1. INTRODUCTION
1.1 Nanoscience
and Nanotechnology
In recent
day’s nanotechnology has induced great scientific advancement in the field of
research and technology. Nanotechnology is the study and application of small
object which can be used across all fields such as chemistry, biology, physics,
material science and engineering. Nanoparticle is a core particle which
performs as a whole unit in terms of transport and property. As the name
indicates nano means a billionth or 10-9 unit. Its size range usually from
1-100nm due to small size it occupies a position in various fields of nano
science and nanotechnology. Nano size particles are quite unique in nature
because nano size increase surface to volume ratio and also its physical,
chemical and biological properties are different from bulk material. So the main
aim to study its minute size is to trigger chemical activity with distinct
crystallography that increases the surface area. Thus in recent years much
research is going on metallic nanoparticle and its properties like catalyst,
sensing to optics, antibacterial activity and data storage capacity. The
concept of nanotechnology emerged on 9th century. For the first time in 1959, Richard Feynmangave a talk on the
concept of nanotechnology and described about molecular machines built with
atomic precision where he discussed about nanoparticles and entitled that “There’s
plenty of space at the bottom”. Professor
Peter Paul Speiserand his research group were first to investigate on
polyacrylic beads for oral administration and target on microcapsule. In the
year of 1960 nanoparticle develop for drug delivery and also vaccine purpose
which change the medicinal scenario. The first paper published in1980 by K. Eric Drexlerof Space Systems
Laboratory Massachusetts Institute of Technology was titled as “An approach
to the development of general capabilities for molecular manipulation”. The
term “nanotechnology” first time used as scientific field by NarioTanigushiin the 1974 his paper
was “Nanotechnology” mainly consists of the processing of, separation,
consolidation, and deformation of materials by one atom or one molecule.
Nanotechnology is a
fast growing area in the field on science which is a interdisciplinary field of
both science and technology that increase the scope of investing and regulating
at cell level between synthetic material and biological system. Nanotechnology
proceeds by three processes - separation, consolidation, deformation of
material by one atom or molecule. It is divided into three types- Wet nanotechnologywhich deals with the
biological system such as enzymes, membrane, cellular components. Dry nanotechnologydeals with the
surface science, physical chemistry & gives importance on fabrication of
structure in carbon, silicon, inorganic materials.Computational nanotechnologywhich deals with modeling&
stimulating the complex nanometer scale structure, these three fields are
interdependent to each other. There are two methods of synthesis of metallic
nanoparticles which are chemical method and physical method. In chemical
approach it includes chemical reduction, electrochemical technique and
photochemical reduction.The chemical process is again subdivided into classical
chemical method where some chemical reducing agent (such as hydrazine, sodium
borohydride, hydrogen) is used, radiation chemical method generated by
ionization radiation. In the physical approach it includes condensation,
evaporation and laser ablation for metal nanoparticle synthesis. The biological
synthesis of nanoparticle is a challenging concept which is very well known as green
synthesis. The biological synthesis of nano material can solve the
environmental challenges like solar energy conservation, agricultural
production, catalysis, electronic, optics, and biotechnological area. Green
synthesis of nanoparticle are cost effective, easily available, ecofriendly,
nontoxic, large scale production and act as reducing and capping agentin
compared to the chemical method which is a very costly as well as it emits
hazardous by-product which can have some deleterious effect on the environment.
Biological synthesis utilizes naturally occupying reducing agent such as plant
extract, microorganism, enzyme, polysaccharide which are simple and viable
which is the alternative to the complex and toxic chemical processes. Plants
can be described as nano factories which provide potential pathway to
bioaccumulation into food chain and environment. Among the different biological
agents plants provide safe and beneficial way to the synthesis of metallic
nanoparticle as it is easily available so there is possibilities for large
scale production apart from this the synthesis route is eco-friendly,the rate
of production is faster in comparison to other biological models such as
bacteria, algae and fungi. From the various literature studies it can be stated
that the amount of accumulation of nanoparticle varies with reduction potential
of ions and the reducing capacity of plant depends on the presence of various
polyphenols and other heterocyclic compounds. Nanoparticle of gold, silver, copper, silicon, zinc,
titanium, magnetite, palladium formation by plants has been reported. Colloid
zinc nanoparticle had exhibited distinct properties such as catalytic,
antibacterial, good conductivity, and chemical stability. Silver nanoparticles
have its application in the field of bio labeling, sensor, antimicrobial,
catalysis, electronic and other medical application such as drug delivery and
disease diagnosis.
Nanotechnology
is defined as a highly developing field because of its vast array of
applications in various fields of medical science, technology and various
research areas. The word ‘Nano’isoriginated from a Greek word whose meaning is
extremely small or dwarfs. The basic concepts behind nanoscience and
nanotechnology was reported with a talk entitled “There’s a Plenty of Room at
the Bottom” by the physicist Richard Feynman at the California Institute of
Technology (Caltech) on December 29, 1959.The term “Nanotechnology” was later
coined by Professor Norio Taniguchi by using Feynman’s explorations of
ultra-precision machining.In 1974, the term nanotechnology was pioneered by
Nori Taniguchi on the talk of Production Engineering at the Tokyo International
Conference. He focused on the ultra-precision machining in his talk and
hisresearch was based on the mechanisms of machining hard and brittle materials
like ceramics of silicon, alumina and quartz crystalsby ultrasonic
machining.Richard Feynman established a talk on the scanning electron
microscope that, it could be developed in resolution and stability whichwill
bevaluable and functional foranyone to “see” the atoms.Scanning tunneling
microscope helps in the proper detection and identification of individual atoms
from whichthe modern nanotechnology began which was developed in 1981.Feynman
also continued to identify the ability to arrange atoms which iswithin the
bounds of chemical stability for the minute structures which in turn would lead
to synthesis of materials in molecular and atomiclevels.
