Multipoint spectroscopic analyzing & imaging method

FACULTY OF ENGINEERING AND SUSTAINABLE DEVELOPMENT .

Multipoint spectroscopic analyzing & imaging method

Provakar Paul

June 2013

Master’s Thesis in Electronics

Master’s Program in Electronics/Telecommunications

Examiner: Dr. José Chilo

Supervisor: Prof. Anders Rydberg and Dr. Dragos Dancila

Provakar Paul Multipoint spectroscopic analyzing & imaging method

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‘’If I have a thousand ideas and only one turns out to be good, I am satisfied’’

-Alfred Nobel


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Abstract

Spectroscopy is a technique as the interaction of different radiation spectrum with matter to analysis of

a sample. This thesis work proposed two methods are multiple pointes spectroscopies analyzing then

imaging detection methods for solid samples. Developed method one is using Ultraviolet (UV),

Visible (Vis) and Infrared (IR) detection. Where detection was assembled with deuterium as well

tungsten-halogen lamp source (which were able to generate 175 nm to 3300 nm wavelength), a manual

X-Y stepper for scan an inhomogeneous biological sample, optical design beside Indium gallium

arsenide (InGaAs) detection unit was used of Lamda 950 by PerkingElmer. Second improved

methodology is Vis detection imaging of samples. In Vis detection imaging was constructed with

Helium-Neon (HeNe) red laser as a source (able to generate 632.8 nm wavelengths), a silicon pin

photodiode detector, lens, multimeter, X-Y positioner stepper motors to scan samples. The work show

successfully detected and imaged of water, fresh leaf, brain phantom in addition 3mm horizontal and

1.5 mm vertical cooper line. The thesis works proposed methods has obtained accurate results of all

the samples detection specifically has devised imaging of samples. This spectroscopic process is

suitable for any type of liquid, solid also gas detecting moreover imaging approach can be applicable

in any type of inhomogeneous matter.


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Acknowledgements

This master thesis research was done at Uppsala University, Department of Engineering Sciences and

I would like to thanks Professor Anders Rydberg who give me chance to work in the Microwave

Engineering group. I would like to express my sincere thanks to Dr. Dragos Dancila, who was always

helpful and available for discussion throughout all the stages of the project and for providing me all

the materials and required facilities to the necessary research work. His constant guidance and

motivation has helped me bring out this thesis work in its current shape. Also I wish to thank and

acknowledge Dr. Robin Augustine of this group for his time, invaluable help, assistance and patience

during the course of this thesis. In addition, I would like to acknowledge Solid State Electronics PhD

student Patrik Ahlberg for help to understand Lamda 950 by PerkingElmer equipment and Dr. Seibt

Wolfgang (Staff retied) for Project in Fiber Optic equipment and Jonatan Bagge for technical service.

Furthermore I would like to thanks Dr. José Chilo for his encouragement and becoming my examiner.

I also like to express my gratitude to Professor Edvard Nordlander, Niclas Björsell, Per Ängskog for

being such nice teacher and Mikael Krigh departmental secretary for all the help in administrative

matters. Thanks to my friends Juan Alfaro, Danial, Erif, Dhayalini and all my classmates of University

of Gävle.

Most of all, I would like to thanks my mum, dad and brother for support, encouragement and praying

for my success in each and every step of my life, I extent my deep appreciation to my beloved partner

Jessica Persson and the most important treasure of my life our daughter Alva.


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Table of contents Abstract ................................................................................................................................................... ii

Acknowledgements ................................................................................................................................ iv

List of Table .......................................................................................................................................... vii

List of Figure ........................................................................................................................................ viii

Chapter 1:Introduction ............................................................................................................................ 1

1.1 Background: ...................................................................................................................................... 1

1.2 Current research and present problem in this area ............................................................................ 2

1.3 Thesis Goal and Outline: ................................................................................................................... 3

Chapter 2:Theory ..................................................................................................................................... 4

2.1 Spectroscopy ................................................................................................................................. 4

2.2 The Electromagnetic Radiation ..................................................................................................... 6

2.3 Radiation energy............................................................................................................................ 7

2.4 Application of UV, VIS and IR radiation ...................................................................................... 8

Chapter 3: Methods and Measurments .................................................................................................. 12

3.1 Method 1: Lamp is a source of UV-Vis-IR Spectroscopy........................................................... 12

3.1.1.1 UV, VIS and IR Sources ....................................................................................................... 13

3.1.1.2 Optical design and Sampling configurations ......................................................................... 16

3.1.1.3 UV, VIS and IR Detectors ..................................................................................................... 16

3.1.2. Measurements, Absorption and Transmittance ...................................................................... 17

3.1.2.1. UV Spectra measurement of: Water, Leaf, Brain Phantom. ................................................ 18

3.2. Method 2:VIS Imaging ............................................................................................................... 28

3.2.1.1 HeNe Laser Spectroscopy setup ............................................................................................ 28

3.2.1.2 (HeNe) Laser Spectroscopy Methord .................................................................................... 28

3.2.1.6 T-LA Series miniature linear actuators with built-in controllers .......................................... 30

3.2.2 Laser base imaging of Leaf, Phantom and hidden object: ........................................................ 33

Chapter 4: Discussion and Conclusion .................................................................................................. 38

References: ............................................................................................................................................ 40

Appendix-A: Matlab Programs ............................................................................................................ 42

Appendix-B: Used equipments and manuals ........................................................................................ 45


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List of Table

Table 2.1. Descristion about energy transitions of different radiation spectra . ...................................... 8

Table 2.2. The Approximate electromagnetic region .............................................................................. 8

Table 2.3. Calculation of stretching frequencies for different types of bonds ...................................... 10

Table 3.1. Descristion about Composite Breadboard Laboratory Table. .............................................. 30

Table 3.2. Description about the actuators. ........................................................................................... 31


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List of Figure

Figure 1.1: Block diagram of an analytical instrument showing of Spectroscopy. ................................. 2

Figure 2.1: Show the connection between Transmittance and Absorbance ............................................ 4

Figure 2.2: Radiation of light on solid .................................................................................................... 5

Figure 2.3: The Electromagnetic Spectrum . ........................................................................................... 6

Figure 2.4: Modes of vibration and bending of water molecule. ............................................................ 9

Figure 3.1.a: Block diagram of lamp is a source of UV-Vis-IR Spectroscopy system . ....................... 14

Figure 3.1.b: The peak wavelength and total radiated amount vary with temperature. ....................... 15

Figure 3.3.a: UV spectra absorbance and transmission measurement of water .................................... 19

Figure 3.3 b: Absorbance coefficients of leaf and water with UV spectrometer. ................................. 19

Figure 3.3.c:. Absorbance and Transmission coefficient leaf with UV radiation of brain phantom ..... 20

Figure 3.4.a: Vis spectra absorbance and transmission measurement of water. .................................... 22

Figure 3.4.b: Absorbance and Transmission coefficient leaf with Vis spectrometer. ........................... 22

Figure 3.4.c:. Lamp VIS spectroscopy of Leaf with specific absorption at five different spot. ............ 23

Figure 3.4.d: Vis Absorbance and Transmission coefficient phantom .................................................. 23

Figure3.5.a: IR spectra absorbance and transmission measurement of water ....................................... 25

Figure3.5.b: IR spectra absorbance and transmission measurement of leaf. ......................................... 26

Figure3.5.c: IR spectra absorbance and transmission measurement of phantom .................................. 27

Figure 3.2.a: Block Diagram of Laser bias VIS spectroscopy setup. .................................................... 29

Figure 3.2.b: Lab setup of Laser bias VIS spectroscopy. ...................................................................... 32

Figure 3.6: (a) HeNe laser image of leaf (b) Brain Phantom. ............................................................... 35

Figure 3.6: (c) Image diagram cooper line ........................................................................................... 36

Figure 3.7: (a) A plat leaf and sample which (b) Absorbance Spectra of leaf . .................................... 36

Figure 3.8: Absorption coefficient leaf and water with UV/Vis/IR spectrometer. ................................ 37

Figure3.9: Absorbance and transmittance measurement ...................................................................... 37


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Chapter 1: Introduction

1. 1Background:

From the beginning of science till today, there is a considerable interest in finding more and more

details in biological sample, objects or areas on the surrounding world as biologists want to know the

construction of the cells, bacteria, and viruses. Major focuses for material engineers are to know the

inhomogeneity and imperfections in metals, crystals, and ceramics. Geologists are curious about the

details of rocks, minerals, and fossils. The important interested of doctors are to examine neurons from

brain cells or detect cancer cells. Much Research in analytic chemistry is to detailed study of some

substances. In order to get solution of those above interest some of the best- known useful methods

are ultrasound imaging, magnetic resonance imaging (MRI), microscopic imaging, imaging using X-

rays and different radiation type spectrometer. In ultrasound imaging, a sound wave of 20 KHz applies

to body and reflected echo is use for effective imaging. For MRI magnetic field and radio wave

63MHz to 85MHz for imaging while x-ray radiation uses x-ray imaging [1].

