Bio-Physics

Unit – VI
SEMESTER – II
Applied Physics
Introduction
• Biophysics:-
Science of understanding physical phenomenon of
biological systems using physics laws. It deals with the
role of physical principles in the organization and
functioning of the living system.

• Molecular Modeling:-
In order to study the molecules, the three dimensional
models were made using available data and various
scientific tools which is called as Molecular modeling.
In order to observe, view or understand atoms/molecules
in the range of few oA various advanced and sophisticated
instruments are used which are generally characterized in
two types-
• Spectroscopic instruments:
• Microscopic instruments:
Spectroscopy:
Spectroscopy is based on the absorption and emission of
electromagnetic radiation in the form of characteristics spectral
lines of bio-molecules. The spectra produced can be used for
chemical analysis, examine atomic and molecular energy levels
and molecular structure etc.
Different ranges of electromagnetic radiations are useful to
probe different accepts of the materials which are summarized
in table below:
Type of source Range of Wavelengths Applications
Diffraction and small angle scattering to study
X-ray 10-12 – 10-8 m internal structure (at atomic level) of material.
Ultraviolet 10-8 – 4 × 10-7 m Electronic structure of atoms/molecule
Visible 4 × 10-7 m – 7.5 × 10-7 m Electronic structure of atoms/molecule
Infrared 7.5 × 10-7 – 10-3 m Vibrational and rotational levels of molecules
Microwaves 10-3 – 1 m Rotational levels of molecules
Radio waves 1 - 103 m NMR for intercellular pH and concentration etc.
UV-VIS-IR Spectroscopy

Schematic of Working Principle

UV-VIS-IR spectrometer
NMR Spectroscopy:
(Nuclear Magnetic Resonance spectroscopy)

• Nuclear magnetic resonance spectroscopy, most commonly

known as NMR spectroscopy, is a research technique that
exploits the magnetic properties of certain atomic nuclei to
determine physical and chemical properties of atoms or the
molecules in which they are contained
• Basic Principle: nuclear magnetic resonance
• Nuclear magnetic resonance (NMR): an effect whereby
magnetic nuclei in a magnetic field absorb and re-emit
electromagnetic (EM) energy. This energy is at a specific
resonance frequency which depends on the strength of the
magnetic field and other factors.
Microscopy
• Microscopy is the technical field of using microscopes to view
samples and objects that cannot be seen with the unaided eye
(objects that are not within the resolution range of the normal
eye).
• There are three well-known branches of microscopy, optical,
electron, and scanning probe microscopy.

Optical Microscopy:
Optical involve the diffraction, reflection, or
refraction of electromagnetic radiation
interacting with the specimen, and the
subsequent collection of this scattered
radiation or another signal in order to create
an magnified image. Eg: compound
microscope.
Electron Microscope
• An electron microscope is a type of microscope that uses a
beam of electrons to illuminate the specimen and produce a
magnified image.
• Electron microscopes have a greater resolving power than a
light-powered optical microscope.
• The electron beam posses the laws of quantum mechanics.
The wavelength of the electron varies inversely to its energy
and is given by the De Broglie’s Hypothesis i.e.
• (The electrons have wavelengths about 100,000 times shorter
than visible light (photons), and can achieve better than
0.2 nm resolution and magnifications of up to 2,000,000x,
whereas ordinary, light microscopes are have resolution
limited to about 200nm and magnifications below 2000x.)
Electron Microscope : Working
• Schematic diagram of an electron
microscope is shown the fig 1. It
generally consists of three parts:
• - Electron gun: it emits the beam of
electron.
• - Anode: to apply desired accelerating
potential.
• - Electrostatic and electromagnetic lens
system: to control the electron beam
and focus it the specimen (condenser
lens) and to form a magnified image by
focusing electron beam on or through
the specimen (objective aperture lens
and projector lens).
Electron Microscope :
Advantages and disadvantages
• Advantages:
- With varying the K.E. of electron beam by changing the
accelerating potential it’s wavelength and hence magnification
power can be altered.
- Highly precised resolution with far superior image is
achieved compare to X-ray diffraction techniques.

