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Q1: Scanning tunneling Microscope (STM)

Answer: The scanning tunneling microscope (STM) is a magnificent microscope ever built.
It was generated in 1981 by Gerd Binning and Heinrich Rohrer of IBM’s Zurih Lab in
Zurich, Switzerland. The invention deserved Nobel Prize for physics in 1986.

 The Scanning Tunneling Microscope is an electron microscope that transmits three-

dimensional images of the electron cloud around the nucleus.
 The STM allows the inspection of the properties of a conductive solid surface at an
atomic size.
 It is a very important technique in determining the atomic structures and electronic
states of the surface under investigation.
 Surfaces can be viewed at the atomic size to the high resolution (0.1 Ǻ) that STM has.

Basic components of STM

Five basic components:

1. Metal tip,

2. Piezoelectric scanner,

3. Current amplifier (nA),

4. Bipotentiostat (bias),

5. Feedback loop (current).

Work Principle of STM

When voltage is generated between a specimen and a tip with a very small distance (a few
angstroms), due to the quantum tunneling, there is an electron transition from the specimen to
tip or from tip to specimen, which corresponds to a tunnel current at the picoamper stage.
 This tunneling current, which occurs when scanning the specimen on the specimen
surface, is measured and is used to obtain surface tomography, since this current is a function
of the distance between the specimen and the tip.

STM Advantages
 STMs are also versatile. They can be used for ultra-high vacuum, air, water and other
liquids and gasses.
 They will activate in temperatures as low as zero Kelvin up to a few hundred degrees
Celsius.
 Scanning Tunneling Microscope works faster than Atomic Force Microscope.
 AFM max sample size is 150x150 µm. On the other hand, STM generates mm size
length and width.
 Lastly, resolution of STM is much better than AFM.

STM Disadvantages
 There are very few disadvantages to using a scanning tunneling microscope.
 STMs can be difficult to use effectively. There is very specific technique that
requires a lot of skill and precision.
 STM requires very stable and smooth surfaces, excellent vibration control and sharp
tips.
 STMs use highly specialized equipment that is fragile and expensive.
 Although, STM analysis only conductive materials, AFM uses for conductive and
insulator materials.
  STM requires vacuum atmosphere but AFM can work even in liquid. For that reason
AFM can be used for biological materials.

Reference :( http://jdetrick.blogspot.com/2012/03/scanning-tunneling-microscope-
and-3-d.html)

Q2: Atomic force microscope (AFM)

Answer: Atomic force microscopy (AFM) was developed when people tried to extend STM
technique to investigate the electrically non-conductive materials, like proteins.  In 1986,
Binnig and Quate demonstrated for the first time the ideas of AFM, which used an ultra-small
probe tip at the end of a cantilever.

AFM is one kind of scanning probe microscope that possesses a very high resolution (on the
order of fractions of nanometers) • Operates by measuring force between its probe and the
sample. Unlike the electron microscope, AFM provides a 3-D surface profile.

The AFM has three major abilities: force measurement, imaging, and manipulation.It is
powerful because an AFM can generate images at atomic resolution with angstrom scale
resolution height information, with minimum sample preparation.

Principle of Atomic force microscope (AFM)

 Surface Sensing
 Detection Method

Surface Sensing
An AFM uses a cantilever with a very sharp tip to scan over a sample surface. As the tip
approaches the surface, the close-range, attractive force between the surface and the tip cause
the cantilever to deflect towards the surface. However, as the cantilever is brought even
closer to the surface, such that the tip makes contact with it, increasingly repulsive force takes
over and causes the cantilever to deflect away from the surface.

Detection Method
A laser beam is used to detect cantilever deflections towards or away from the surface. By
reflecting an incident beam off the flat top of the cantilever, any cantilever deflection will
cause slight changes in the direction of the reflected beam. A position-sensitive photo diode
(PSPD) can be used to track these changes. Thus, if an AFM tip passes over a raised surface
feature, the resulting cantilever deflection (and the subsequent change in direction of
reflected beam) is recorded by the PSPD.
AFM is better than STM

It give information about the conducting and non-conducting surfaces. 3-D image of surfaces
are obtained by this techniques. Sample can be analyzed in open air.

Application
The AFM has been applied to problems in a wide range of disciplines of the natural sciences,
including solid-state physics, semiconductor science and technology, molecular engineering,
polymer chemistry and physics, surface chemistry, molecular biology, cell biology, and
medicine.

