Ms.

SINDU DIVAKARAN
ASSISTANT PROFESSOR
DEPARTMENT OF BIOMEDICAL ENGINEERING
SCHOOL OF BIO AND CHEMICAL ENGINEERING
 Measurement instruments for absorption,
scattering and emission.
 Excitation light sources
 High Pressure Arc Lamp
 LED’s
 LASERS
 Optical Filters
 Optical Detectors- Time and Phase resolved
detectors.
 Many light sources exist.
 Most common source of light- SUN
 High pressure mercury lamp.
 Low pressure mercury lamp.
 Medium pressure mercury lamp.
 Low pressure sodium lamp.
 High pressure sodium lamp.
 Laser.
 Light emitting diode
 A type of electric lamp which produces light by creating an arc in
the space between two electrodes when electrical energy is
supplied.
 In a carbon arc lamp, the electrodes are in contact at first which
is in air.
 This causes a low voltage to attain an arc. Electrodes are
detached slowly.
 As a result of this, the electric current gets heated and the arc is
maintained between the electrodes. By the process of heating,
the tip of the carbon electrodes gets evaporated.
 The high-intensity light is produced by this carbon vapor which is
highly luminous in the arc.
 The color of the light produced depends on the temperature,
time and electrical characteristics.
 Used in searchlights, movie projectors (high-intensity light),
Microscope lighting (and other research applications)
,Therapeutics, Endoscopy.
 Produce light by passing electricity through
ionized xenon gas at high pressure.(30
atmospheres)
 It produces a bright white light that closely
mimics natural sunlight.
 Two thoriated tungsten made electrodes are
placed face to face with a small gap in an
airtight transparent envelope of fused
silica(Quartz).
 The envelope or bulb is filled with xenon gas in
very high pressure. The pressure inside the bulb
is about 30 bars.
 Xenon arc lamps typically have a lifetime
between 400 and 600 hours.
 When voltage is applied across the electrodes, the gas
discharge phenomenon starts in the xenon gas in the gap
between electrodes.
 The free electrons get accelerated and collide with xenon
atoms producing excited atoms.
 When in the excited atoms, the electrons return from its
higher energy level to its previous energy state, the extra
energy is released as a photon.
 Wavelength of energy emitted through photons is within
visual range. Color of the light of xenon arc light is like
daylight.
 Three categories: continuous-output xenon short-arc
lamps, continuous-output xenon long-arc lamps, and xenon
flash lamps.
 Xenon arc lamps of smaller sizes, down to 10 watts, are
used in optics and in precision illumination
for microscopes.
 Contain a mixture of liquid mercury and an inert gas (such
as argon or xenon), housed within a glass envelope
together with a pair of closely spaced electrodes.
 When current is applied to the electrodes, a discharge
electrical arc occurs in the gap between them, which
produces enough heat to vaporize the mercury and create
a high-pressure internal atmosphere.
 Emit a very intense light having a color temperature
around 5500K.
 Arc is very small (about 1 or 2 millimeters in length), and
the image of the arc must be positioned along the optical
axis of the microscope, at the center of the condenser
aperture in the vertical illuminator, to ensure even
illumination.
 The average lifetime of a mercury arc discharge lamp
varies between 200 and 300 hours.
 Spectral output of mercury lamp is a line type
while for Xe lamp its continuum type.
 Xe lamp is commonly used as it produces a
smooth spectral output.
 Xe arc lamp used for steady state spectrometers
 Continuous light output from 250-700nm.
 Operation care-
 Reduce excitation stray light using
monochromators
 Use regulated DC power supply
 Removal of heat by lamp.
 Warm up period.
 A two LED semiconductor light source.
 PN junction opto- semiconductor that emits a
monochromatic (single color) light when operated in
a forward biased direction.
 Emits light when activated by a suitable voltage is
applied to the leads.
 When a suitable voltage is applied to the leads,
electrons recombine with electron holes within the
device, releasing energy in the form of photons. This
effect is called electroluminescence
 Color of the light (corresponding to the energy of the
photon) is determined by the energy band gap of the
semiconductor.(doping )
 Small in size and consume very little power.
