieving the atomic resolution
in 3D electron tomography cannot be considered as end but start to new path in the field of multi-
dimensional electron microscopy. [24]

2.5 TEM combined with Spectroscopic Techniques

Transmission electron microscopy has been combined with various spectroscopic instruments for better
analyzing the materials. STEM combined with EELS and EDS can provide complete mapping of material
in two dimensions. Other spectroscopic techniques like Fourier transform infrared spectroscopy (FTIR),
Nuclear magnetic resonance (NMR) and Raman spectroscopy etc. Researchers characterize the fine
particulate material (PM) by combining TEM with NMR, FTIR and Raman spectroscopy. Images
obtained from TEM revealed that particulate material mainly composed of organic airborne constituents,
along with trace elements from combustion sources. NMR showed the percentage of carbon in both PM
and its probable sources along with their variances and resemblances. FTIR exhibits the peak due to
NH4NO3 that confirmed the existence of secondary mechanisms. Certain compounds like sulfates and
nitrates were discovered by Raman spectroscopy. Obtained results displayed that combination of
nondestructive methods is effective to study sources of PM. [25]
 2.6 TEM for functional characterization
Automated crystal orientation mapping (ACOM) technique when combined with TEM can determine the
crystalline materials of nanoscale.[26] It is time saving as compared to conventional diffraction, bright/dark
field imaging.[27] A specimen from cast iron as studied using ACOM-TEM technique. This technique
collects the patterns created through diffraction of electrons and compare them to prefeed data templates.
Results help in determination of graphite crystal growth patterns and structure. Materials containing
carbon are difficult to analyze through conventional tools but can be easily evaluated through this
technique.[28]

To achieve better performance from TEM in characterizing nano biomaterials, a novel carbon nanotube
grid for TEM is developed. It is nano sized, is very thin but strong and very conductive. Due to these
properties, performance of TEM is improved.[29]

Gold (Au) loaded hydroxyapatite particles were prepared by coating collagen for drug delivery.
Characterization was done using TEM-EDX that revealed spherical shaped particles. Gold is in darker
shade the HAp nanoparticles. High resolution TEM showed the structure of nanoparticles and calculated
the inter atomic space between Au and HAp as 0.203nm and 0.213nm respectively. The nanoparticle size
falls in 24nm to 32nm range mostly.[30]

Polypeptides based on lysine and α-aminoisobutyric acid (Aib) were prepared to use in biomaterials due
to their biocompatibility and biodegradable nature. Polymer characterization was done using various
techniques including TEM along with dynamic light scattering that confirmed nanospheres of varying
morphology were formed.[31]

Egg shells are used by scientist for preparation of bio ceramic (calcium salicylate nanomaterial) for use in
tissue scaffolds. These scaffolds are used for tissue regeneration devices. The calcium salicylate
nanoparticles were evaluated by many instruments including TEM. Results exhibited formation of
polycrystal structured nanoparticles of calcium salicylate that are less than 50nm in size.[32]

3. Conclusion and future prospect

To conclude transmission electron microscope is a very crucial tool that can determine the shape, size,
morphology and structure to the atomic resolution in spaciotemporal planes. It can give the versatile
information about nano materials. When combined with other spectroscopic techniques and precalculated
software it overcome its limitations and produce remarkable results. More improvements are required in
terms of electron beam sensitive nanomaterials characterization on which many researchers are working.
Overall, it is great and vital instrument that has been in use for studying nanomedical materials and it will
be of great use in future too.

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