The
study about Nanoscience and nanotechnology provides the
well-developedapplication of exceptionallyminiature things and be capable of
the encroachment of all the fields of scientific research and development likePhysics,
Chemistry, Materials andMetallurgy engineering, Biology and also in
Biotechnology. Bio-nanotechnology is the conjunction between biotechnology and
nanotechnology for developingvarious biosynthetic and environmental
ecofriendlytechnologies for the synthesis of various nanomaterials. Multiple of
research on synthesis of nanoparticles has put forth great interesttowards the
emerging field of science due to their distinguishing physical and chemical
properties than the macroscopic particles.The vast array of development of
novel synthesis protocols and various characterization techniques are the
evidences for the vastadvancement for the nanotechnology. A brief and general
definition of nanotechnology by the US National Science and Technology Council
states: “The essence of nanotechnology has the capability to work at the
molecular level such as atom by atomfor the creation of large structures with
essentiallyinnovative molecular organization. The aim is to exploit these
properties by gaining control of structures and devices at atomic, molecular,
and supramolecular levels and to become skilled atwell-organized manufacture
and use these devices.” The United States National Science Foundation (USNSF)
defines nanoscience or nanotechnology is the study which deals with materials
and systems deserving the following key properties:
1.
The dimension must be at least one dimension from 1-100 nm.
2. The process can be designed with various methodologies which
show elementary control over the physical and chemical properties of structures
that can be measure by the molecular-scale.
3. According to the building block property, larger structures
form by the combination of smaller one. According to the Microbiological study,
the Nanoscience leds to the sizes of
different types of bio particles which deals with bacteria, viruses, enzymes,
etc. fall within the nanometer range.
Nanotechnology
has the capability at atomic precision which is useful formaking of materials,
various instruments and systems. Nanotechnology is the science and
engineeringof recent well established technology which refers at the
nanoscale.It is concerning 1 to 100 nanometer size. Nanotechnology can also be
defined as the study and investigation about the synthesis, characterization, exploration
and application of nanosizedmaterials which is of 1-100nm in size and will be
valuable and functional for the development of science.A recent advance in the
emerging field of nanotechnology has thecapability for the preparation of the
highly ordered nanoparticles of various different size and shape which led to
the development of new biocidalagents. The prefix nano means a factor of one
billionth (10-9) and can be applied, e.g., to time (nanosecond), volume
(nanoliter), weight (nanogram) or length (nanometer or nm). In
thiscommon use “nano”refers to the length and the nanoscale refers to a
length from the atomic level of around 1nm up to 100nm. Nanotechnology is useful in the
techniques for various diagnostic processes, drug delivery, sunscreens, antimicrobial
bandages, disinfectant and a friendly manufacturing process which reduces waste
products thateventuallymost important to atomically precise molecular
manufacturing with less waste products which is also serves as catalyst for
greater efficiency in present manufacturing process by minimizing or
eliminating the use of toxic materials for reduction of pollution such aswater
and air, and, as an alternative energy production which issolar and fuel
cells.The goal of nanotechnology is to close sizegap between the smallest
lithographically fabricated structures and chemicallysynthesized large
molecules.
The successful
use of nanotechnology in the food industry can take a number of forms. These
include the use of nanotechnology in packaging of materials. It is usefulfor
developing the antimicrobial packaging of the food products. The nanoparticles
are dispersed throughout the plastic and are capable to block reachingoxygen,
carbon dioxide and moisture from fresh meats or other foods. Packaging can able
to contribute the control of microbial growth in foodstuffswhich can lead to
spoiling or in the case of a range of pathogenic microorganisms and appearance
of disease due to spread of infection. They are also being investigated for
their potential use as fungicides in agriculture, as anticancer drugs, and
imaging in biomedical applications. An additional advantage of
nanobiotechnology is the development of consistent and more reliable processes
for thesynthesis of nanomaterial excess of a range of sizes with
superiormonodisperse character and chemicalcomposition.
1.2. Nanomaterials
The
improvement of reliable experimental and investigational protocols for the
synthesis of nanomaterialsmore over a range of chemical compositions, sizes and
high monodispersity is one of the most challenging issues in current
nanotechnology.The term ‘Nanomaterial’ is now frequently used for the
conventional materials which are consciously and deliberately engineered to
nanostructure of modern application of nanotechnology. Nanomaterials are the
forms of matter at nanoscale. Generallynanomaterials are referred as the
infrastructure or building blocks element for nanotechnology.The “Building
blocks” for nanomaterials consist of carbon-based components and
organics, semiconductors, metals and metal oxides.Nanomaterials with structural
features at the nanoscale can be found in the form of clusters, thin films,
multilayer and nanocrystalline materials which are often expressed by the
dimensionality of 0, 1, 2 and 3. The materials include metals, amorphousand
crystalline alloys, semiconductors, oxides, nitride and carbide ceramics in
theform of clusters, thin films, multilayer and the bulk nanocrystalline
materials, etc.
Fig.1.1.
Classification of nanomaterials according to the dimension
Table.1.Classification
based on dimensionality
|
Dimension
|
Example of nanomaterials
|
|
0D
|
Colloids, nanoparticles,
nanodots, nanoclusters
|
|
1D
|
Nanowires, nanotubes,
nanobelts, nanorods
|
|
2D
|
Quantum wells, super lattices,
membranes
|
|
3D
|
Nanocomposites, filamentary
composites, cellular materials, porous materials, hybrids, nanocrystal
arrays, block polymers
|
The familiar nanomaterial ‘carbon
black’ was used in industrial production over a century ago. The other early
nanomaterials are fumed silica, a form of silicon dioxide (SiO2), titanium
dioxide (TiO2) and zinc oxide (ZnO). Nanomaterials can have different
properties at nanoscale which is also established by the Quantum effects. Among
all nanomaterials, some are having better conductivity towards heat and
electricity, different magnetic properties, light reflection and also change
colors according to their size which is also changed. These properties are a
little bit different from the bulk materials. Nanomaterials also have larger surface
areas than similar volumes of larger-scale materials which signify the meaning
of more surfaces is available for interactions with other materials around
them.Nanomaterials are being controlled to nano crystalline size which is less
than 100 nm which can show atom-like behaviors. This results from its higher
surface energy due to their large surface area and wider band gap between
valence and conduction band. It occurs when they are divided to near atomic
size. Nanomaterials are also referred as ‘‘a wonder of modern medicine’’. It
signifies the importance of antibiotics which kill atleast six different
disease-causing organisms whether the nanomaterials can kill at least
650cells.Nanomaterials are being enthusiastically researched for specific
function like microbial growth inhibition,carriers of antibiotics and also act
as killing agents.