 PN junction of an LED is surrounded by a
transparent, hard plastic epoxy resin
hemispherical shaped shell or body which
protects the LED from both vibration and
shock.
 LEDs have their cathode, ( – ) terminal
identified by a flat spot on the body or by
the cathode lead being shorter than the
other as the anode ( + ) lead is longer than
the cathode (k).
 LEDs - exotic semiconductor compounds such
as Gallium Arsenide (GaAs), Gallium
Phosphide (GaP), Gallium Arsenide Phosphide
(GaAsP), Silicon Carbide (SiC) or Gallium
Indium Nitride (GaInN) all mixed together at
different ratios to produce a distinct
wavelength of colour.
 In optical fiber combination.
 Surface emitter
 Edge emitter
 Super luminescent LED.
 Not so commonly used-
 Planar and dome LED.
 SLED operates at 850nm wavelength. SLED is a five
layered double hetrojunction on device consisting of a
GaAs and GaAlAs layers.
 Massive electron injection into a thin active layer for
recombination of electron and holes and enhanced
focus of emitted light into the optical fiber.
 Plane of the active light emitting region is oriented
perpendicularly to the axis of the fiber. From the
substrate of the device, a well is etched. Fibers are
connected in the well to accept the emitted light.
 The circular active area in practical surface emitters in
normally 50μm in diameter and up to 2.5μm thick SLED.
 Only active region near the surface will emit the
significant amount of light while absorbing from the
other parts. Hence it is known as surface emitting LED.
 Demand for optical sources for longer distance,
higher bandwidth systems operating at
longer wavelengths led to the development of edge-
emitting.
 Primary active region of the ELED is a narrow stripe,
which lies below the surface of the semiconductor
substrate. The semiconductor substrate is cut or
polished(facets) so that the stripe runs between the
front and back of the device.
 Rear facet is highly reflective and the front facet is
antireflection-coated. The rear facet reflects the
light propagating toward the rear end-face back
toward the front facet. By coating the front facet
with antireflection material, the front facet reduces
optical feedback and allows light emission.
 ELEDs emit light only through the front facet.
A semiconductor device that emits low-
coherence light of a broad spectrum like LED
but high brightness like Laser Diode.
 Emission occurs by flowing forward current
to a p-n junction. When a power supply is
connected to the p-layer positive and the n-
layer negative, electrons enter from the n-
side and holes from the p-side. When the two
meet at the junction, an electron drops into
a hole and light is emitted.
 Light is reflected at the end surface of the
active layer.
 Optical coherence tomography (OCT), e.g.
for cornea and retina diagnostics, for
cardiovascular imaging, for other biomedical
purposes or for biology research
 Testing of optoelectronic components, e.g. in
terms of transmission or reflection spectra,
amplification factors, dispersion.
 White light interferometry
 Fiber-optic gyroscopes,(FOG) for navigation
of large airplanes, benefit from a broadband
light source.
 Simplest of the structures that are available.
 Fabricated by liquid or vapour phase
epitaxial processe
 s over GaAs surface.
A hemisphere of n-type GaAs around p-
region.
 Higher external power efficiency than planar
LED.
 LASER — Light Amplification by Stimulated Emission of
Radiation
 Source of highly directional, monochromatic, coherent
light.
 Operates under a “stimulated emission” process.
 The semiconductor laser differs from other lasers (solid,
gas, and liquid lasers):
 small size (typical on the order of 0.1 × 0.1 × 0.3 mm3)
 high efficiency
 output is easily modulated at high frequency by controlling
the junction current
 low or medium power particularly suitable for fiber optic
communication
 Important applications of the semiconductor lasers:optical-
fiber communication, video recording, optical reading,
high-speed laser printing. high-resolution gas spectroscopy,
atmospheric pollution monitoring.
 The principle of laser is based on the
stimulated emission of light.