Reference :( https://www.nature.com/articles/s41598-018-19379-x)

Q3: Electron Microscope

Answer: An electron microscope is a microscope that uses a beam of accelerated electrons as
a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter
than that of visible light photons, electron microscopes have a higher resolving power than
light microscopes and can reveal the structure of smaller objects. A scanning transmission
electron microscope has achieved better than 50 pm resolution in annular dark-field imaging
mode[1] and magnifications of up to about 10,000,000× whereas most light microscopes are
limited by diffraction to about 200 nm resolution and useful magnifications below 2000×.

Electron microscopes use shaped magnetic fields to form electron optical lens systems that
are analogous to the glass lenses of an optical light microscope.

Construction
An electron microscope is similar to that of an optical microscope. Here the focusing of
electrons can be done either by magnetic lens or by electrostatic lens. Normally in electron
microscope magnetic lenses are used for focusing.

The electron microscope consists of an electron gun to produce the stream of electrons.
Similar to the condensing lens, objective and eye piece in an optical microscope here three
magnetic lenses are used.

 Magnetic condensing lens

 Magnetic objective lens

 Magnetic projector lens

The whole arrangement is kept inside a vacuum chamber to allow the passage of electron
beam.

Working

 Stream of electrons are produced and accelerated by the electron gun.

 The electron beam is made to pass through the center of the doughnut shaped
magnetic condensing lens.

 These electrons are made as parallel beam and are focused on to the object.

 The electrons are transmitted more in the less dense region of the object and is
transmitted less (i.e.,) absorbed by the denser region of the object.

 Thus the transmitted electron beam on the falling over the magnetic objective lens,
resolves the structure of the object to form a magnified real image of the object.
Further the image can be magnified by the magnetic projector lens and the final image
is obtained on the fluorescent screen.

 In order to make a permanent record of the image of the object, the final image can
also be obtained on a photographic plate.
TYPES OF ELECTRON MICROSCOPE

SCANNING ELECTRO MICROSCOPE

SEM focuses on the sample’s surface and its composition. Scan a gold-plated specimen to
give a 3-D view of the surface of an object which is black and white. Used to study surface
features of materials, cells and viruses. Scanning Electron microscope has resolution 1000
times better than Light microscope.

TRANSMISSION ELECTRON MICROSCOPE

Stream of electrons is formed. Accelerated using a positive electrical potential Focused by
metallic aperture and Electro magnets  Interactions occur inside the irradiated sample which
are detected and transformed into an image. Scanning electron microscopy allows for higher
magnification and better resolution than standard light microscopy. Since the sample is
bombarded with electrons rather than light, the level of detail in a smaller area is much
greater than a light microscope.

Reference:(https://www.slideshare.net/kalyanacharjya/electron-microscopy-sem-tem?
from_action=save)

Q4: Fluorescence Methods

Answer: Fluorescence is the emission of light by a substance that has absorbed light or other
electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a
longer wavelength, and therefore lower energy, than the absorbed radiation. The most
striking example of fluorescence occurs when the absorbed radiation is in the ultraviolet
region of the spectrum, and thus invisible to the human eye, while the emitted light is in the
visible region, which gives the fluorescent substance a distinct colour that can be seen only
when exposed to UV light. Fluorescent materials cease to glow nearly immediately when the
radiation source stops, unlike phosphorescent materials, which continue to emit light for
some time after.

Fluorescence imaging

Fluorescence imaging is the visualization of fluorescent dyes or proteins as labels for

molecular processes or structures. It enables a wide range of experimental observations
including the location and dynamics of gene expression, protein expression and molecular
interactions in cells and tissues.

Time Resolved Fluorescence

Fluorescence is the emission of light by a molecule upon excitation by light at a shorter

wavelength than the emitted one.

Fluorescence detection can be mostly divided in two types of measurements: steady state and
time-resolved fluorescence. The main difference between these two methods is based on the
nature and properties of the fluorescent molecules (fluorophores) used and the consequent
time of detection.

Steady state is the most widespread fluorescent detection mode and is commonly referred to
as “fluorescence intensity”. Standard fluorophores (e.g. fluorescein, rhodamine, etc.) emit
light at a specific wavelength within nanoseconds upon excitation. This very short time gap
between excitation and emission allows the almost concomitant detection of the fluorescent
emission signal with the excitation of the sample.