• Disadvantages:
- Needs ultrahigh vacuum to avoid the excess of scattering and
absorption of electrons through air molecules.
- Sample preparation is very critical. It evolves very precise
and sophisticated techniques.
Scanning Electron Microscope
Basic arrangement:
• The Schematic diagram of a scanning electron
microscope is shown in fig 2 and is described below:
• An electron beam is thermionically emitted from an
electron gun.
• Anode is used as limiting aperture as well as to
accelerate the electrons.
• The electron beam, (energy ~ 0.5 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,
which deflect the beam in the x and y axes to scans
a rectangular area of the sample surface.
• The objective lens is used to focus the electron
beam on the sample.
• Secondary and back scattered electrons produced
by sample due to the electron beam interaction is
be detected by specialized detectors and analyzed
carefully to creates a pseudo three-dimensional
image or spectrum of the unique elements that
exist in the sample analyze
Scanning Electron Microscope
• Working:
• SEM functions by scanning the sample by a highly energized
electron beam.
• When finally focused primary electron beam interacts with the
sample. The electrons lose energy by repeated random scattering
and absorption by the atoms/ molecules on the surface of the
sample. The resultant scattering is depends on atomic number
and density of the sample as well as surface topography.
• The energy exchange between the electron beam results into
results in the reflection of high-energy electrons by elastic
scattering (back scattered electrons), the emission of secondary
electrons by inelastic scattering and the emission of
electromagnetic radiation each of which can be detected by
specialized detectors and imaging devices.
Scanning Electron Microscope

Pollen Grains

Silicon Wafer
Scanning Tunneling Microscope
• The ability of electrons to tunnel through a potential
barrier is used in the scanning tunneling microscope
(STM). It is used to study the surfaces on an atomic scale
of size.

• As with any transmission illumination scheme, the

electrons pass through a sufficiently thin specimen.
However, STEM is distinguished from
conventional transmission electron microscopes by
focusing the electron beam into a narrow spot which is
scanned over the sample in a raster (scanning x-y plane).
Scanning Tunneling Microscope
Construction and working:
Most advance microscope
based on quantum
mechanical concept
tunneling of electrons
through a potential barrier.
In this technique, a sharp
metal probe with a point so
fine that its tip is a single
atom is brought close to the
surface (~ few oA ) of the
conduction or
semiconducting material.
Adventages:
The ultimate elemental mapping resolution of any analytical technique.
Sub 0.2 nm image resolution.
Small area crystallographic information
Limitaions:
Significant sample preparation time.
Requirement of very thin sample <100nm
Possibility of sample damage due to the absorption of high energy electrons.
Not suitable for biophysics due to the need
Scanning Tunneling
Microscope C60 nanoparticles

Atomic arrangement
Atomic Force Microscope
Atomic force microscopy (AFM) or scanning
force microscopy (SFM) is a very high-
resolution type of
scanning probe microscopy, with
demonstrated resolution on the order of
fractions of a nanometer, more than 1000
times better than theoptical diffraction limit.
Working :
The AFM consists of a cantilever with a sharp
tip (probe) at its end that is used to scan the
specimen surface. The cantilever is typically
siliconor silicon nitride with a tip
radius of curvature on the order of
nanometers. When the tip is brought into
proximity of a sample surface,forces
between the tip and the sample lead to a
deflection of the cantilever according to
Hooke's law.
Atomic force microscope
Atomic Force Microscope
• Advantages
 Provide a three dimensional surface profile
 Sample viewed by AFE do not required any special
treatments.
 Can work in ambient air or even a liquid environment. No
need of vacuum system.
• Disadvantages:
• Scanning area is limited.
• Less resolution compare to STM
• Not able to measure steep walls or overhanges