 Components of a typical laser are:-
 1. Gain Medium for Population energy
 2. Laser Pumping energy
 3. Cavity
 4.Reflector
 5.Laser Beam
 Two energy levels E1(lower) and E2(higher)
 Three main process for laser action:
1- Photon absorption
Photon with energy hγ1 incident on the atom in E1,atom
excited into E2 through absorption of photon
2- Spontaneous emission
Atom return to E1 by random manner.
3- Stimulated emission
Photon have equal energy b/w two states(E1-E2) interact with
atoms causing it to lower state with creation of second photon. Give
coherent radiation.
 Horizontal line denotes current and vertical
line shows the optical power of light
produced.
 A gradual increase in power is noticed until a
threshold point is reached.
 After the threshold value, a rapid increase in
power is noticed even for a small increase in
the current.
 The power produced by the laser diode also
depends on the temperature associated with
the device.
 Coherence: Crucial property of laser, due to stimulated
emission. Denotes that the wavelength of the waves of
emitted light is in phase.
 Monochromaticity: Single wavelength. Waves having single
wavelength denotes that the emitted radiation is of a
single colour.
 Brightness: Determined by the power per unit surface area
per unit solid angle. Due to continuous reflections, a light
of high intensity and more power is produced by the laser
diode. Results in generation of bright light through the
device.
 Directionality: Highly directional No divergence.
Directionality in a laser diode is achieved because the
emitted photons undergo multiple reflections through the
mirror. Any time the light deviates from its axis it gets
skipped. Only a highly focused light beam is achieved.
A semiconductor laser diode is a device
capable of producing a lasing action by
applying a potential difference across a
modified p-n junction.
 This modified pn-junction is heavily doped
and contained within a cavity thus providing
the gain medium for the laser.
 A feedback circuit is also implemented in
order to control the amount of current sent
to the laser diode.
 Components of laser diode-
 Metal contact
 P-type material
 Active region (n-type material)
 N-type material
 Metal contact
 The frequency of the emitted light depends
on the length of the optical cavity along the
optical axis.
 Semiconductor laser diodes come in many
shapes and sizes.
 They maybe round, square, or rectangular,
and have a few to many leads.
 Materials include: GaAlAs,AlGaInP, InGaAsP.
 Reasons for using one material over another are for
the specifications needed, especially desired
wavelength.
 Beam quality - fair to high quality depending on
design.
 Raw beam is elliptical or wedge shaped
 Proper correction requires additional optics .
 A typical S.L.D.’s output power ranges from
approximately 0.1 mW to 5.0 mW.
 Semiconductor laser diodes are cheap and easy to use
compared to other types of lasers.
 Precisely controlled current source is needed to
regulate the amount of current to the laser diode.
 Advantages
 Operational power in case of laser diodes is
less as compared to other light emitting
devices.
 Small in size thus allows better handling.
 Laser diodes generate light of high efficiency.
 Disadvantages
 Light of high density, puts adverse effects on
eyes.
 Expensive.
 Optical filters - vital component in optical
systems, Iike fiber optic splitters, anti-reflective
coatings, laser filtering, and optical detectors.
 Transmit only the desired wavelengths and either
absorb or reflect light of unwanted wavelengths.
 Utilized to select a portion of the incoming light
for use as an input for an optical system.
 Used to select a portion of the light to be used
as an output .
 Used to split light into multiple components with
differing wavelengths.
 Filter out background noise from the rest of the
optical spectrum
 Long pass filters transmit wavelengths above
a cut-on wavelength
 Short pass filters transmit wavelengths below
a cut-off wavelength
 Band pass filters transmit wavelengths in a
narrow range around a specified wavelength
 Band width can be specified
 Neutral Density filter is a nondiscriminant
intensity reducing filter
 Absorption Filter is colored glass that absorbs
unwanted light
 Central Wavelength - CWL used in defining
bandpass filters, describes the midpoint of
spectral bandwidth over which the filter
transmits.
 Bandwidth - a wavelength range used to denote
a specific part of the spectrum that passes
incident energy through a filter.