The electromagnetic spectrum Ultraviolet (UV), Visible (Vis) and Infrared (IR) is covered the

radiation wavelength region from 175nm to 3300nm. And spectroscopy is the study of interaction of

light or radiation with matter. The word spectroscopy has two parts: spectro which refers to

electromagnetic spectrum and skopeo which in Greek means the eye can see [2]. The instruments

packages which are used in spectroscopy analysis are called spectrometer. There are five essential

components required for most spectrometers. These are as follows:

I. A source or sources of electromagnetic radiation.

II. A means for selecting a narrow band filter of wavelengths.

III. A stepper or holder facilities for holding the sample

IV. A device or devices capable of measuring the intensity of the radiation beam transmitted

through the sample.

V. A display or output device to show the image or to recode the measured data in a suitable

form.

The approaches have used in this study aims to use nanometer wavelength radiation to detect examine

detail inside a biological sample and other object detection.


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Figure 1.1: Block diagram of an analytical instrument showing of Spectroscopy.

1.2 Thesis goal

The IR radiation was discovered in 1800 by Herschel, UV radiation was the first to come into general

use during the 1930s, 1947 Wright and Hersher developed double beam dispersive IR

spectrophotometer and in 1983-spectra Tech and Digitab developed the commercial microspectro

photometer [2]. However in Uppsala University, Microwave group Johannes Hjerdt June 8, 2011 did a

research title: ‘’near field measurement of biological tissues using millimeter wave/THz frequencies’’.

In Johannes research Biological tissues have been imaged using terahertz and millimeter wave

radiation generated by millimeter wave extenders and focused by collimating polyethylene lenses,

dielectric probe and a resonator. But there were several limitation of that work like, it was took too

much time to scan a matter, could not detect extreme detail inside a biological sample. At KTH under

supervision of Professor Faris Gelmukhanov a research tittle ‘’Ultrafast X-ray spectroscopy with

applications in molecular and material science’’ in Period: 2006-01-01 - 2008-12-31 had done. Where

free electron lasers was used a source, the work was focused of material detection but not for

biological sample detection [3].

Although at present there are different spectrometers commercially available with market to examine

single point biological sample. Previous work has focused only on one point spectroscopic detection

of biological sample [4]. Unfortunately current biological or molecule observing instrument are not

capable to scan certain area, instruments are big in size, expensive in addition take too much time to

detect an object further more they cannot detect very small molecules and often technically difficult to

perform. An alternative approach of spectroscopy is necessary. In this report the two type of

spectroscopic instrument setup is use for detect and scan a biological. In the first approach is different

lamp base UV, Vis and IR spectroscopic detection and second approach is laser source base Vis

spectroscopic detection and. These spectroscopy techniques fall into the category of absorption or

transmittance spectroscopy. As this term suggests, these analytic techniques or imaging involve

absorption of specific energies of electromagnetic radiation which correspond exactly in energy to

specific excitations within the sample molecule being examined.


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1.3 Thesis Outline:

This master thesis work is about developed two methods or instruments setup for multipoint

inhomogeneous biological sample detection and imaging. In this work one setup using Ultraviolet

(UV), Visible (Vis) and Infrared (IR) spectra to analysis for characterizing different sample is used,

another instruments setup is for imaging different biological sample and hidden objects using Vis

Spectra. The primary focus of the thesis was choose correct instruments and settled instruments setup

to detecting biological sample (neuron from brain tissue). The research work presented in this thesis is:

I. Method of Biological sample imaging using Laser Vis radiation and 2D scan imaging using

manual or motor control stepper.

II. Method of Biological sample analyzing using UV, Vis, IR radiation and 2D scan using manual

stepper.

Chapter 2 is presupposed about physical background on spectroscopic analyzing system and related

theory about electromagnetic spectrum. Different objects or molecules absorb different wavelengths of

electromagnetic radiation and undergo different energetic radiation, range is non-ionizing. Suppose

Water has a high dielectric constant at IR wavelengths, which makes it suitable for spectroscopic

imaging of tissue or other material.

Chapter 3 is describes contribute two methods of the thesis and measurements. At first part of this

describe first method is multipoint UV, Vis and IR detection for spectroscopy analyzing

instrumentation, setup and measurements. Method one UV/Vis/IR multipoint spectroscopy is support

both liquid and solid sample measurement. Then we took water, a green fresh leaf, brain phantom

sample and observed UV, Vis and IR spectra full analyzing is showed. Second method is about

Helium Neon laser base Vis Spectroscopy technique for imaging of any kind of biological sample or

hiding object. In this method light source of different wavelength of the spectroscopic system pass

through the sample and PIN photo diode detector measures the power, MATLab program is used to

plot an image of the sample.

Finally chapter 4 of the report is conclusion where summary of the work, mapping achievements also

idea of future research are given and challenges to consider.


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Chapter 2: Theory

This master thesis used two methods to solve the given task so one needs to understand the physical

background to these approaches.

2.1 Spectroscopy

Spectroscopy is a way of analyzing spectra, basics of the technique is radiation. This method is

broadly used to examine and characteristic of atoms, molecules and ions. In this thesis, UV/VIS/IR

radiation has been used for recognition of solid and liquid. The spectroscopy measuring instrument

setup is called spectrometer [4]. Spectrometer has two sections, source and detector. Spectrum is a set

of lines resulting from the decomposition of a complex light; more generally, it shows the intensity

distribution of an electromagnetic wave as a function of frequency energy. When an atom is heated,

one of its electrons from its outermost layers (or more rarely the internal layers) goes from ground

level to a higher energy level which is called level of the excited state [5]. When the electron goes to

lower energy level, it re-emits the energy as light (photon).

2.1.1 UV, Vis and IR Spectroscopy for liquid

If we take any kind of liquid sample using this spectroscopy system, we can measure the percentage of

transmittance. It means percentage of light beam transmitted through the sample from source to

detector. By use the value of transmittance to calculate the absorbance of that liquid sample. Beer's

law is to calculate the absorption of light. Figure 2.1 shows a beam from source radiated with power

P0, after passing through a sample solution absorption occurred the beam of radiation leaving the

sample has radiant power P. So we can easily calculate absorbance from percentage transmittance

data. So, there is no absorption, then absorbance is zero, and then transmittance is 100%.

If the entire beam is fully absorbed, then transmittance is zero.

Figure 2.1: Show the connection between Transmittance and Absorbance


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Figure 2.2: Radiation of light on solid

2.1.2 UV-Vis-IR Spectroscopy for solid

Several numbers of situations happen when a radiation or beam of light falls on a solid sample. Figure

2.2 showed the situating may happen.

2.1.2.1 Transmittance measurements

There are several barriers we have to face during transmittance measurements of a solid. If the surface

of the solid sample is convex or concave then the beam will deviate. If the surface is not smooth then

also the beam will be deviated. Another important reason for beam deviating is refraction. Because of

the property of a solid sample beam can be diffused in different direction. Use a special kind of

equipment called integrating sphere we can measure the direct transmittance, diffused and deviated

beam power. Figure 2.2 is diagram of transmittance measurements of solid, where 𝐼0 is incident beam,

that passes through a solid sample and we got 𝐼1, which is transmittance beam.

Transmittance, T = P / P0

% Transmittance, %T = 100 T

P= incident beam power.

P0 =Radiated beam power.

Absorbance,

A = log10 P0 / P

A = log10 1 / T

A = log10 100 / %T

A = 2 - log10 %T


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Figure 2.3: The Electromagnetic Spectrum [6].

2.1.2.2 Measuring absorbance for a solid sample

I. When an incident beam fall on a solid sample the beam will ether reflected or transmitted.

II. We can calculated that %Absorbance+%R+%T=100% (R=reflectance and T= transmittance)

III. So Absorbance will be, %Absorbance=100%-%R-%T.

2.2 The Electromagnetic Radiation

It is known that moving electrical charges induce magnetic fields and, inversely, that changes of the

magnetic field create an electric field [7]. Vibrating electrical charges therefore cause a periodic

change of electromagnetic fields, which propagate as electromagnetic waves linearly into space with

the speed of light [8]. Depending on their appearance or their effect on material and on human senses,

one speaks of various types of radiation (e.g. light, heat, X-rays) which differ from one another only

with regard to wavelength or frequency but which are identical physically. The Wavelength range of

the electromagnetic spectrum comprises a wide scale ranging from radio waves to gamma rays (figure

2.3).

In the classical treatment, electromagnetic radiation can be considered as a propagating wave of

electrical energy with an orthogonal magnetic component oscillating exactly the same frequency.