1.2.1Metal oxide nanoparticles
Different types of nanoparticles
developed by the chemical and physical methods show poor morphology.Mostly
toxic chemicals are used in these processes and elevated temperaturewhich
isbeing helpfulfor the synthesis of respective nanoparticles is toxic to our
environment.Metal oxides have also been serves as sorbents for various
environmental pollutants. But the biological methods of synthesis are more
favorable than the chemical and physical methods of synthesis since these
methods are eco-friendly.The biological methods for synthesisof nanoparticles
by using various microorganisms, enzymes, plants,and their extracts have been
suggested as the probable and promisingecofriendly alternatives to the chemical
and physical methods of synthesis. The biocompatibility of nanoparticles is
more essential for specific biomedical applications and researches.
The metal nanoparticles
have various functions which are not observed in bulk phase. All these
arealreadybeen studied broadlydueto their exclusive electronic, magnetic,
optical, catalyticallyand antimicrobial properties of wound healing and also
for anti-inflammatory properties.The metal nanoparticles have the surface
Plasmon resonance absorption in the UV–visible region. Over the past few
decades, the structure of inorganic nanoparticles exhibit appreciable, drastic,
novel and highly improved physical, chemical and biological properties with well
recognized function due to their nanoscale size. Recent studies reported that
nanoparticles of some materials including metal oxides can also induce the cell
death in eukaryotic cells and the growth inhibition in prokaryotic cells due to
cytotoxicity nature. The transition metal oxides with nanostructure and
semiconductors with dimensions in the nanometer have capacity to attract
towards the areas belonging to Physics, Chemistry, Materials and Metallurgy
engineering, Biotechnology, Information technology and Environmental science
with their respective technologies in several aspect of development and
research. Different types of specific applications can be usedfor synthesis of
metal oxide nanoparticles as major components like sensors, pigments and various
medical materials.The dispersion of metal oxide nanoparticles in physiological
solutions is also important for biological in vitro and in vivo studies.
1.3Lemon
Leaves:
The lemon leaves
are dark green in color and arranged alternately on the stem. What appears to
be a slender second leaf (called a wing) is present on the petiole.Lime leaves
are small, pale green, and oblong in shape.
Fig:
Lemon leaves
Common
noun :Lemon tree
Scientific
noun:Citrus limonumRisso, Citrus limon (L.) Burm
Family.Citrus
family-Rutaceae
Habitat:
Cultivated because of its fruits and as a garden tree in warm Mediterranean
places next to thesea. It probably descends from the species "
Citrusmedica L. ", native from India.
Characteristics
:
Perennial tree of the Citrus family - rutaceae- up to 3 m. Toothed, elliptical
or lanceolateleaves, pointed. Flowers white inside, rosy at the margin of the petals.
The fruit is a hesperidium till 12,5 cm.wide, with a thick rind, dark yellow
when fully ripe.
1.3.1Active
components:
The
main components are:
Flavonoids: hesperidoside,
limocitrin in the pericarp of the Spanish lemons.
Acids:
Ascorbic (vitamin C), citric; caffeic ( fruit )
Essential
oil : rich in isopulegol, alpha-bergamotene, alpha-
pinene, alpha- terpinene, alpha- thujene, betabisolobene, beta- bergamotene,
beta- phelandrene, citral, limonene and sabinene, ( in the fruit , specially
inlemons from California)
Caffeine (leaves, flowers)
Pectin
Minerals:
potassium and calcium.
1.4 Silver nitrate :
Silver nitrate is an inorganic compound with chemical formula AgNO3. This compound is a versatile precursor to many other silver compounds, such as
those used in photography.
It is far less sensitive to light than the halides. It was once
called lunar caustic because silver was called luna by the ancient alchemists, who
believed that silver was associated with the moon.
Silver nitrate can be prepared by
reacting silver, such as a silver bullion or silver foil, with
nitric
acid, resulting in silver nitrate, water, and oxides of nitrogen. Reaction
byproducts depend upon the concentration of
nitric
acid used.
3 Ag + 4 HNO3 (cold and diluted) → 3 AgNO3 + 2 H2O + NO
Ag + 2 HNO3 (hot and concentrated) → AgNO3 + H2O + NO2
This is performed under a fume hood
because of toxic nitrogen oxide(s) evolved during the reaction.
1.4.1 USES
Precursor to
other silver compounds
Silver nitrate is the least expensive
salt of silver; it offers several other advantages as well. It is non-
hygroscopic,
in contrast to
silver fluoroborate and
silver perchlorate. It is relatively stable to
light. Finally, it dissolves in numerous solvents, including water. The nitrate
can be easily replaced by other ligands, rendering AgNO
3 versatile. Treatment with solutions of
halide ions gives a precipitate of AgX (X = Cl, Br, I). When making
photographic
film, silver nitrate is treated with
halide salts of sodium or potassium to form
insoluble
silver
halide in situ in
photographic
gelatin, which
is then applied to strips of tri-
acetate or
polyester. Similarly, silver nitrate is used to prepare some silver-based
explosives, such as the
fulminate,
azide,
or
acetylide,
through a
precipitation reaction.
Treatment of silver nitrate with base
gives dark grey
silver
oxide
2 AgNO3 + 2 NaOH → Ag2O + 2 NaNO3 + H2O
Halide
abstraction
The silver cation,
Ag+, reacts
quickly with halide sources to produce the insoluble silver halide, which is a
cream precipitate if Br- is used, a white precipitate if
Cl− is used and a yellow precipitate if
I−is used.
This reaction is commonly used in
inorganic chemistry to abstract halides:
Ag+ + X−(aq) →
AgX
where X− = Cl−, Br−,
or I−.
The same reaction is used on board
ships in order to determine whether or not
boiler
feedwater has been
contaminated with
seawater. It
is also used to determine if moisture on formerly dry cargo is a result of
condensation from humid air, or from seawater
leaking through the hull.
[13]
Organic
synthesis
Silver nitrate is used
in many ways in
organic
synthesis, e.g. for
deprotection and oxidations.
Ag+
binds
alkenes reversibly, and silver nitrate has
been used to separate mixtures of alkenes by selective absorption. The
resulting
adduct can be decomposed with
ammonia to release the free alkene.
[14]
Biology
1.5
Agar
Agar is derived
from the polysaccharide agarose, which forms the supporting structure in the
cell walls of certain species of algae, and which is released on boiling. These
algae are known as agarophytes and belong to the Rhodophyta (red algae) phylum.