 Full Width-Half Maximum -FWHM describes the
spectral bandwidth over which a bandpass filter
will transmit.
 Blocking Range -A wavelength interval used to
denote a spectral region of energy that is
attenuated by the filter
 Optical Density - OD describes the amount of
energy blocked or rejected by a filter. A high
optical density value indicates low transmission,
and low optical density indicates high
transmission. Optical densities of 6 or greater
are used for extreme blocking needs such as
Raman spectroscopy or fluorescence microscopy.
 Cut-On Wavelength - Denotes the wavelength at
which the transmission increases to 50%
throughput in a longpass filter.
 Cut-Off Wavelength - Denote the wavelength at
which the transmission decreases to 50%
throughput in a shortpass filter.
 Absorbance filters -Colour glass filters
-absorb the unwanted wavelengths
 Interference filters -Dichroic, Dielectric,
reflective filters
 Reflect the unwanted wavelengths
 Composed of transparent glass or quartz
substrate on which multiple thin layers of
dielectric material, sometimes separated by
spacer layers .
 Consists of multiple thin layers
of dielectric material having different refractive
indices.
 The thickness of the dielectric materials ranges
from 1/4 to 1/2 of the targeted wavelength.
 May be metallic layers.
 Permit great selectivity.
 wavelength-selective
 They used to direct light in different spectral
region to different detectors.
 They are interference filters , long pass or
short pass.
 Angle Sensitive
 Di- is Greek for two, and -chroic is Greek for
color - from Greek dikhroos, bicolored.
 Reflects unwanted wavelengths, and
transmits the desired portion of the
spectrum.
 Constructive and destructive interference
occurs between reflections from various
layers
 Transmission determined by :
 thickness of the dielectric layers
 number of these layers
 angle of incidence light on the filters
 Advantages
 Used as reflectors in two and three color
analysis.
 Do not produce fluorescence by themselves.
 Available in short pass versions.
 Excellent as primary barrier filters.
 Disadvantages
 Lower blocking properties
 Reduced passing properties
 Light is blocked based on the absorption properties of
the glass substrate used.
 Light that is blocked does not reflect off the filter;
rather, it is absorbed and contained within the filter.
 Light can be incident upon the filter from a wide
range of angles and the filter will maintain its
transmission and absorption properties.
 Are colored glass filters which absorb unwanted light.

 Consist of dye molecules uniformly suspended in glass

or plastic.
 Remove much more of the unwanted light than do
the interference filters
 Will often fluoresce
 Advantages-
 They are inexpensive.
 They have very good blocking properties.
 They have very good transmission properties.

 Disadvantages -
 They can only pass long wavelengths -hence,
can only block short wavelength
 Since they are made of solution of dye and
glass, they can themselves produce
fluorescence.
 An optical detector converts the optical
signal into an electrical signal, which can
then be further processed.
 Generates an electrical current
proportional to the intensity of incident
optical radiation.
 Relationship between the input
optical radiation and the output electrical
current is given by the detector
responsivity.
 High sensitivity at the operating wavelength
 Large electrical response to the optical signal
 Short response time to obtain a suitable
bandwidth
 A minimum noise introduced by the detector
 Stability of performance characteristics
 Small Size
 Low bias voltage
 High reliability
 Low cost
 Diodes
•PN-diodes
•Pin diodes
•Avalanche photodiodes
•Schottky diodes
 Photo conductors
 Photo transistors
 The photo- generated electron hole pairs
in the depletion region of the diode
contribute to the overall photocurrent.
 Operated under short circuit conditions or
under reverse bias voltage.
 Quantum efficiency of the diode might be
slightly higher for reverse bias voltage.
 Depletion region is very thin, hence the
quantum efficiency of a pn-diode is usually
not very high.
 The depletion region is extended across
the intrinsic or lightly doped layer and
therefore more photo-generated carriers
contribute to the photocurrent.
 All three layers (p-,i- and n-region) have
the same optical bandgap.