Electromagnetic radiation can be described by either its frequency or wavelength. These values are

inversely proportional to each other being related by the following equation :

U

V

Gamma Rays

X-

Rays V

I

S

Infrared Microwave Radio

wave

BL GN YL OR RD

0.0001nm 10 nm 100 0nm 1 m 1 mm

400 nm 700 nm 600 nm


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𝜆𝑣 = 𝑐 (2.1)

Where,𝜆 = 𝑤𝑎𝑣𝑒𝑙𝑒𝑛𝑔𝑡ℎ,

𝑣 = 𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑜𝑓 𝑡ℎ𝑒 𝑒𝑙𝑐𝑡𝑟𝑜𝑚𝑎𝑔𝑛𝑒𝑡𝑖𝑐 𝑟𝑎𝑑𝑖𝑎𝑡𝑖𝑜𝑛,

𝑐 = 𝑠𝑝𝑒𝑒𝑑 𝑜𝑓 𝑙𝑖𝑔ℎ𝑡 3 × 108𝑚𝑠−1 [9].

Electromagnetic spectrum has been divided in many bands of frequencies (figure 2.3) which are of

great importance in technological applications. The radio band has been widely used for radio,

television, radar etc., and the use of optical band is now underway [8].

a. Ultraviolet (UV) radiation

UV radiation naturally occurring radiation is more energetic and shorter wavelength than visible light.

The human eye cannot perceive it. There are three forms of UV radiation: UV-A (315-380 nm long

wave), UV-B (280-315 nm short wave) and UV-C (100-280 nm extremely short waves). All three

come in different proportions before the sunlight. Normally found only UV-A and UV-B rays to the

earth's surface [9], the UV-C fraction is absorbed by the ozone layer.

b. Visible (VIS) radiation

VIS radiation is electromagnetic radiation, which is visible to humans. The wavelength range is

between 380 nm and 780 nm. Each of these wavelengths is corresponding to a color. Red: 620-700

nm, Orange: 592-620 nm, Yellow: 578-592 nm, Green: 500-578 nm, Blue: 446-500 nm and Violet:

400-446 nm.

c. Infrared (IR) radiation

The range of electromagnetic radiation with wavelengths of 780 nm to 1 mm is known as infrared

radiation. Infrared (IR) radiation is also called heat radiation, is part of the optical radiation. Infrared

radiation is divided in the short-wave IR-A radiation with a wavelength range of 780 to1400

nanometers, the IR-B radiation 1400 to 3000 nanometers and the long-wave portion of the IR-C

radiation 3000 nanometers to 1 millimeter. The most important natural source of IR radiation is the

sun [10]. IR radiation has a 50 percent share of the solar radiation that reaches the ground. The

absorption of the radiation by the atmosphere contained in the gases, such as natural and artificial

water, carbon dioxide, ozone, methane, and chlorofluorocarbons (CFCs) leads to the additional heating

of the earth. This process is crucial for the heat balance of the earth.

2.3 Radiation energy

The electromagnetic radiation is the emission of radiation in the visible region by heated material such

as e.g. the radiation of a white-hot tugsten wire of a lamp. Then too, energy released during a chemical

reaction can be emitted as radiation. Thus, electromagnetic radiation is an energy carrier. Energy and

radiation frequency have the following relationship:

ℎ. 𝑣 = ℎ.𝑐

𝜆 (2.2)

The parameter ℎ = 6.626 × 10−34𝐽𝑠 [13] is the planck constant and c the speed of light.


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Table 2.1.Descristion about energy transitions of different radiation spectra [2] .

Radiation spectrum Energy Transitions

X-rays Bond Breaking

Ultraviolate (UV) Electronic

Visible (Vis) Electronic

Infrared (IR) Vibrational

Microwave Rotational

Radio frequencies Nuclear Spain, Electron Spin

Radiation frequency and energy are therefore directly proportional to one another. The position of

various types of radiation in the total electromagnetic spectrum also reflects their energy: the short-

wave or high-frequency gamma rays posse high energy, while the long-wave radio waves have low

energy.

2.4 Application of UV, VIS and IR radiation

Since every different type of band has a different nature frequency of vibration and since the same

type of band is two different compounds is the slightly different environment, no two molecules of

different structure will have exactly the same electromagnetic absorption pattern or spectrum.

Although some of the frequencies absorbed in the two cases might be the same, in no case of two

different molecules will their infrared spectra is identical. Thus the electromagnetic spectra or UV, Vis

or IR spectrum can be used for molecular much as a fingerprint can be used for humans. By

comparing the infrared spectra of two substances thought to identical, one can establish whether or not

they in fact are identical. It their infrared spectra coincide peak for peak (absorption to absorption) in

most cases two substance will be identical.

Second and more important use of the infrared spectrum is that it gives structural information about a

molecule. The absorptions of each type of bond (N-H, O-H, C-X, C-O, C-C etc.) are regularly found

only in certain small portions of the vibrational infrared region.

Table2.2. The Approximate electromagnetic region where various common type of bonds absorb [6].

Frequency (cm-1)

4000 -2500 2501-2000 2001-1800 1801-1650 1651-1550 1551-650

O-H

N-H

C-H

C⁼C

C⁼N

X⁼C⁼Y

Very

few band C⁼O C⁼N

C⁼N

C-C1

C-O

C-N

2500-4000 4000-5000 5000-5500 5500-6100 6100-6500 6500-15.4

Wavelength𝝀 (nm)


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Figure 2.4: Modes of vibration and bending of water molecule.

The potential to extract material characteristics that are unavailable using other frequency bands is one

of the primary motivations for the development of UV, VIS and IR radiation sources. Astronomy and

space research has been one of the strongest driver for UV, VIS and IR radiation research due to the

vast amount of information available concerning the presence of abundant molecules such as oxygen,

water and carbon monoxide in stellar dust clouds, comets and planets [11].

2.4.1 The Modes of Vibration and bending

The simples types or modes of vibrational motion in a molecule which are IR, Vis or UV radiation

active, that is, give rise to absorptions, are the stretching and bending modes.

The vibration described in figure 2.4 above is the fundamental absorptions of water molecule. They

arise from excitation from the ground state to the lowest energy excited state. Usually the spectrum is

complicated because of the presence of weak overtone, combination and difference. Overtone result

from excitation from the ground state to higher energy states, which correspond to integral multiples

of the frequency of the fundamental (v). When two vibrational frequencies in a molecule couple to

give rise to a vibration of a new frequency within the molecule, and when such a vibration is radiation

active, it is called a combination band.

Difference bands are similar to combination bands. The observed frequency, in this case, results from

the difference between the two interacting bands. Overtone, combination and difference bands can be

calculated by using manipulations of frequencies in wavenumbers by multiplication, addition and

subtraction respectively.

2.4.2 Material characterization

Almost any compound having covalent bonds, whether organic or inorganic, will be found to absorb

various frequencies of electromagnetic radiation in the different electromagnetic region of the

spectrum. A major application of UV, VIS and IR radiation is the material characterization using

spectroscopy systems. UV, VIS and IR radiation spectroscopy is capable of highly sensitive gas

detection down to part-per-million sensing of methyl chloride [12] and determining the carrier

concentration and mobility of doped semiconductors such as GaAs and silicon wafers. It may even be

used for ‘watching paint dry’. Another important application of UV, VIS and IR spectroscopy is high

temperature superconductor characterization [13].


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We will now consider how bond strength and masses of the atoms bonded together affect the UV, Vis

and IR absorption frequency. For the sake of simplicity, we will restrict the discussion to a simple

heteronuclear diatomic molecule (two different atoms) and its stretching vibration. A diatomic

molecules can be considered as two vibration masses connected by a spring. The bond distance

continually changes, but equilibrium or average bond distance can be defined. Whenever the spring is

stretched or compressed beyond this equilibrium distance, the potential energy of the system will

increase.

The Hooke’s Law of expression given above may be transformed in the following way to a very useful

equation:

�̅� =1

2πc√

𝑘

𝜇 (2.3)

�̅�=frequency in cm-1

c=velocity of light =3x1010cm/sec

k= force constant in dynes/cm

𝜇 =𝑚1 ∗𝑚2

𝑚1+𝑚2; 𝑚𝑎𝑠𝑠𝑒𝑠 𝑜𝑓 𝑎𝑡𝑜𝑚𝑠 𝑖𝑛 𝑔𝑟𝑎𝑚𝑠 (2.4)

𝑜𝑟 𝑀1 𝑀2

(𝑀1+𝑀2)(6.02𝑥1023); 𝑚𝑎𝑠𝑠𝑒𝑠 𝑜𝑓 𝑎𝑡𝑜𝑚𝑠 𝑖𝑛 𝐴𝑀𝑈 (2.5)

6.02𝑥1023= Avogrado’s number. From the denominator of the reduced mass expression 𝜇 by taking

its square root, one obtains the expression:

�̅� =7.76x1011

2πc√

𝑘

𝜇 (2.6)

�̅�(𝑐𝑚−1) = 4.12√𝑘

𝜇 (2.7)

𝑀1 𝑀2

(𝑀1+𝑀2); Where 𝑀1 𝑎𝑛𝑑 𝑀2 are atomic weights, K= force constant in dynes/cm.