Agar is actually the resulting mixture of two components: the linear
polysaccharide agarose, and a heterogeneous mixture of smaller molecules called
agaropectin.
1.5.1
Agar is used:
·
As an impression material in dentistry.
·
To make salt bridges for use in electrochemistry.
·
In formicariums as a transparent
substitute for sand and a source of nutrition.
·
As a natural ingredient to form
modelling clay for young children to play with.
·
Gelidium agar is used primarily for
bacteriological plates. Gracilaria agar is used mainly in food applications.
1.6
Dextrose
Dextrose is the
name of a simple sugar chemically identical to glucose (blood sugar) that is
made from corn. While dextrose is used in baking products as a sweetener, it
also has medical purposes. Dextrose is dissolved in solutions that are given
intravenously, which can be combined with other drugs, or used to increase a
person’s blood sugar. Dextrose is also available as an oral gel or tablet.
Because dextrose is a “simple” sugar, the body can quickly use it for energy.
Dextrose is used
in various concentrations for different purposes. For example, a doctor may
prescribe dextrose in an IV solution when someone is dehydrated and has low
blood sugar. Dextrose IV solutions can also be combined with many drugs, for IV
administration. These solutions may be used to reduce the sodium level in the
blood. The extra dextrose in a person’s body can cause sodium to go into the
cells, reducing the amount in the bloodstream.
Dextrose is a
carbohydrate, which is one part of nutrition in a normal diet. Solutions
containing dextrose provide calories and may be given intravenously in
combination with amino acids and fats. This is called total parenteral
nutrition (TPN) and is used to provide nutrition to those who can’t eat normally.
1.7
Potato dextrose agar :
Potato dextrose agar and potato dextrose broth are common microbiological growth media made from potato infusion,
and dextrose.
Potato dextrose agar (abbreviated "PDA") is the most widely used
medium for growing fungi and bacteria
Figure:
Potato Dextrose Agar
1.7.1. Required components for PDA :
|
grams
|
ingredient
|
|
1000
|
water
|
|
200
|
potatoes
(sliced washed unpeeled)
|
|
20
|
Dextrose
|
|
20
|
agar powder
|
Potato
infusion can be made by boiling 200 grams of sliced (washed but unpeeled)
potatoes in ~ 1 liter (0.22 imp gal; 0.26 US gal) distilled
water for 30 minutes and then decanting or straining the broth through cheesecloth. Distilled water is added such that the total volume of the suspension
is 1 liter. 20 grams (0.71 oz) dextrose and 20 grams (0.71 oz) agar
powder is then added and the medium is sterilized by autoclaving at 15 pounds per square
inch for 15 minutes.
1.8 Centrifugation
Centrifugation is a process that
involves the use of the centrifugal force for the sedimentation of
heterogeneous mixtures with a centrifuge, used in industry and in laboratory
settings. This process is used to separate two immiscible liquids. More-dense
components of the mixture migrate away from the axis of the centrifuge, while
lessdense components of the mixture migrate towards the axis. Chemists and
biologists may increase the effective gravitational force on a test tube so as
to more rapidly and completely cause the precipitate ("pellet") to
gather on the bottom of the tube. The remaining solution is properly called the
"supernate" or "supernatant liquid". The supernatant liquid
is then either quickly decanted from the tube without disturbing the
precipitate, or withdrawn with a Pasteur pipette.
1.8.1.Centrifugation
in biological research
1 Micro
centrifuges
2 High-speed
centrifuges
3 Fractionation
process
4
Ultracentrifuges
Figure:
Centrifugation
1.11.
UV-Vis Spectroscopy Ultraviolet-visible
spectroscopy or ultraviolet-visible spectro photometry (UV-Vis or UV/Vis)
refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. This means it uses light in
the visible and adjacent (near-UV and near-infrared [NIR]) ranges. The absorption or reflectance in the
visible range directly affects the perceived color of the
chemicals involved. In this region of the electromagnetic
spectrum, molecules undergo electronic. This technique is complementary
to fluorescence
spectroscopy,
in that fluorescence deals with transitions from
the excited state to the ground state, while absorption measures transitions from the ground
state to the excited state.
Fig.1.3.
UV/Vis spectroscopy
1.11.1. Working Principle
Molecules containing π-electrons or
non-bonding electrons (n-electrons) can absorb the energy in the form of
ultraviolet or visible light to excite these electrons to higher anti-bonding
molecular orbitals. The more easily excited the electrons (i.e. lower
energy gap between the HOMO and the LUMO), the longer the wavelength of light
it can absorb.
1.11.2. Applications UV/Vis spectroscopy is routinely
used in analytical chemistry for the quantitative determination of different
analyses, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is
commonly carried out in solutions but solids and gases may also be studied.
·
Solutions
of transition metal ions can be colored (i.e., absorb visible light)
because electrons within the metal atoms can be
excited from one electronic state to another. The color of metal ion solutions
is strongly affected by the presence of other species, such as certain anions
or ligands. For instance, the color of a dilute solution of copper sulfate is a very light blue; adding ammonia intensifies the color and changes the wavelength of
maximum absorption (λmax).
·
Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of
the electromagnetic
spectrum. The
solvents for these determinations are often water for water-soluble compounds,
or ethanol for organic-soluble compounds. (Organic solvents may
have significant UV absorption; not all solvents are suitable for use in UV
spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent
polarity and pH can affect the absorption spectrum of an organic compound.
Tyrosine, for example, increases in absorption maxima and molar extinction
coefficient when pH increases from 6 to 13 or when solvent polarity decreases.
·
While charge transfer
complexes also
give rise to colors, the colors are often too intense to be used for
quantitative measurement.
1.11.3.Beer Lambert’s Law The method is most often used in a
quantitative way to determine concentrations of an absorbing species in
solution, using the Beer-Lambert law:
,
Where A is the
measured absorbance, in Absorbance Units
(AU),
is the intensity of the incident light at a given wavelength,
is the transmitted intensity, L the
path length through the sample, and c the concentration of the absorbing species. For each species and
wavelength, ε is a constant known as the molar absorptivity or extinction coefficient.
This constant is a fundamental molecular property in a given solvent, at a
particular temperature and pressure, and has units of
or often
.