 The thicker the i-layer the further the
sensitivity can be extend in the near
infrared part of the optical spectrum.
 Typical materials used for the three optical
communication bands:
 Short wave band (800nm – 900nm)
Silicon pin diodes are the best choice for the short wave band.
The diodes are very inexpensive, reliable and easy to handle.
 Medium Wave band (1250 nm - 1350nm)

Indium gallium arsenide phosphide (InGaAsP) is an

alternative. The material has an optical bandgap of 0.89eV
(depending on the composition of the material) and is perfectly
suitable for the medium wavelength band.
 Long Wave Band (1500nm - 1600 nm)
Optical bandgap of the material has to be already very small.
Material used is InGaAs (indium gallium arsenide). InGaAs has a
bandgap energy of 0.77 eV.
 The sensitivity of the detector has to be
matched with the optical spectrum of the
incident light.
 A thin metal layer replaces either the p- or
the n-region of the diode. Depending on the
semiconductor and the metal being involved
a barrier is formed at the interface of the two
materials. This barrier leads to a bending of
the bands. Due to the applied voltage the
bands can be bended more or less. In the
region of the band bending electron hole
pairs can be separated.
 Sensitivity of the receiver can be increased by
amplification.
 APDs amplify the signal during the detection
process.
 The operating principle of a APD is based on the
avalanche effect, where a highly accelerated
electron excites another electron due to “impact
ionization”.
 However, in the first step a photon has to be
absorbed and a electron-hole pair has to be
generated. The device consists of two regions. In
region 1 of the device the electron hole pairs are
generated and separated. In region 2 of the device
the carriers are accelerated and impact ionized.
 Arriving photons pass through thin n+p- junction. The
carriers are absorbed in a - region. The absorption leads
to the generation of electron-hole pairs in this region.
 The electric field in the -region is high enough to
separate the carriers. The electric field across the -region
is not high enough for the charge carriers to gain enough
energy for multiplication to take place.
The electric field, however, in the n+p-region the electric
field is significantly higher, so that the charge carriers (in
this case electrons only) are strongly accelerated and pick
up energy.
The electrons collide with other atoms in the lattice, which
leads to the production of new electron-hole pairs (“impact
ionization”). The newly released charge carriers again will
collide with the lattice to produce more electron-hole pairs.
 Simplest available detector structure.
 In the case of a photoconductor the resistivity of
the device is changed as a function of the
intensity and not photocurrent.
 A voltage is applied to the detector to measure the
change in current flow.
 The photoconductive detector is formed by two
adjacent finger contact which are placed on a
semiconducting material.
 The photoconductive detector is an unipolar
device, which means that the current flow is
either completely dominated by electrons or by
holes. Diodes are bipolar devices, because
electrons and hole contribute to the current
transport.
 Pulse excitation source is used.
 Width of the excitation is generally much shorter than the
emission process of interest, i.e., much shorter than the lifetime
(decay time) of the samples.
 To measure the lifetime, the time-dependent intensity is
measured following the excitation pulse, and the decay time is
calculated from the slope of a plot of log I (t) vs. t, or from the
time at which the intensity decreases to 1/e of the initial
intensity value I (t = 0).
 To measure the emission intensity free from the excitation pulse
intensity, one can gate the detection after a delay time when the
excitation pulse has decreased to zero .
 Different compounds having different decay times can be
differentiated by using different delay times (dT) and gate times
 Source of noise is the DC noise from the background.
Improvement in the S/N ratio can be achieved by using multiple
excitation pulses.
 Also called frequency domain techniques.
 Sample is excited with intensity-modulated light.
 The intensity of the incident light changes with a
very high frequency compared with the
reciprocal of the decay time .
 Following excitation with a modulated signal,
the emission is also intensity-modulated at the
same modulation frequency.
 Because the emission from the sample follows a
decay profile, there is a certain delay in the
emission relative to the excitation .
 This delay is measured as a phase-shift , which
can be used to calculate the decay time.
 The finite time response of the sample also
results in demodulation of the emission.