Table2.3. Calculation of stretching frequencies for different types of bonds

𝐶 = 𝐶

�̅�(𝑐𝑚−1) = 4.12√𝑘

𝜇 [From equation 2 7]

𝑘 = 10𝑥105(double bond)

𝜇 =𝑀𝐶 𝑀𝐶

(𝑀𝐶 + 𝑀𝐶)=

(12 )(12)

(12 + 12)= 6

�̅�(𝑐𝑚−1) = 4.12√10𝑥105

6= 1682𝑐𝑚−1 𝑐𝑎𝑙𝑐𝑙𝑢𝑎𝑡𝑒𝑑

�̅� = 1682𝑐𝑚−1

𝐶 − 𝐻

�̅�(𝑐𝑚−1) = 4.12√𝑘

𝜇 [From equation 2 7]

𝑘 = 5𝑥55 (for single bond)


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𝜇 =𝑀𝐶 𝑀𝐻

(𝑀𝐶 + 𝑀𝐻)=

(12 )(1)

(12 + 1)= 0.923

�̅�(𝑐𝑚−1) = 4.12√5𝑥55

6= 3032 𝑐𝑚−1 𝑐𝑎𝑙𝑐𝑙𝑢𝑎𝑡𝑒𝑑

�̅� = 3032 𝑐𝑚−1

2.4.3 UV, VIS and IR radiation for imaging

Pulsed UV, VIS and IR radiation imaging is use for imaging semiconductors, organic solvents,

cancerous tissue, flames and many more [14]. Phase-sensitive spectroscopic images, which hold the

potential for material identification or functional imaging is the main attraction of UV, VIS and IR

imaging. Imaging dry dielectric substances including paper, plastic and ceramics is done well using

UV, VIS and IR imaging systems as these materials are non-absorbing in this frequency range.

Different materials may be easily discriminated on the basis of their refractive index, which is

extracted from the UV, VIS and IR phase information. UV, VIS and IR imaging systems find

important application in security screening and manufacturing quality control as many materials that

are opaque at optical frequencies. High resolution imaging of insulation used to insulated spaces

shuttle fuel tanks can be obtained using UV, VIS and IR imaging.

2.4.4 UV, VIS and IR radiation for Biomedical Application

UV, VIS and IR radiation have broad applicability in biomedical fields of research like cancer

detection, drug discovery, genetic analysis and many others [16]. The collective vibrational modes of

many proteins and DNA molecules are predicted to occur in the UV, VIS and IR radiation range. The

complex refraction index of pressed pellets consisting of DNS and other bimolecular have been

determined and found to show absorption consistent with a large density of low frequency IR active

modes [18]. UV, VIS and IR Spectroscopy have shown the capability to differentiate between single

and double stranded DNA due to associated changes in refractive index. It has also been demonstrated

that a UV, VIS and IR radiation sensing system is capable of detecting DNA mutations of a single

base pair with femtomol sensitivity. A future biomedical application of UV, VIS and IR radiation has

find broad application in trace gas sensing and proteomics [17].


12

Chapter 3: Methods and Measurements:

The experiments were conducted two different instrument setup, method 1: was lamp source UV, Vis

and IR and method 2: was laser base Vis setup. Both experiment methods, used equipment, why the

equipment are selected and result are described in this chapter; following is the flowchart of work:

3.1 Method 1: Lamp is a source of UV-Vis-IR

Spectroscopy

Spectroscopic imaging and analysis was carried out using two different lamp sources and a detector

unit. The lamp light source generated UV, Vis and IR wavelength radiation and they was focused

using optical lens setup lastly transmitted radiation beam was measured. The sample holder was

placed immediately after the optical lens setup. And the detection unit was next to the sample holder.

4. Measerments

3. Selection equipment and Develop controlling, data reading and imaging Matlab programs

2. Propose Methors of given task:

1. Thesis's given task: Multipoint spectroscopic analyzing & imaging method of Biological Samples

a. Multipoint Spectroscopic analysis

Matlab Program

Calibration

b. Laser base imaging

Matlab Program Calibration

Uv, Vis & IR measurement

of: Water, Leaf, Brain

Phantom.

Leaf, Brain Phantom, Hiding

objects detection imaging.


13

Two different lamp sources were used to generate different wavelength range. The instrumental

foundations of UV, Vis and IR spectroscopy was for observing the electromagnetic absorption spectra.

One needs to measure electromagnetic radiation transmitted through the sample. The transmittance of

a medium is defined as the ratio of transmitted to incident radiation power. UV, Vis and IR

spectroscopy is important because of the information content in the spectrum. The UV, Vis and IR

spectroscopy has become most important analytic methods for analytical chemists and other imaging

process. The UV light or Vis /NIR radiation are very resourceful accessories for transmittance and

reflectance measurements on almost any solid or biological sample. In this part, the transmittance

measurement is made between 175 to 3300nm. Figure 3.1 show the block diagram of lamp is a source

of UV-Vis-IR Spectroscopy system. Following equipment ware used in this method:

i. UV, Vis and IR source. Tungsten-halogen visible lamp and Deuterium ultraviolet lamp.

ii. Optical design and sampling configuration.

iii. Detection unit InGaAs photodiode.

The advantage of using above equipment and more detail about method of analyzing the biological

sample and hidden object are descried in bellow.

3.1.1.1 UV, VIS and IR Sources

A perfect source is very important for spectroscopic imaging and analyzing. There are different

instrument are found to generate short wavelengths. But we have to investigate before select a product

to use as a UV, Vis and IR source. There are number of reason consider before chose tungsten-halogen

visible Lamp and Deuterium ultraviolet Lamp in this method, which are describe bellowt. Production

and detection of UV, VIS and IR radiation was a challenge till the 1990’s due to lack of high power

and cost. However rapid development and research on high-speed electronics, laser, visible Lamp, UV

lamp and materials enabled to generate UV, VIS and IR radiation. Different radiation sources use

different techniques to generate them and they can be classified as broadband pulses techniques,

narrowband techniques or continues wave (CW) techniques. Two sources are used in UV/Vis/NIR

spectrometers cover the range of wavelength from 175 to 3300 nm.


14

Figure3.1.a block diagram of lamp is a source of UV-Vis-IR Spectroscopy system [18].

a. Tungsten-halogen visible Lamp

A commercial Tungsten-halogen visible Lamp is used to generate 360 to 3300 nm wavelength.

According to the specification tungsten lamp have guarantee of thermal radiators. The advantage of

thermal radiators: wave is generated by warming metal. When the metal is warmer brighter the light

produced. Changing the warm level different wavelength can be generated. Introducing current inside

the solid metal body more or less bright light is generated. The main principal of generating different

wavelength is same as blackbody radiation. As to the method of the blackbody emission, current go

through the metal body of the lamp and radiation increase the fourth power. When the current follow

increases simultaneously the temperature increases. In figure 3.2 the relation between peak wavelength

and ration amount of temperature is described. At the highest temperature 5500k can achieve red light

or 750nm wavelength radiation.

During read light emission the current flow inside the metal body is most high. By controlling the

current follow inside metal body or tuning inside the lamp different wavelength radiation are

generated. According to the figure 3.2 at temperature 5000k the green light wavelength is shifting and

going lower wavelength then red radiation. It is change is continuing which is shown in the figure. For

measurements above 360nm compact tungsten halogen sources in a quantz envelope are nowadays

preferred. Tungsten halogen lamps contain a small quantity of iodine vapor within the quartz envelope

housing the tungsten filament. When molecules of this compound strike the hot tungsten filament

decomposition occurs and tungsten metal is redeposited. The life time of this lamp is about 900 to

10,000 hours. Detailed Specifications:

i. Wavelength Range 360 to 3300 nm.

ii. Dimensions9.0 x 5.0 x 3.2 cm.

iii. Bulb Lifetime900 hrs. (Standard), 10,000 hrs. (long-life).

iv. Power Output 6.5 W.


15

Figure 3.1.b the peak wavelength and total radiated amount vary with temperature [18].

b. Deuterium ultraviolet Lamp

An available commercial deuterium ultraviolet Lamp is use to generate 175 to 400 nm wavelength.

According to the specification inside deuterium lamp there is deuterium gas. When the deuterium gas

inside the lamp is warmer higher the UV wavelength is produced. Changing the warming level

different UV wavelength can be generated. Introducing current inside the cathode of the lamp more or

less bright light is generated. For measurements below 320nm a deuterium arc source is used as this

emits has continues spectrum below 400nm. Special filters are often included in the optical path when

a tungsten halogen lamp is being used below 400nm.

Detailed Specifications

i. Wavelength Rang 175 to 400 nm.

ii. Dimensions9.0 x 5.0 x 3.2 cm.

iii. Bulb Lifetime900 hrs. (Standard), 10,000 hrs. (long-life).

iv. Power Output 6.5 W.