The absorbance and extinction ε are
sometimes defined in terms of the natural logarithm instead of the base-10 logarithm. The
Beer-Lambert Law is useful for characterizing many compounds but does not hold
as a universal relationship for the concentration and absorption of all
substances. A 2nd order polynomial relationship between absorption and
concentration is sometimes encountered for very large, complex molecules such
as organic dyes (Xylenol Orange or Neutral Red, for example).
1.11.4. UV-spectro photometer
The instrument used in ultraviolet-visible
spectroscopy is called a UV/Vis spectrophotometer.
It measures the intensity of light passing through a sample(
), and compares it to the intensity of light before it passes
through the sample (
). The ratio
is called the transmittance, and is usually
expressed as a percentage (%T). The absorbance
, is based
on the transmittance:
The UV-visible spectrophotometer can
also be configured to measure reflectance. In this case, the spectrophotometer
measures the intensity of light reflected from a sample (
), and compares it to the intensity of light reflected from a
reference material (
) (such as a white tile). The ratio
is called the reflectance, and is usually
expressed as a percentage (%R). The
basic parts of a spectrophotometer are a light source, a holder for the sample,
a diffraction grating in a monochromator or a prism to separate the different wavelength of light, and a
detector. The radiation source is often a Tungsten filament (300-2500 nm), a deuterium arc lamp, which is continuous over the
ultraviolet region (190-400 nm), Xenon arc lamp, which is continuous from 160-2,000 nm; or more
recently, light emitting diodes (LED)[8] for the visible wavelengths. The detector is typically
a photomultiplier tube, a photodiode, a photodiode array or a charge-coupled
device (CCD).
Single photodiode detectors and photomultiplier tubes are used with scanning
monochromators, which filter the light so that only light of a single
wavelength reaches the detector at one time. The scanning monochromator moves
the diffraction grating to "step-through" each wavelength so that its
intensity may be measured as a function of wavelength. Fixed monochromators are
used with CCDs and photodiode arrays. As both of these devices consist of many
detectors grouped into one or two dimensional arrays, they are able to collect
light of different wavelengths on different pixels or groups of pixels
simultaneously.
Fig.1.4. Schematic UV- visible
spectrophotometer.
A spectrophotometer can be
either single beam or double beam. In a single beam
instrument (such as theSpectronic 20), all of the light passes through
the sample cell.
must be measured by removing the sample. This was the
earliest design and is still in common use in both teaching and industrial
labs.
In a double-beam instrument, the
light is split into two beams before it reaches the sample. One beam is used as
the reference; the other beam passes through the sample. The reference beam
intensity is taken as 100% Transmission (or 0 Absorbance), and the measurement
displayed is the ratio of the two beam intensities. Some double-beam
instruments have two detectors (photodiodes), and the sample and reference beam
are measured at the same time. In other instruments, the two beams pass through
abeam chopper, which blocks one beam at a time.
The detector alternates between measuring the sample beam and the reference
beam in synchronism with the chopper. There may also be one or more dark
intervals in the chopper cycle. In this case, the measured beam intensities may
be corrected by subtracting the intensity measured in the dark interval before
the ratio is taken. Samples
for UV/Vis spectrophotometry are most often liquids, although the absorbance of
gases and even of solids can also be measured. Samples are typically placed in
a transparent cell, known as a cuvette. Cuvettes are typically rectangular in shape, commonly with
an internal width of 1 cm. (This width becomes the path length,
, in the Beer-Lambert law.) Test tubes can also be used as cuvettes in some instruments. The
type of sample container used must allow radiation to pass over the spectral
region of interest. The most widely applicable cuvettes are made of high
quality fused silica or quartz glass because these are transparent throughout the UV,
visible and near infrared regions. Glass and plastic cuvettes are also common,
although glass and most plastics absorb in the UV, which limits their
usefulness to visible wavelengths. Specialized
instruments have also been made. These include attaching spectrophotometers to
telescopes to measure the spectra of astronomical features.
UV-Visible micro spectrophotometers
consist of a UV-Visible microscope integrated with a UV-Visible
spectrophotometer. A complete spectrum of the absorption at all wavelengths of
interest can often be produced directly by a more sophisticated
spectrophotometer. In simpler instruments the absorption is determined one
wavelength at a time and then compiled into a spectrum by the operator. By
removing the concentration dependence, the extinction coefficient (ε) can be
determined as a function of wavelength.
1.12. FTIR
Spectra analysis FT-IR stands for
Fourier Transform Infrared, the preferred method of infrared spectroscopy.
Ininfrared spectroscopy, IR radiation is passed through a sample. Some of the
infrared radiation isabsorbed by the sample andsome of it is passed
through(transmitted). The resultingspectrum represents the molecularabsorption
and transmission,creating a molecular fingerprintof the sample. Like a
fingerprintno two unique molecularstructures produce the sameinfrared spectrum.
This makesinfrared spectroscopy useful forseveral types of analysis.
Fig.1.5.Fourier
transform infrared spectroscopy
Fig.1.6.
Spectrometer
So,
FT-IR will provideinformation
•
It can identify unknown materials
•
It can determine the quality or consistency of a sample
•
It can determine the amount of components in a mixture
1.12.1.
Infrared Spectroscopy
Infrared spectroscopy has been a workhorse technique
for materials analysis in the laboratory for overseventy years. An infrared
spectrum represents a fingerprint of a sample with absorption peaks
whichcorrespond to the frequencies of vibrations between the bonds of the atoms
making up the material.Because each different material is a unique combination
of atoms, no two compounds produce theexact same infrared spectrum. Therefore,
infrared spectroscopy can result in a positive identification(qualitative analysis) of every different kind of
material. In addition, the size of the peaksin thespectrum is a direct
indication of the amountof
material present. With modern software algorithms,infrared is an excellent tool
for quantitative analysis.
1.12.2.
Older Technology
The original infrared instruments were of the dispersivetype. These instruments
separated theindividual frequencies of energy emitted from the infrared source.
This was accomplished by the useof a prism or grating. An infrared prism works
exactly the same as a visible prism which separatesvisible light into its
colors (frequencies). A grating is a more modern dispersive element which
betterseparates the frequencies of infrared energy. The detector measures the
amount of energy at eachfrequency which has passed through the sample. This
results in a spectrumwhich is a
plot ofintensity vs. frequency.