 At low frequency, the emission closely follows
the excitation. Hence, the phase angle is near 0
and the modulation is near 1.
 As the modulation frequency is increased, the
finite lifetime of the emission prevents the
emission from closely following the excitation.
This results in a phase delay of the emission and
a decrease in the peak-to-peak amplitude of the
modulated excitation.
 The shape of the frequency response is
determined by the number of decay times
displayed by the sample. If the decay is a single
exponential, the frequency is simple.
 For detection and analysis of the optical signal.
 Measure spectroscopic properties related to the
molecular composition and structure of
biochemical species in the sample of interest.
 Types of spectroscopic measurements:
absorption, scattering (elastic and inelastic),
and emission.
 Light at a certain wavelength (or frequency v =
c/λ) is used to irradiate a sample of interest.
This process is called “excitation.” Some
of the properties of the light that emerges from
the sample are measured and analyzed.
 We can measure the light emitted and scattered by the
sample, process that occur at wavelengths different from
the excitation wavelength; the techniques involved are
fluorescence, phosphorescence, and inelastic scattering
(Raman scattering).
 A basic spectrophotometer generally consists of the
following components:
1. An excitation light source
2. Dispersive devices (optical filters, monochromators, or
polychromators)
3. A sample (usually in a compartment with a sample
holder)
4. A photometric detector (equipped with a read-out
device).
 The recorded spectra (absorption, emission, or excitation)
represent the photon emission rate or power recorded at
each wavelength, over a wavelength interval determined
by the slit widths, and dispersion of the monochromator.
 Collimated output of a light source is focused on
the entrance slit of an excitation
monochromator for wavelength scanning.
 Output of the excitation monochromator is
directed to the sample inside the sample
compartment.
 The light transmitted by the sample is collected
through appropriate optics and focused onto a
detector.
 Used in a single-beam absorption spectrometer.
 Double-beam instruments include a reference
beam, which is used to correct spectral
fluctuations in the lamp automatically in order
to reduce electronic drift and lamp warm-up
periods.
DISPERSIVE
ELEMENT
 Elastic scattering (ES) technique involves detection of the
backscattering of a broadband light source irradiating the sample
of interest.
 A spectrometer records the backscattered light at various
wavelengths
and produces a spectrum that is dependent on sample structure,
as well as chromophore constituents.
 Sample is illuminated with the excitation light, which is selected
with a dispersive element and then directed to a specific point
location (e.g., via an optical fiber) of the sample.
 The scattered light is measured at the same wavelength as the
excitation wavelength.
 With inelastic scattering measurements, one measures the
scattered light from the sample in a spectral region different
from the excitation wavelength.
 In this case, the basic setup is similar to the ES setup but has an
additional dispersive element to analyze the scattering emission
from the samples
 The excitation light source is usually a laser or
high-intensity xenon arc lamp.
 The collimated output of the light source is
focused on the entrance slit of an excitation
monochromator whose output is directed to the
sample.
 When a laser is used as the excitation source,
the excitation monochromator is not required.
 The emission from the sample is collected
through appropriate optics and focused onto the
entrance slit of an emission monochromator.
 The excitation beam and the emission beam are
usually focused at right angles for minimum
interference from scattered light.
 3 basic classes of spectrophotometers:
 Filter instruments
 Monochromator instruments,
 Multichannel devices.
 The first type of device uses optical filters,
whereas the latter two systems use prisms or
gratings as dispersive elements.
 3 major features to consider are
 Intensity of the excitation light source
 Resolution and throughput of the
monochromators
 Sensitivity of the detector.
 For fluorescence measurements at room
temperatures, the spatial resolution need not be
excessively high.
 IR absorption and Raman instruments require
very high spectral resolution.
 The spectrometer used for phosphorescence
measurements has a phosphoroscope attachment
for isolating the long-lived phosphorescence
from shorter-lived emissions.
 The sensitivity of the spectrometer is strongly
dependent on the choice of the detector, such as
the photomultiplier (PM) tube or the charge-
coupled device (CCD).