16

3.1.1.2 Optical design and Sampling configurations

The UV, Vis and IR Spectroscopy operate on the ‘single beam’ principle, with one beam passing

through the sample (figure 3.1). The spectrometer has two source lamps, one emitted light at UV range

and other in range of Vis /IR. The light emitted by the source first passes through a monochromatic

diffraction grating. That is breaking down light into its components wavelengths; in the same way as a

prism. The radiation beam is then focused onto a detector which measures the ration of the intensities

of the two beams and the difference is automatically converted and plotted out as the absorbance.

Instruments usually have a full scale deflection for A=2, which means that the strongest ration of 𝐼0

𝐼

that the instrument can tolerated is 100. For optical design lamda 950 which has made by

perkingElmer is used. Inside Lamda 950 has a 200 mm mix sphere optical design (Figure 3.1). There

is a holder for liquid sample and it is transmittance. The light beam can easily pass through the holder

and measure at predictable detector. The integrating sphere will detect all direction light which has

passed by liquid sample.

3.1.1.3 UV, VIS and IR Detectors

High sensitive detection method was needed to detect the low output power of UV, VIS and IR

radiation sources which was affected by high levels of thermal background radiation in the spectral

range. Direct detectors based on thermal absorption which is a common method used for broadband

detection require cooling to reduce the thermal background. Advance in superconductor research has

produced extremely sensitive bolometer which can be used to detect UV, VIS and IR radiation wave.

Although detection speeds are currently limited to 1ms, high speed designs are proposed. Heterodyne

sensors are used in applications where very high sensor spectral resolution is required. Narrow band

detectors such as electronic resonator detectors, based on the fundamental frequency of plasma waves

in field effect transistor have been demonstrated up to UV, VIS and IR [5].

The function of the detector is to respond to radiation falling on the sensing surfaces and to provide an

electrical signal which is proportional to the intensity of that radiation. Two main types of detectors

are currently used in UV/Vis/IR spectrometers. Silicon photodiodes are now replacing the phototubes

and photovoltaic cells incorporated in older instruments. Early silicon photodiodes had poor sensitivity

below 400 nm but modern developments have improved their sensitivity so that they can now be used

to below 250nm. For maximum sensitivity at low energies the photomultiplier tube is used in more

expensive instruments. Photomultiplier tube is used in more expensive instruments. Photomultipliers

have the advantage that they can be made to respond over the whole range from 190 to 950nm. They

need a high-voltage supply connected to the various dynodes within the tube that are used to amplify

the initial electron emission from the photocathode surface. Many modern instruments now used diode

array detectors.


17

a. InGaAS photodiode

Indium gallium arsenide (InGaAs) photodiode meet the most demanding requirements of read the data

from 400nm to 1700nm. The advantages of InGaAs photodiode was their low noise and the achievable

bandwidths. Key Features

I. It was achieved high sensitivity

II. high shunt resistance

III. low noise

IV. low capacity

V. high reliability

And typical applications of photodiode there for it was choose:

I. IR sensors

II. Optical Communications

III. Measurement & Control Technology

IV. Process Instrumentation

V. Temperature Measurement

3.1.2. Measurements, Absorption and Transmittance spectra of water,

leaf and Phantom:

Water is a crucial element and it is the main constituent for all living organisms. It is the most

significant and essential factor for all kind of biological cell, inside living cell water present is 60% to

95%. So it is important to understand absorption spectrum of water molecule. Its unique structure

gives it properties that subsequently give it many various biological roles. Water molecules are

dipolar, have covalent bond, electron are not equally shared and this all create hydrogen bonding

between atoms [8]. So the structure gives many important properties such as waters thermal, high

surface tension, incompressibility and cohesiveness. Those give it many useful biological roles such as

being a solvent, a coolant, an insulator, as support a lubricant and reagent. Plants have been in

operation since life first began, leaves are the core of plants life: main functions of leaves are

photosynthesis (food manufacture system for plants), interchange of gases (exchange of oxygen and

carbon dioxide occurs during photosynthesis and respiration), vaporization of water, storage food, and

vegetative properties (some leaf produce buds and that can produce new plants). Inside leaf there is

organic solvent which is chlorophyll. There are two types of chlorophyll one is chlorophyll a (chl a)

another is chlorophyll b (chl b). Inside all leaf in the world we will found either chl a or chl b, they

have extremely characteristic absorption spectra. Both chl a and chl b have maximum absorption at the

wavelength 640 and 660 nm of visible region. It is important to absorbance measure of leaf sample.

We will do Lamp VIS spectroscopy of Leaf at five different spot. We used brain phantom as a sample

because it is interesting to analyzing brain sample with our instrument setup. Spectroscopic analyzing

technology development could be useful in a therapeutic context, i.e. addressing diseases for which no

curative therapeutic route exists today (such as Alzheimer's disease, ALS and Parkinson's disease,PD).

In the same time, the results could be used to better monitor treatment and trials on new drugs.


18

3.1.2.1. UV Spectra measurement of: Water, Leaf, Brain

Phantom.

a. Water:

Full spectrum UV spectra radiation of water is shown in Figure 3.3 a. We took a transparent

disposable plastic cuvette. This cuvette was transparent in UV radiation. Fill the cuvette with water

then was placed that in holder inside UV/Vis/IR spectrometer system. Different wavelength’s

radiation was penetrated the water in cuvette after that detector measure the energy level of the signal.

At figure 3.3 a 𝜆 200 to 380 nm absorptions is almost zero from 225 nm which mean close to 100%

transmission. During water transmission experiment the result shows, the energy levels of UV

radiation were absorbed by water molecules. There is a certain rule at losing this energy level. UV

radiations had loosed individual energy level because water has its own absorption spectra. Figure. 3.3

strong absorptions tailing off into the normally inaccessible region below 225nm are detected because

a property which is made good use of by photosynthesis and allowing production of both biomass and

oxygen.

b. Leaf

When a beam from UV source fall on leaf surface, the incident beam will ether reflected or

transmitted. And we can easily calculate absorbance (A) from percentage of transmittance (T) data,

which is A =2 - log10 %T. The absorbance result of leaf at 175nm to 350 nm is shown in figure-3.3 b.

If we observed the UV region absorbance spectra of leaf sample, there is remarkable high absorption

at UV region compare to IR and VIS region. Chlorophyll which is dominant in leaf has strong

absorbance of UV spectrum. Leaf has other compound like cellulose, lignin and carbohydrates this

make UV radiation limited then VIS radiation. For leaf UV Spectra 175-350 nm, absorption of

Radiated Energy level was more compounds.

c. Brain Phantom:

A peak in the spectrum at a given wavelength corresponds to absorption of energy at this wavelength

by the type of molecules; for some molecules, more than one area of absorption is observed. If we

observed the UV region absorbance spectra of brain phantom sample, there is remarkable high

absorption at UV wavelength 200nm to 250nm regions figure 3.3 c. There is much different type of

compounds in biological tissue and this different type of molecules which is dominant in phantom has

strong absorbance of visible spectrum. Water is the most abundant organic in any part biological

sample. The absorption spectrum of brain phantom is shown in Figure over the wavelength 175-

400nm. Between 175nm to 200 nm the absorption coefficient increased and a peak at about 180 nm,

over the wavelength 300 to 400nm region is relatively low absorption and continuously decreasing.


19

Figure 3.3.a: UV spectra absorbance and transmission measurement of water

Figure 3.3 b:. Absorbance coefficients of leaf and water with UV spectrometer.


20

Figure 3.3.c:. Absorbance and Transmission coefficient leaf with UV radiation of brain phantom

3.1.2.2. Vis Spectra measurements of: Water, Leaf, Brain

Phantom

a. Water

Full spectrum Vis spectra radiation of water is shown in Figure 3.4.a. We were taken a transparent

disposable plastic cuvette. This cuvette was transparent in Vis radiation. Fill the cuvette with water

then place that in holder inside Vis spectrometer. Vis wavelength’s radiation were penetrated the water

in cuvette after that detector measure the energy level of the signal. At Figure 3.4.b 𝜆 380 to 800 nm

absorptions is almost zero and transmission is almost 100%. Water is color less, so wave beam do not

absorb in the visible region. During water transmission experiment the result shows, the energy levels

of Vis radiation are absorbed by water molecules. There is a certain rule at losing this energy level.

Water transmission is almost 90% to 'visible' light region, a property which is made good use of by

photosynthesis and allowing production of both biomass and oxygen. Vis spectra region absorption

and transmission spectra are almost uniform.

b. Leaf

The Vis absorbance result of leaf is shown in figure-3.4.b. We observed there is remarkable high

absorption at VIS region compare to IR region. Chlorophyll which is dominant in leaf has strong

absorbance of visible spectrum. Leaf has other compound like cellulose, lignin and carbohydrates this


21

make UV and IR radiation limited then VIS radiation. For leaf Vis Spectra 650 to 680 nm, absorption

of Radiated Energy level was more compounds, and we saw 1 peak at 659nm, because of chlorophyll

A inside the leaf we have that peak. A fresh leaf contains 80- 90 % water of its weight. Figure 4.6

showed combined absorption coefficient of leaf and fresh water.