Fourier transform infrared spectroscopy is
preferred over dispersive or filter methods of infrared spectral analysis for
several reasons
• It is a non-destructive technique
• It provides a precise
measurement method which requires no external calibration
• It can increase speed, collecting a scan every
second
• It can increase
sensitivity – one second scans can be co-added together to ratio out random
noise
• It has greater optical throughput
• It is mechanically simple with only one moving
part
1.12.3.
Importance FT-IR
Fourier
Transform Infrared (FT-IR) spectrometry was developed in order to overcome the
limitationsencountered with dispersive instruments. The main difficulty was the
slow scanning process. A methodfor measuring all of the infrared frequencies simultaneously, rather than
individually, was needed.A solution was developed which employed a very simple
optical device called an interferometer.The
interferometer produces a unique type of signal which has all of the infrared
frequencies “encoded”into it. The signal can be measured very quickly, usually
on the order of one secondor so.
Thus,the time element per sample is reduced to a matter of a few seconds rather
than several minutes.
Most interferometers employ a beam splitter which takes the incoming
infrared beam anddivides it into two optical beams. One beam reflects off of a
flat mirror which is fixed in place. Theother beam reflects off of a flat
mirror which is on amechanism which allows this mirror to move a veryshort
distance (typically a few millimeters) away fromthe beam splitter. The two
beams reflect off of theirrespective mirrors and are recombined when they
meetback at the beam splitter. Because the path that one beamtravels is a fixed
length and the other is constantlychanging as its mirror moves, the signal
which exits the interferometer is the result of these two beams “interfering”
with each other. The resulting signalis called an interferogramwhich has the unique property that every data point
(a function of themoving mirror position) which makes up the signal has
information about every infrared frequencywhich comes from the source.
Fig.Interferogram
This
means that as the interferogram is measured; all frequencies are being measuredsimultaneously. Thus, the use of the
interferometer results in extremely fast measurements. Because
the analyst requires a frequency
spectrum (a plot of the intensity at each individualfrequency) in order
to make identification, the measured interferogram signal cannot be
interpreteddirectly. A means of “decoding” the individual frequencies is
required. This can be accomplished via awell-known mathematical technique
called the Fourier transformation.
This transformation isperformed by the computer which then presents the user
with the desired spectral information for analysis.
Fig. Conversion of
signal into spectrum by using FFT
1.12.4.
The Sample Analysis Process
The
normal instrumental process is as follows:
1.
The Source: Infrared energy is emitted from a
glowing black-body source. This beam passesthrough an aperture which controls
the amount of energy presented to the sample (and, ultimately, to the
detector).
2.
The Interferometer: The beam enters the interferometer where
the “spectral encoding” takesplace. The resulting interferogram signal then
exits the interferometer.
3.
The Sample: The beam enters the sample compartment
where it is transmitted through or reflectedoff of the surface of the sample,
depending on the type of analysis being accomplished. This is where specific
frequencies of energy, which are uniquely characteristic of the sample, are
absorbed.
4.
The Detector: The beam finally passes to the detector
for final measurement. The detectors usedare specially designed to measure the
special interferogram signal.
5.
The Computer: The measured signal is digitized and
sent to the computer where the Fouriertransformation takes place. The final
infrared spectrum is then presented to the user forinterpretation and any
further manipulation.
Fig.1.9.Sample analysis
process
Because there needs to be a relative scale for the
absorption intensity, a background spectrummust
also be measured. This is normally a measurement with no sample in the beam.
This can becompared to the measurement with the sample in the beam to determine
the “percent transmittance.”This technique results in a spectrum which has all
of the instrumental characteristics removed.Thus all spectral features which
are present are strictly due to the sample. A single backgroundmeasurement can
be used for many sample measurements because this spectrum is characteristic
ofthe instrumentitself.
1.12.5.
Advantages of FT-IR
Some
of the major advantages of FT-IR over the dispersive technique include:
•
Speed: Because all of the frequencies are measured simultaneously, most
measurements by FTIRare made in a matter of seconds rather than several
minutes. This is sometimes referred to as theFelgett Advantage.
•
Sensitivity: Sensitivity is dramatically improved with FT-IR for many
reasons. The detectorsemployed are much more sensitive, the optical throughput
is much higher (referred to as theJacquinot
Advantage) which results in much lower noise levels, and the fast scans
enable theaddition of several scans in order to reduce the random measurement
noise to any desired level(referred to as signal averaging).
•
Mechanical Simplicity: The moving mirror in the interferometer is the
only continuouslymoving part in the instrument. Thus, there is very little possibility
of mechanical breakdown.
•
Internally Calibrated: These instruments employ a HeNe laser as an
internal wavelengthcalibration standard (referred to as the Connes Advantage). These instruments
are self-calibratingand never need to be calibrated by the user.
These advantages, along with several others, make
measurements made by FT-IR extremelyaccurate and reproducible. Thus, it a very
reliable technique for positive
identificationofvirtuallysample. The sensitivity benefits enable
identification of even the smallest of contaminants. Thismakes FT-IR an
invaluable tool for quality controlor
quality assurance applications whether it isbatch-to-batch comparisons to
quality standards or analysis of an unknown contaminant. In addition,the
sensitivity and accuracy of FT-IR detectors, along with a wide variety of
software algorithms, havedramatically increased the practical use of infrared
for quantitative analysis.
Quantitative methodscan be easily developed and calibrated and can be
incorporated into simple procedures for routine analysis.
Thus, the Fourier Transform Infrared (FT-IR)
technique has brought significant practicaladvantages to infrared spectroscopy.
It has made possible the development of many new samplingtechniques which were
designed to tackle challenging problems which were impossible by
oldertechnology. It has made the use of infrared analysis virtually limitless.
1.13. SEM measurement
The surface morphology of the as-synthesized silver
nanoparticles was observed by Scanning Electron Microscope (SEM) (Zeiss
Scanning electron microscope). The sample was coated on the conductive carbon
tape.
Fig.1.10. SEM Microscope
A scanning electron microscope (SEM) is a type of electron microscope that produces images of a
sample by scanning it with a focused beam of electrons. The electrons interact with atoms in the sample, producing
various signals that can be detected and that contain information about the
sample's surface topography and composition. The electron
beam is generally scanned in a raster scan pattern, and the beam's position is combined with the
detected signal to produce an image. SEM can achieve resolution better than 1
nanometer. Specimens can be observed in high vacuum, in low vacuum, in wet
conditions (in environmental SEM), and at a wide range of cryogenic or elevated
temperatures. The
most common mode of detection is by secondary electrons emitted by atoms
excited by the electron beam. On a flat surface, the plume of secondary
electrons is mostly contained by the sample, but on a tilted surface, the plume
is partially exposed and more electrons are emitted. By scanning the sample and
detecting the secondary electrons, an image displaying the topography of the
surface is created.