The second type, time-resolved fluorescence (TRF), is monitored as a function of time upon
excitation. In contrast to steady state fluorescence intensity, time-resolved fluorescence is
based on the detection of intensity decays and/or on the delayed detection of the emission
signal upon excitation. In time-resolved fluorescence measurements, the excitation light pulse
is shorter than the decay time of the fluorescent signal.
Time-resolved fluorescence detection can only be achieved when the emission signal of the
fluorophore is prolonged to the micro- or even milli-second range and not short-lived within
nanoseconds as for common labels

Lanthanides (Ln) are a group of uniquely fluorescent metallic chemical elements often
collectively known as “rare earth elements”. Lanthanides have very low absorption
(excitation) coefficients and slow emission rates. This results in prolonged fluorescence
decay times between 0.5 to 3 milliseconds (long lifetimes). All lanthanide elements form
trivalent cations (Ln3+) and display emission in aqueous solution. Additionally, their emission
peaks are very sharp and narrow with a large Stokes shift.

How is time-resolved fluorescence detected?

Similar to fluorescence intensity detection, the setup for time-resolved fluorescence

measurements consists of a light source, an optical device for wavelength selection and a
photomultiplier tube (PMT) detector.

Advantages of time-resolved fluorescence and lanthanides

 Reduction of background noise

 A large Stokes shift

 High intensity signals

 Long assay stability

Disadvantages of time-resolved fluorescent

 Higher costs
  Multiple steps

Fluorescence Anisotropy

Measurement of steady-state and particularly time-resolved fluorescence anisotropy offers

fascinating possibilities to study molecular orientation and mobility as well as processes that
affect them. Except of special cases, anisotropy does not depend on the concentration of
fluorophores, thus on the detected signal intensity. Owing to this similarity with the behavior
of fluorescence lifetime, we can regard anisotropy as a yet another dimension of fluorescence
information. Molecules with their absorption transition moment (ATM) aligned with the
polarization plane of excitation are preferentially excited. This is called photo-selection,
because at the same time molecules with ATM oriented perpendicular to the excitation
polarization plane are not excited, they remain in their ground state.

Subsequently, the polarization plane of an emitted fluorescence photon is defined by the

orientation of the emission transition moment of the molecule, thus by the orientation of the
molecule itself at the moment of emission. The degree of fluorescence polarization expressed
as a dimensionless quantity, anisotropy, is usually the highest at the moment of excitation and
then normally decreases in time. Common reason for this is the random molecular motion,
e.g. Brownian rotation or conformational flexibility that tends to randomize the initially well
aligned photo selected fluorophore population. The so called anisotropy decay can be
observed sometimes even in a solid sample (e.g. rigid, frozen or very viscous environment)
owing to intermolecular processes or energy transfer between molecules. Time-resolved
anisotropy measurement is more informative than its steady-state counterpart, the latter
reporting time averaged values only, without direct insight into the dynamics of the process.

Figure
Anisotropy measurements are typically performed with standard fluorescence spectrometers
using plane-polarized excitation light. Lasers usually emit polarized light; the output of LEDs
or steady-state lamps is made polarized using high quality polarizers. The fluorescence
emission is then detected through a second polarizer (sometimes called analyzer). In standard
fluorescence spectrometers the excitation is usually polarized vertically in respect to the plane
of the optics (L-geometry). Anisotropy (-decay) is then calculated from consecutively
measured vertical and horizontal polarized intensities (decays). In microscopes it is more
common to use two detectors and a polarizing beam splitter inserted between them. [7]

Fluorescent label

Fluorescent labeling is the process of covalently binding fluorescent dyes to biomolecules

such as nucleic acids or proteins so that they can be visualized by fluorescence imaging.

Fluorescent Sensors

Fluorescence sensors can be used to study chlorophyll and to measure dissolved oxygen
concentrations. In this article an explanation of the processes in a fluorescence sensor is given
as well as an explanation of the sensor design.

Sensors Design

Until recently, a submersible fluorometer consisted of 5 main optical components: a light

source, a lens system to convey the exciting light to the sample volume, a second lens system
to gather the emitted fluorescence, one or more optical filters to separate the excitation and
emission wavelengths, and a photo detector. This arrangement is shown schematically in
Figure 1. The first submersible instruments used xenon flash lamps and photomultiplier
detectors, but these have been replaced by light-emitting diodes and silicon detectors for most
applications with considerable savings in instrument size and power consumption. The main
optical problem encountered is the tendency of ambient light, or excitation scattered back
from the sample volume, to interfere with the measurement of the relatively weak
fluorescence signal. Modern compact fluorometers use a wide range of proprietary optical
configurations, including coaxial arrangements of the excitation and collection optics or light
pipes rather than lenses.