Figure 3.4.c is result of absorbance VIS spectroscopy with Lamp source. We had scanned the leaf with

manual stepper and step size was 5mm. Reason of took such a big step size is the spot size of the VIS

lamp source beam. The spot size of tungsten-halogen visible Lamp beam after passing the optical

design of Lamda 950 by perkingElmer was 5 x 10 mm. We scan about 25x10 mm area of leaf sample

and took transmittance data of 5 different spot’s which is plotted at figure 3.4.c. We observed leaf had

absorbs most energy from wavelengths of orange-red light region then the violet-blue light region. So

the main one peak we can see at orange-red light region which is around 632.8 nm. Leaf absorbance

spectra is minimal after 700nm at Vis range.

c. Phantom

The absorbance measurement of brain phantom, wavelength (λ [nm]) against absorbance (A) is plotted

in figs 3.3.d is plotted. The absorbance (A) is the logarithm of the ration of the intensity of the incident

radiation (𝐼0) to that of the transmitted radiation (𝐼). When a beam from Vis source fall on brain

Phantom, the incident beam will ether reflected or transmitted. And we can easily calculate absorbance

(A) from percentage of transmittance (T) data, which is A =2 - log10 %T. Measurements of brain

Phantom have been described because it is present almost same characteristic of biological brain

sample. So it is very important to understand the absorbance of Vis radiation of phantom. A peak in

the spectrum at a given wavelength corresponds to absorption of energy at this wavelength by the type

of molecules; for some molecules, more than one area of absorption is observed. If we observed the

Vis region absorbance spectra of brain Phantom sample, there is remarkable high absorption at VIS

wavelength 632.8 nm regions. There is much different type of compounds in biological tissue and this

different type of molecules which is dominant in phantom has strong absorbance of visible spectrum.

Water is the most abundant organic in any part biological sample. The absorption spectrum of brain

phantom is shown in Figure 3.4.d over the wavelength 400-1000nm. Between 580nm to 650 nm the

absorption coefficient increased and a peak at about 630 nm, over the wavelength 650 to 1000 nm

region is relatively low absorption and continuously decreasing. The region of 400 to 580 nm is

slightly decreasing but there is no peak at that part.


22

Figure 3.4.a. Vis spectra absorbance and transmission measurement of water.

Figure 3.4.b:. Absorbance and Transmission coefficient leaf with Vis spectrometer.


23

Figure 3.4.c:. Lamp VIS spectroscopy of Leaf with specific absorption at five different spot.

Figure 3.4.d:. Vis Absorbance and Transmission coefficient phantom


24

3.1.2.3. IR Spectra measurements of: water, leaf, brain

phantom.

a. Water:

IR spectra radiation of water is shown in Figure 3.3. We had taken a transparent disposable plastic

cuvette. This cuvette is transparent in UV to IR radiation. Fill the cuvette with water then place that in

holder inside IR spectrometer. Different wavelength’s radiation penetrated the water in cuvette after

that detector measure the energy level of the signal. At figure 3.5.a 𝜆 800 to 3300 nm absorptions

spectra, this is very complex. In IR region we found two spikes at 1000nm and 1100 nm region, which

are due to water molecules. After 1200 nm wave beam is absorb in the IR region, the energy levels are

absorbed by water molecules. There is a certain rule at losing this energy level. IR radiations have

loosed individual energy level because water has its own absorption spectra. Figs. 3.5a strong

absorptions tailing off into the normally inaccessible region below 1200nm are detected. Water

transmission is almost 0% to 1400nm to 3300nm region, a property which is made worst use of by

photosynthesis and allowing production of both biomass and oxygen. At IR Spectra, absorption of

Radiated Energy level is more complain to VIS and UV spectra.

b. Leaf:

When a beam from IR source fall on leaf surface, the incident beam will ether reflected or transmitted.

And we can easily calculate absorbance (A) from percentage of transmittance (T) data, which is A =2 -

log10 %T. The absorbance result of leaf at 705nm to 3300 nm is shown in figure-3.5.b. If we observed

the IR region absorbance spectra of leaf sample, there is remarkable high absorption at VIS region

compare to IR region. Chlorophyll which is dominant in leaf has less absorbance of IR spectrum. Leaf

has other compound like cellulose, lignin and carbohydrates this make IR radiation limited then VIS

radiation. For leaf IR Spectra 2000-3300nm, absorption of Radiated Energy level was more

compounds, but we saw 2 peaks at 1400nm and 1800nm. A fresh leaf contains 80-90 % water of its

weight. Figure 3.5.b showed combined absorption coefficient of leaf and fresh water.

c. Brain phantom

The absorbance measurement of brain phantom, wavelength (λ [nm]) against absorbance (A) is plotted

in figs 3.5a is plotted. The absorbance (A) is the logarithm of the ration of the intensity of the incident

radiation (𝐼0) to that of the transmitted radiation (𝐼). When a beam from Vis source fall on brain

Phantom, the incident beam will ether reflected or transmitted. And we can easily calculate absorbance

(A) from percentage of transmittance (T) data, which is A =2 - log10 %T. Measurements of brain

Phantom have been described because it is present almost same characteristic of biological brain

sample. So it is very important to understand the absorbance of IR radiation of phantom. A peak in the

spectrum at a given wavelength corresponds to absorption of energy at this wavelength by the type of

molecules; for some molecules, more than one area of absorption is observed. If we observed the IR

region absorbance spectra of brain Phantom sample, there is remarkable high absorption at IR

wavelength 1375 nm, 1800nm, 3200nm regions. There is much different type of compounds in

biological tissue and this different type of molecules which is dominant in phantom has strong

absorbance of IR spectrum. Water is the most abundant organic in any part biological sample. The

absorption spectrum of brain phantom is shown in Figure 3.5.c over the wavelength 800-3300nm.

Without the peak the absorption coefficient is mostly constant


25

Figure3.5.a: IR spectra absorbance and transmission measurement of water


26

Figure3.5.b: IR spectra absorbance and transmission measurement of leaf.


27

Figure3.5.c: IR spectra absorbance and transmission measurement of phantom


28

3.2. Method 2:VIS Imaging

Detection of an item inside an unclear cover is a key challenge today. One solution of detection an

object behind obscuring layers is Vis spectroscopy. Red laser which has under Vis spectra can use for

detection of a hidden object, mine detection, and imaging through scattering biological medium. In

this section, we discuss methods of VIS spectroscopy or laser spectroscopy. Laser Spectroscopy

(figure 3.1) means a process for getting information about transmission laser beam. Set of equipment

was used in this method. First we discuss about the setup of the experiment followed the equipment

used in this experiment.

3.2.1.1 HeNe Laser Spectroscopy setup

For the laser spectroscopy a single beam HeNe laser was used. Laser was place inside the metric laser

holder. The holder contained M6 x 1.0 hole. Inside the hole with Socket head cap Screws HeNe Laser

was fixed. Then the laser with metric laser holder was placed on the composite breadboard laboratory

table. And we fixed the metric laser holder on composite breadboard laboratory Table. DC power was

need for the HeNe laser. State line to laser beam a stepper was placed. Stepper was used here is

Edmund Optics 6" XY positioer and motor is use T-LA Series Miniature Linear Actuators. AC current

was used for linear actuators. Actuator was controlled by a Matlab program (program is given in

Appendix B). An USB connector actuator was connected with a Laptop computer. After laser a

valumax objective lens is placed. Lens is place on a holder. The lens was used to focus the laser beam

to the sample. Then we used our detector which is a Silicon PIN Photodiode. Fame used stick the

diode and connects with power cable. We was used a XY move able holder to fix the PIN photodiode.

PIN diode was connected with a Fluke 45 Multimeter. Multimeter had a RS-232 connector at output.

A RS-232 to USB to serial converter cable was use to connect the multimeter with laptop computer.

And matlab interface was use for the extracting data from the multimeter.

3.2.1.2 (HeNe) Laser Spectroscopy Method

When we switched on the power supply of the laser, it create spontaneous light emission of a red beam

which had output Power 0.8 mW and Beam Diameter (1/e²) 0.57 mm. Simultaneously the stepper start

working. A 12 V DC power supply is attach with an actuator. And from both the actuator were

connected each other. Among the two actuators only one was connected with power supply and with

laptop to get the command. Through first connected actuator second actuator will get command and

get the power supply. Step size of the positioned was controlled by program. At first experiment we

was used 1 mm step size. According to our demand the step size of the stepper could be controlled.