1.13.1. History
An account of the early history of
SEM has been presented by McMullan Although Max Knoll produced a photo with a 50 mm object-field-width
showing channeling contrast by the use of an electron beam scanner, it
was Manfred von Ardennewho in 1937 invented a true microscope
with high magnification by scanning a very small raster with a demagnified and
finely focused electron beam. Ardenne applied the scanning principle not only
to achieve magnification but also to purposefully eliminate the chromatic aberration otherwise inherent in the
electron microscope. He further discussed the various detection modes,
possibilities and theory of SEM, together with the construction of
the first high magnification SEM. Further work was reported by
Zworykin's group, followed by the Cambridge groups in the 1950s and early
1960s headed by Charles Oatley, all of which finally led to the marketing of the first
commercial instrument by Company as the "Stereo scan" in 1965
(delivered to DuPont).
1.13.2. Working principle
The types of signals produced by a
SEM include secondary electrons (SE), back-scattered electrons (BSE), characteristic
X-rays, light (cathodoluminescence) (CL), specimen current and transmitted
electrons. Secondary electron detectors are standard equipment in all SEMs, but
it is rare that a single machine would have detectors for all possible signals.
The signals result from interactions of the electron beam with atoms at or near
the surface of the sample. In the most common or standard detection mode,
secondary electron imaging or SEI, the SEM can produce very high-resolution
images of a sample surface, revealing details less than 1 nm in size. Due to the very narrow electron beam, SEM
micrographs have a large depth of field yielding a characteristic three-dimensional appearance
useful for understanding the surface structure of a sample. This is exemplified
by the micrograph of pollen shown above. A wide range of magnifications is
possible, from about 10 times (about equivalent to that of a powerful
hand-lens) to more than 500,000 times, about 250 times the magnification limit
of the best light microscopes. Back-scattered electrons
(BSE) are beam electrons that are reflected from the sample by elastic scattering. BSE are often used in analytical
SEM along with the spectra made from the characteristic X-rays, because the
intensity of the BSE signal is strongly related to the atomic number (Z) of the
specimen. BSE images can provide information about the distribution of
different elements in the sample. For the same reason, BSE imaging can
image colloidal gold immune-labels of 5 or 10 nm diameter,
which would otherwise be difficult or impossible to detect in secondary
electron images in biological specimens. Characteristic X-rays are emitted when the electron
beam removes an inner shell electron from the sample, causing
a higher-energy electron to fill the shell and release
energy. These characteristic X-rays are used to identify the composition and
measure the abundance of elements in the sample.
1.13.3. Sample preparation
All samples must also be of an appropriate size to fit in
the specimen chamber and are generally mounted rigidly on a specimen holder
called a specimen stub. Several models of SEM can examine any part of a 6-inch
(15 cm) semiconductor wafer, and some can tilt an object of that size to
45°.For conventional imaging in the SEM, specimens must be electrically
conductive, at least
at the surface, and electrically
grounded to
prevent the accumulation of electrostatic charge at the surface. Metal objects
require little special preparation for SEM except for cleaning and mounting on
a specimen stub. Nonconductive specimens tend to charge when scanned by the
electron beam, and especially in secondary electron imaging mode, this causes
scanning faults and other image artifacts. They are therefore usually coated
with an ultrathin coating of electrically conducting material, deposited on the
sample either by low-vacuum sputter coating or by high-vacuum evaporation.
An alternative to coating for some
biological samples is to increase the bulk conductivity of the material by
impregnation with osmium using variants of the OTO staining method (O-osmium, T-thiocarbohydrazide, O-osmium). Nonconducting
specimens may be imaged uncoated using environmental SEM (ESEM) or low-voltage
mode of SEM operation. Environmental SEM instruments place the specimen in a
relatively high-pressure chamber where the working distance is short and the
electron optical column is differentially pumped to keep vacuum adequately low
at the electron gun. The high-pressure region around the sample in the ESEM neutralizes
charge and provides an amplification of the secondary electron signal.
Low-voltage SEM is typically conducted in an FEG-SEM because the field emission guns (FEG) is capable of producing
high primary electron brightness and small spot size even at low accelerating
potentials. Operating conditions to prevent charging of non-conductive
specimens must be adjusted such that the incoming beam current was equal to sum
of outcoming secondary and backscattered electrons currents. It usually occurs
at accelerating voltages of 0.3–4 kV. Embedding in a resin with further polishing to a
mirror-like finish can be used for both biological and materials specimens when
imaging in backscattered electrons or when doing quantitative X-ray
microanalysis. The
main preparation techniques are not required in the environmental SEM outlined below, but some biological specimens can
benefit from fixation.
1.13.4. Scanning process and image
formation
Fig.1.11. Schematic of an SEM
In a typical SEM, an electron beam
is thermionically emitted from an electron gun fitted with a tungsten filament cathode. Tungsten is normally used in thermionic electron guns
because it has the highest melting point and lowest vapor pressure of all
metals, thereby allowing it to be heated for electron emission, and because of
its low cost. The electron beam,
which typically has an energy ranging from 0.2 keV to 40 keV, is focused by one or two condenser lenses
to a spot about 0.4 nm to 5 nm in diameter. The beam passes through
pairs of scanning coils or pairs of deflector plates in the electron column,
typically in the final lens, which deflect the beam in the x and y axes
so that it scans in a raster fashion over a rectangular area of the sample surface. When the primary electron beam
interacts with the sample, the electrons lose energy by repeated random
scattering and absorption within a teardrop-shaped volume of the specimen known
as the interaction volume, which extends from less than 100 nm to
approximately 5 µm into the surface. The size of the interaction volume
depends on the electron's landing energy, the atomic number of the specimen and
the specimen's density. The energy exchange between the electron beam and the
sample results in the reflection of high-energy electrons by elastic scattering, emission of secondary electrons
by inelastic scattering and the emission of electromagnetic
radiation, each of
which can be detected by specialized detectors. The beam current absorbed by
the specimen can also be detected and used to create images of the distribution
of specimen current. Electronic amplifiers of various types are used to
amplify the signals, which are displayed as variations in brightness on a
computer monitor (or, for vintage models, on a cathode ray tube). Each pixel of computer video memory is synchronized with
the position of the beam on the specimen in the microscope, and the resulting
image is therefore a distribution map of the intensity of the signal being
emitted from the scanned area of the specimen. In older microscopes image may
be captured by photography from a high-resolution cathode
ray tube, but in modern machines image is saved to computer data
storage.