Fluorescent probes

Fluorescent probes are molecules that absorb light of a specific wavelength and emit light of
a different, typically longer, wavelength (a process known as fluorescence), and are used to
study biological samples. The molecules, also known as fluorophores, can be attached to a
target molecule and act as a marker for analysis with fluorescence microscopy.

Fluorescence Microscopy
A fluorescence microscope is much the same as a conventional light microscope with added
features to enhance its capabilities.

 The conventional microscope uses visible light (400-700 nanometers) to illuminate

and produce a magnified image of a sample.

 A fluorescence microscope, on the other hand, uses a much higher intensity light
source which excites a fluorescent species in a sample of interest. This fluorescent
species in turn emits a lower energy light of a longer wavelength that produces the
magnified image instead of the original light source.

Fluorescent microscopy is often used to image specific features of small specimens such as
microbes. It is also used to visually enhance 3-D features at small scales. This can be
accomplished by attaching fluorescent tags to anti-bodies that in turn attach to targeted
features, or by staining in a less specific manner. When the reflected light and background
fluorescence is filtered in this type of microscopy the targeted parts of a given sample can be
imaged. This gives an investigator the ability to visualize desired organelles or unique surface
features of a sample of interest. Confocal fluorescent microscopy is most often used to
accentuate the 3-D nature of samples. This is achieved by using powerful light sources, such
as lasers, that can be focused to a pinpoint. This focusing is done repeatedly throughout one
level of a specimen after another. Most often an image reconstruction program pieces the
multi-level image data together into a 3-D reconstruction of the targeted sample.

How does Fluorescent Microscopy Work?

In most cases the sample of interest is labeled with a fluorescent substance known as a
fluorophore and then illuminated through the lens with the higher energy source. The
illumination light is absorbed by the fluorophores (now attached to the sample) and causes
them to emit a longer lower energy wavelength light. This fluorescent light can be separated
from the surrounding radiation with filters designed for that specific wavelength allowing the
viewer to see only that which is fluorescing.

The basic task of the fluorescence microscope is to let excitation light radiate the specimen
and then sort out the much weaker emitted light from the image. First, the microscope has a
filter that only lets through radiation with the specific wavelength that matches your
fluorescing material. The radiation collides with the atoms in your specimen and electrons are
excited to a higher energy level. When they relax to a lower level, they emit light. To become
detectable (visible to the human eye) the fluorescence emitted from the sample is separated
from the much brighter excitation light in a second filter. This works because the emitted
light is of lower energy and has a longer wavelength than the light that is used for
illumination.

Most of the fluorescence microscopes used in biology today is epic-fluorescence

microscopes, meaning that both the excitation and the observation of the fluorescence occur
above the sample. Most use a Xenon or Mercury arc-discharge lamp for the more intense
light source.

FRET

Fluorescence resonance energy transfer (FRET)* is a distance-dependent physical process by

which energy is transferred nonradiatively from an excited molecular fluorophore (the donor)
to another fluorophore (the act Fluorescence resonance energy transfer (FRET)* is a
distance-dependent physical process by which energy is transferred nonradiatively from an
excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means of
intermolecular long-range dipole–dipole coupling. FRET can be an accurate measurement of
molecular proximity at angstrom distances (10–100 Å) and highly efficient if the donor and
acceptor are positioned within the Förster radius (the distance at which half the excitation
energy of the donor is transferred to the acceptor, typically 3–6 nm).

The efficiency of FRET is dependent on the inverse sixth power of intermolecular

separation, making it a sensitive technique for investigating a variety of biological
phenomena that produce changes in molecular proximity. Technological advances in light
microscopy imaging, combined with the availability of genetically encoded fluorescent
proteins provide the tools necessary to obtain spatial and temporal distribution of protein
associations inside living cells by means of intermolecular long-range dipole–dipole
coupling. [12]

Nanoparticles and dots

Recent breakthroughs of nanotechnology in biology and medicine have provided new
approaches to address and solve problems either in basic research or clinics. Many consider
that the applications of various nanostructures and Nano devices could significantly
contribute to fighting cancer and neurodegenerative diseases. Depending on the properties of
the nanostructures used, their function could be due to their intrinsic properties or due to the
functionalization of nanomaterial that render them biologically active.5,6 Unfortunately some
nanoparticles can constitute threats to living cells, animals or humans.