Laser beam would penetrated according to the characteristic of the sample was used as testing plate.

After that sample transmitted beam will fall on to PIN diode. And PIN diode could read the power

lave of the beam signal. That could be showed by the multimeter. Multimeter will pass the data

through the RS-232 connecter to storage device in this case laptop MatLab program. For each step

Mutimeter was read a new value and that was stored in a matrix.


29

Figure 3.2.a:. Block Diagram of Laser bias VIS spectroscopy setup.

The steppers were worked in our case forward and return backward. If our stepper had to scan a

sample then it would work like first forward then one step down and then come backward. Advantage

of this type of scanning was actuator need not to go 0 modes again and again. This process saves time.

In Matlab programming we had used a special command to make shifting. So when we was put the

value from the multimeter to matlab it was give to right place. When we was read the image it was not

be reversed.

3.2.1.3 Helium-Neon (HeNe) Laser

In this experiment a Helium-Neon (HeNe) Laser was used as a source. HeNe Laser (Figure shown at

Appendix A) can emit red light beam which has wavelengths of 632.8 nm with 1 mW power. HeNe

laser built with Helium and Neon gas which ratio is 10:1. In experiment the laser beam passes through

test plates and detector measured power level of the signal. HeNe laser has very good power and

thermal stability because of its robust tube design. The red laser is TEM00 mode at 632.8 nm.

Metric Laser Holder: to mount the Newport’s Helium-Neon (HeNe) Laser a metric laser holders of

Edmund Optics was used. This holder is fully metallic made. It had two mount ring (Mounting hole

are M6 x 1.0) which was fixed and easily adjustable. Figure is shown at Appendix A.

3.2.1.4 Composite Breadboard Laboratory Table

A Stainless Steel composite breadboard ultra-light table was used to fixe all the equipment during this

experiment. This was a light table. The weight of the table was about 20kg. Top part of the steel table

was full with XY tapped holes. Each hole was M6 x 1.0.


30

Table 3.1Descristion is about composite breadboard laboratory table.

Length (inches) 47

Width (inches) 19

Thickness (mm) 50

Flatness (mm) 0.13

Core Cell Area (cm2) ≤ 3

Construction Stainless Steel (400 Series) Top and Bottom

Plates, Plated Steel Honeycomb Core, High

Pressure Laminate Core Sidewalls

Mounting Threads (765) 1/4-20 Tapped on 1" Centerlines, on 1.5"

Corners

Weight (lbs) 62

3.2.1.5 X Y Positioner and Stepper Motors

XY positioned was used during the measurement it was required to scan a particular area. For

measurement with high spatial resolution, equipment with good accuracy was required. Choosing a

XY positioner main considering fact was the pitch distance. Small pitch length can give better spatial

resolution to scan a test plates. If the pitch length was very small then it was difficult to use it

manually. So smart choose was a motor stepper. With motor stepper it was easy to get accuracy step

size. In this laser spectroscopy experiment Edmund Optics 6" XY positioner was used. To scan (sweep

objects with the single beam, probe or resonator) the device under testing an XY positioner was

constructed by using equipment of two actuators, two translation stages and on Z-axis bracket. Feature

of Edmund Optics 6" XY positioner:

i. 2" Travel in each positioner.

ii. It was smooth, ball Bearing Movement

iii. Controlled by Zaber's T-LA60 actuators

The preloaded ball design permits low friction linear movement with no backlash or side play, on

hardened stainless steel balls and ways [7].

3.2.1.6 T-LA Series miniature linear actuators with built-in controllers

X -Y Positioner was controlled by two Zaber’s T-LA60 Series Miniature Linear Actuators. It was

translation distances of 60 mm with a resolution of 0.1 µm (depending on translation speed). Built in

controllers, possibility to connect in a daisy chain and was used only one power supply for up to 3

actuators were some of the abilities of the T-LA60. T-LA Series Miniature Linear Actuators with

Built-in controllers Matlab program was used to control the actuators. In bellow table the specification

of the actuators is give.


31

Table3.2. Description about the actuators.

Specification Value

Microstep Size (Default Resolution) 0.09921875 µm

Integrated Controller Yes

Travel Range 60 mm

Accuracy +/- 16 µm

Repeatability < 1 µm

Backlash < 6 µm

Maximum Speed 4 mm/s

Minimum Speed .0009302 mm/s

Speed Resolution .0009302 mm/s

Maximum Current Draw 300 mA

Power Supply 12-16 VDC

Motor Steps Per Rev 48

Motor Type Stepper (2 phase)

Axes of Motion 1

Power and Com LEDs Yes

Weight 0.15 kg

3.2.1.7 Valumax Objective Lens

The MV-40X Valumax Microscope Objective Lens is was to reduce and focus the beam point. The

lens was placed in contact with the X Y Positioner and Stepper. The lens was 40x magnification, 0.65

numerical aperture, 4.4 mm focal length, and 5.7 mm clear aperture. It was expose conjugate at 188

mm. It was antireflection at Vis radiation area.

i. The exposed effective focus 1 mm.

ii. Could be used at radiation range 350 to 750 nm.

iii. It was worded at the distance -1 mm.

3.2.1.8 Silicon PIN Photodiode

In detector part a high speed and high sensitive PIN photodiode BP104 by Vishay Semiconductors

was used [8]. BP104 photodiode was detect the electromagnetic energy of HeL Laser which was pass

through the sample. This diode was contained large active area combined with flat case. Also it was

highly sensitive. This diode was stitched to a plate and attach with move able holder. To read the

electromagnetic energy it was connected with a digital multimeter. Features:

i. Sensitive Area was 7.5mm2

ii. It was design for high photo sensitivity.

iii. Fast response was obtained from this photodiode.

iv. Pin diode was delivered in a plastic case.

http://www.zaber.com/support/?tab=Glossary#microstep_distance

http://www.zaber.com/support/?tab=Glossary#integrated_controller

http://www.zaber.com/support/?tab=Glossary#travel_range

http://www.zaber.com/support/?tab=Glossary#accuracy

http://www.zaber.com/support/?tab=Glossary#repeatability

http://www.zaber.com/support/?tab=Glossary#backlash

http://www.zaber.com/support/?tab=Glossary#max_speed

http://www.zaber.com/support/?tab=Glossary#min_speed

http://www.zaber.com/support/?tab=Glossary#speed_resolution

http://www.zaber.com/support/?tab=Glossary#currentdraw

http://www.zaber.com/support/?tab=Glossary#powersupply

http://www.zaber.com/support/?tab=Glossary#motor_steps_per_rev

http://www.zaber.com/support/?tab=Glossary#motor_type

http://www.zaber.com/support/?tab=Glossary#axes

http://www.zaber.com/support/?tab=Glossary#leds

http://www.zaber.com/support/?tab=Glossary#weight


32

Figure 3.2.b: Lab setup of Laser bias VIS spectroscopy.

3.2.1.8 Digital Multimeter

To read the signal power level from the PIN photodiode accurately a digital mulimeter was used. In

the experiment fluke 45 digital duel display multimeter was selected. The multimeter was combined

with a RS interface. Therefore it was easy to connect with laptop to control and read the values. A RS

-232 to usb convert connector was used for laptop interface. And Matlab program was controlled and

read value from the multimeter. Features of the digital multimeter:

i. It was providing different function with dual display

ii. It was possible to measure true-RMS Voltage and Current (AC as well as DC)

iii. The facilitation of RS-232 was available.

iv. Measurements value was displayed in dB.


33

3.2.2 Laser base imaging of Leaf, Phantom and hidden

object:

a. Leaf:

Laser spectroscopy image of leaf sample demonstrate at figure 3.6.a Sample diameter was 10x10 mm

middle of the sample leaf the major vein midrib. We had scan the leaf sample with step size .25 mm.

Reason of took step size .25mm was the spot size of the VIS HeNe laser source beam. The diameter of

HeNe red laser beam spot size was 2 mm after passing Valumax Objective Lens diameter of beam

spot become .25 mm, 40x40 steps was needed to cover the full scanning of the sample. Midrib had

less water concentration, for .25mm laser beam it was not possible to penetrate. But leaf’s lamina (the

flattened green extended part) had more water concentration and thin so laser beam were

transmittance. We got a 40x40 pixels matrix of data by 3 hours to complete full spectroscopy imaging.

We took a green color plant leaf for experiment, green color leaf contain more chlorophylls. At Figure

3.7 𝜆 175 to 2000 nm absorptions spectrum is shown and we observed wave beam’s fluctuating

absorbance spectra at visible region. At IR region absorbance spectra is all most constant. Spectral

bandwidth varied 1nm at Vis region and 10 nm at IR region during the measurement. We have

compared the absorption spectra of leaf with water. In comparison graph we clearly found water have

almost zero absorbance because UV/Vis/IR radiation are not absorbed by water molecules. But for

leaf UV/Vis/IR radiation are absorbed by leaf cell because at leaf cell chlorophylls present. In figure

3.7 b at 450nm to 600 nm we see a valley at absorbance which is due to present of chlorophylls at leaf.