1.13.5. Magnification Magnification in a SEM can be controlled over a range of up to
6 orders of magnitude from about 10 to 500,000
times. Unlike optical and transmission electron microscopes, image
magnification in the SEM is not a function of the power of the objective lens. SEMs may have condenser and objective lenses, but
their function is to focus the beam to a spot, and not to image the specimen.
Provided the electron can generate a beam with sufficiently small diameter,
a SEM could in principle work entirely without condenser or objective lenses,
although it might not be very versatile or achieve very high resolution. In a
SEM, as in scanning probe
microscopy, magnification
results from the ratio of the dimensions of the raster on the specimen and the
raster on the display device. Assuming that the display screen has a fixed
size, higher magnification results from reducing the size of the raster on the
specimen, and vice versa. Magnification is therefore controlled by the current
supplied to the x, y scanning coils, or the voltage supplied to the x, y
deflector plates, and not by objective lens power.
1.13.6. Resolution of SEM
Fig.1.12.
Resolution of SEM
A video illustrating a typical
practical magnification range of a scanning electron microscope designed for
biological specimens. The video starts at 25x, about 6 mm across the whole
field of view, and zooms in to 12000x, about 12 μm across the whole field of
view. The spherical objects are glass beads with a diameter of 10 μm, similar
in diameter to a red blood cell.
The spatial resolution of the SEM depends on the size of the
electron spot, which in turn depends on both the wavelength of the electrons
and the electron-optical system that produces the scanning beam. The resolution
is also limited by the size of the interaction volume, the volume of specimen
material that interacts with the electron beam. The spot size and the
interaction volume are both large compared to the distances between atoms, so
the resolution of the SEM is not high enough to image individual atoms, as is possible
in the shorter wavelength (i.e. higher energy) transmission
electron microscope (TEM).
The SEM has compensating advantages, though, including the ability to image a
comparatively large area of the specimen; the ability to image bulk materials
(not just thin films or foils); and the variety of analytical models available
for measuring the composition and properties of the specimen. Depending on the
instrument, the resolution can fall somewhere between less than 1 nm and
20 nm. By 2009, The world's highest SEM resolution at high-beam energies
(0.4 nm at 30 kV) is obtained with the Hitachi SU-9000.
Transmission electron microscopy (TEM) is a microscopy technique in which 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 focusedonto
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.
At smaller magnifications TEM image contrast is due to absorption of electrons in
the material, due to the thickness and composition of the material. At higher
magnifications complex wave interactions modulate the intensity of the image,
requiring expert analysis of observed images. Alternate modes of use allow for
the TEM to observe modulations in chemical identity, crystal orientation,
electronic structure and sample induced electron phase shift as well as the
regular absorption based imaging.
The first TEM was built by Max Knoll and Ernst Ruska in 1931, with this group developing
the first TEM with resolution greater than that of light in 1933 and the first
commercial TEM in 1939.
A TEM image of the polio virus. The polio virus is 30 nm in
size.
Electrons
Theoretically, the maximum resolution, d, that
one can obtain with a light microscope has been limited by the wavelength of
the photons that are being used to probe the sample, λ and
the numerical aperture of the system, NA.
Early twentieth century scientists theorized ways of getting
around the limitations of the relatively large wavelength of visible light (wavelengths of 400–700 nanometers) by using electrons. Like all matter, electrons have both
wave and particle properties (as theorized by Louis-Victor de
Broglie), and
their wave-like properties mean that a beam of electrons can be made to behave
like a beam of electromagnetic radiation. The wavelength of electrons is
related to their kinetic energy via the de Broglie equation. An additional
correction must be made to account for relativistic effects, as in a TEM an
electron's velocity approaches the speed of light, c.
where, h is Planck's constant, m0 is
the rest mass of an electron and E is
the energy of the accelerated electron. Electrons are usually generated in an
electron microscope by a process known asthermionic emission from a filament, usually
tungsten, in the same manner as a light bulb, or alternatively by field electron
emission. The
electrons are then accelerated by an electric potential(measured in volts) and focused by electrostatic and electromagnetic
lenses onto the sample. The transmitted beam contains information about
electron density, phase and periodicity; this beam is used to form an image.
Source
formation
Layout of optical components in a
basic TEM
Single crystal LaB6 filament
Hairpin style tungsten filament
From the top down, the TEM consists of an emission source,
which may be a tungsten filament, or a lanthanum hexaboride (LaB6) source.[19] For tungsten, this will be of the form of either a
hairpin-style filament, or a small spike-shaped filament. 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 almost always aided by the use of a Wehnelt
cylinder to provide preliminary focus by consolidating and
directing the electrons in these initial phases of forming the emitted
electrons into a beam. The upper lenses of the TEM then further focus the
electron beam to the desired size and location for subsequent interaction with
the sample.[20]
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 left hand rule, thus allowing for electromagnets to manipulate the electron beam. The use of magnetic
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, subsequently this is
extremely important toSTEM. From these two effects, as well as
the use of an electron imaging system, sufficient control over the beam path is
possible for TEM operation[citation
needed]. The optical configuration of a TEM
can be rapidly changed, unlike that for an optical microscope, as lenses in the
beam path can be enabled, have their strength changed, or be disabled entirely
simply via rapid electrical switching, the speed of which is limited by effects
such as the magnetic hysteresis of the lenses.
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 condenser lenses, the objective lenses, and the projector
lenses. The condenser lenses are responsible for primary beam formation, while
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.[21] Additional quad or hexapole lenses allow for the
correction of asymmetrical beam distortions, known asastigmatism. It is noted that TEM optical configurations differ
significantly with implementation, with manufacturers using custom lens
configurations, such as in spherical aberration corrected instruments, or
TEMs utilizing energy filtering to correct electron chromatic aberration.