Currently, metal-containing nanoparticles, including quantum dots, are mainly part of the
fluorescent toolbox for biological applications but they may not be a clinical solution for
improving diagnosis or therapy. Other types of nanoparticles such as micelles and FloDots
are mostly used for delivering different agents in a controlled manner and/or as a detection
system for environmental pollutants when applied to medical or detection applications.

Q-Dots

 Q-dots are fragments of semiconductors made of hundreds of thousands of atoms with

optical and electrical properties strongly dependent on the size of these fragments.

 Q-dots are unique because their emission maxima shift to higher wavelengths when
their size increases. No matter what their size, Q-dots can be excited within a
relatively large range of wavelengths.

 https://www.nature.com/subjects/fluorescence-imaging
 https://www.bmglabtech.com/time-resolved-fluorescence/

 https://www.picoquant.com/applications/category/life
 science/fluorescence-anisotropy-polarization#tab-4
 https://www.nature.com/subjects/fluorescent-labelling
 http://www.coastalwiki.org/wiki/Fluorescence_sensors
 https://www.nature.com/subjects/fluorescent-probes

Q5: Synchrotron Radiation

Answer: Synchrotron radiation is the electromagnetic radiation emitted when charge
particles travel in curved path. In Synchrotron the charge particle moves with constant
relativistic speed on a circular arc. A synchrotron produces light by using radio frequency
waves and powerful electro-magnets to accelerate electrons to nearly the speed of light.

 Energy is added to the electrons as they accelerate so that, when the magnets alter
their course, they naturally emit a very brilliant, highly focused light
Synchrotron radiation was named after its discovery in a General Electric synchrotron
accelerator built in 1946 and announced in May 1947 by Frank Elder.

Synchrotron light (also known as synchrotron radiation) is electromagnetic radiation that is

emitted when charged particles moving at close to the speed of light are forced to change
direction by a magnetic field. It produces not only visible light, but also infrared light,
ultraviolet light and X-rays. The Light produced by synchrotrons are about 100 million
times brighter than the X-rays produced by an X-ray machine in a hospital. There are more
than 50 synchrotron light sources around the world.

1. MICROTRON

An electron gun inside a microtone generates electrons. Radio waves then accelerate the
electrons to an energy level of 22MeV

2. BOOSTER RING

The electrons enter a booster ring where magnets force them to travel in a circular path and
radio waves accelerate electrons to 800 MeV.

3. STORAGE RING

The electron beam travels to a storage ring where it races around fo5r hours, reaching 2.5
GeV

3A. BENDING MAGNETS

Bending magnets adjust the path of the electron beam to keep it inside the storage ring.

3B. WIGGLERS/UNDULATORS

Magnets called wigglers and undulators force to emit a concentrated beam of light.
3C. RADIO-FREQUENCY CAVITIES

Radio-frequency cavities add energy to the circulating electrons to replace the energy that
was lost as light.

4. BEAM LINE/ EXPERIMENTAL STATION

The light travel down a beam line, which sends the beam to an experimental station, where
optics focus or filter the light to allow scientist to investigate their samples.

Properties of Synchrotron Radiation

 High intensity
 Continuous Spectrum
 Excellent Collimation (Brilliance)
 Low Emittance
 Pulsed-Time Structured
 Polarization

Advantages of Synchrotron Radiation

The intensity of synchrotron X-rays is more than a million times higher that of X-rays from a
conventional X-ray tube. Experiments that took a month to complete can now be done in only
a few minutes.

• With synchrotron radiation, molecular structures that once baffled researchers can now be
analyzed precisely, and this progress has opened up many new research fields over the last
few years

Applications
 Life sciences: protein and large-molecule crystallography
 LIGA based micro fabrication
 Drug discovery and research
 X-ray lithography
 Analyzing chemicals to determine their composition
 Observing the reaction of living cells to drugs
 Inorganic material crystallography and microanalysis
 Fluorescence studies
 Semiconductor material analysis and structural studies
 Geological material analysis
 Medical imaging
 Particle therapy to treat some forms of cancer
Reference :( https://www.slideshare.net/MuhammadAzhar10/synchrotron-radiation )