At 450nm to 600nm absorbance is less so more transmission. But we can see some pick at seatrain

wave range. At near-infrared range which is 780 to 3300 nm absorbance is very strong but it is not

uniform. Laser spectroscopy image of leaf showed at figure 3.6 a Sample diameter was 10x10 mm.

The diameter of HeNe red laser beam spot size was 2 mm after passing Valumax Objective Lens

diameter of beam spot become.25 mm, 40x40 steps was needed to cover the full scanning of the

sample.

b. Brain Phantom

Laser spectroscopy image of brain phantom sample demonstrate at figure 3.6 (b) Sample diameter was

10x10 mm middle. We had scan the sample with step size .25 mm. Reason of took step size .5 mm

was the spot size of the VIS HeNe laser source beam. The diameter of HeNe red laser beam spot size

was 2 mm after passing Valumax Objective Lens diameter of beam spot become .25 mm, 40x40 step

was needed to cover the full scanning of the sample. We took a white color brain phantom for

experiment. At figure 3.4.c 𝜆 300 to 800 nm absorptions spectrum is shown and we observed

fluctuating absorbance spectra at visible region. It was due to water and white pigment. Spectral

bandwidth varied 1nm at Vis region during the measurement. At 450nm to 800nm absorbance is less

so more transmission. But we could see some pick at seatrain wave range but it is not uniform. Laser

spectroscopy image of phantom showed at figure 3.6.b Sample diameter was 10x10 mm. The diameter

of HeNe red laser beam spot size was 2 mm after passing Valumax Objective Lens diameter of beam

spot become .25 mm, 40x40 step was needed to cover the full scanning of the


34

c. Object detection

Detect of an object behind obscuring layer is one of the challenge, in this thesis work HeNe Laser

spectroscopy system is use to detection and imaging. It has been noted that a short pulse which

penetrates through obscuring layers may be scattered by the layers and the object. Then, the received

signal scattered from the object and from the layers can be separated in time and therefore, detection

of the object hidden behind the scattering layer may be possible.

Vis imaging with HeNe laser source:

The detection and imaging of 1.5 mm vertical cooper line was experimented. When a beam from Vis

source fall detection object, the incident beam will ether reflected or transmitted. Figure 3.6 c.is result

of VIS spectroscopy with laser source. Laser spectroscopy of hidden object diameter was 3mm

horizontal and 1.5 mm vertical cooper line on a glass plate which dimension is 72 mm in horizontal

and 26mm in vertical.

We had scan the object sample with step size .25 mm. Reason of took step size .25mm was the spot

size of the VIS HeNe laser source beam. The diameter of HeNe red laser beam spot size was 2 mm

after passing Valumax Objective Lens diameter of beam spot become .25 mm, 40x40 step was needed

to cover the full scanning of the sample. Glass is transparent so laser beam can full transparent

through the glass but cooper is not transparent to laser beam, for laser beam it was not possible to

penetrate. We got a 40x40 pixels matrix of data. It took 3 hours to complete full spectroscopy imaging

because Fluke 45 digital multimeter took time to read data from detector.


35

(a)

(b)

Figure 3.6: (a) HeNe laser (at wavelength 632.8 nm) image process diagram of carolina poplar leaf sample

(10x10mm) with step size 0.25mm. (b) HeNe laser (at wavelength 632.8 nm) image process diagram of tissue

Brain Phantom (18x20mm) with step size 0.5mm.


36

(c)

Figure 3.6: (c) image process diagram to detect object ( about 3mm horizontal and 1.5 mm vertical cooper line)

with step size 0.25mm.

Figure 3.7: (a) A plat leaf and sample which is 52 x 52 mm. (b) Absorbance Spectra of leaf sample at 174nm to

3300nm.


37

Figure 3.8 Absorption coefficient leaf and water with UV/Vis/IR spectrometer.

Figure3.9: UV, Vis & IR radiation absorbance and transmittance measurement of water.


38

Chapter 4:Discussion and Conclusion

UV, Vis and IR Spectroscopy and Vis imaging are important because of the high information content

and it has variety of possibilities for sample measurement and imaging. Scientists are always

interested to see detail inside an object; special interest is on biological organs. There are number of

ways to observed biological organs or objects, like microscopic or vivo imaging or spectroscopic

imaging. In this thesis work, two methods have been proposed spectroscopic imaging of different

biological sample and hidden object with different wavelength have been experimented. UV/Vis/IR

Spectroscopy cover the radiation wavelength region from 175nm to 3300nm, in this work He-Ne laser

can emit visible red light 632.8 nm, tungsten-halogen visible Lamp can emits 360 nm to 3300 nm and

Deuterium ultraviolet Lamp can emits 175 to 400 nm wavelength is used. Light source of different

wavelength of the spectroscopic system pass through the sample and detector measures the power,

after using image plotting an image of the sample is generated.

4.1 Mapping about Method 1 measurements:

(a).Water: Water spectroscopy, we have scanned the water sample at IR, VIS and UV wavelength

from 200nm to 3300nm. In different wavelength water sample reacted differently. At UV spectrum

from 200 to 400nm water is almost 100% transparent or almost 0% absorbance. Water has no color so

it is normal; at Vis spectra from 300 to 800nm water is more that 90% transparent. However IR reason

is from 800nm to 3300 nm, water absorption spectra at IR is much complex. IR spectra have some

common sub division like near Inferred, short wavelength infrared, mid wavelength and far

wavelength. In this sub division water reacts differently, at short wavelength from 1450 nm water

absorption increase significantly. But at near infrared from 750 nm to 1450 nm water transmission are

different and have found two peaks due to water molecules.

(b) Leaf: For leaf spectroscopy, we know chlorophyll is main organic molecule inside leaf. And in a

fresh leaf there is almost 80% water. So leaf spectroscopy characteristic is maintained by this two

chlorophyll and water. We have scanned the leaf sample at IR, VIS and UV wavelength from 200nm

to 3300nm. In different wavelength leaf sample reacted differently. At UV spectrum from 200 to

400nm water is almost 0% transparent or almost 100% absorbance. IR spectra sub division near

Inferred, short wavelength infrared, mid wavelength and far wavelength leaf absorption is almost

same, from 800m to 3300 nm. However there is two peaks pick due to water molecules at 1450 nm

and 1750nm. Water is a colorful object due to present chlorophyll. And due to sunlight and other in

some leaf we can find chlorophyll A and other chlorophyll B. Vis spectra from 300 to 800nm we

found a peak at 632.5 due to chlorophyll A. which is red spectra of visible reason.

(c) Brain Phantom: We took a white color brain phantom for experiment. 𝜆 300 to 800 nm

absorptions spectrum have been observed fluctuating absorbance spectra at visible region. It is due to

water and white pigment. Spectral bandwidth varied 1nm at Vis region during the measurement. At

450nm to 800nm absorbance is less so more transmission. But we can see some pick at seatrain wave

range but it is not uniform


39

4.2 Mapping about Method 2 measurements:

HeNe laser (at wavelength 632.8 nm) image process diagram of carolina poplar leaf sample

(10x10mm) with step size 0.25mm had been imaged. And a diagram of tissue Brain Phantom

(18x20mm) with step size 0.5mm successfully imaged. Lastly we had detected object (about 3mm

horizontal and 1.5 mm vertical cooper line) with step size 0.25mm.

4.3 Achievement and Future work:

This thesis work is perfect in the field of spectroscopic imaging and detection of any type of biological

and chemical liquid, solid or gas. He-Ne laser, tungsten-halogen visible Lamp and Deuterium

ultraviolet Lamp source photo detector diode used and perfectly worked. This is a very simple and use

full spectroscopic analyzing and detection method also cost effective, efficient, robust technique for

biological sample. This is an improved detection system for detect a hidden object also. Main focus of

the thesis is detection and imaging: which significantly had done in this thesis work. One important

contribution of this work is appropriate data accusation in UV, Vis and IR spectra.

The limitation about this spectroscopic system is big spot size of source wave: we cannot make

imaging of an object less than 0.25mm. For leaf, brain phantom and object detection we have scan the

sample 0.25mm step so it ought not possible to detect anything less than that. If we can use better

optical lens after source and focus that source beam small then we can get data of very small area. And

we can make image of an object more detail. In future we can use a lens after the source such we can

make the source spot size small. Also we can make the instrument setup such a way so we can

measure the reflection power from the sample. This work we have only measure the transmittance, it

will be interesting to measure reflection from a sample.


40

References: [1] J.-Ming Jin, Theory and Computation of Electromagnetic Fields, Wiley-IEEE Press, Nov 2010, pp.

12-30.

[2] D. M. Pozar, Microwave Engineering, 4th ed, New York: Princeton; Jan. 2012, pp.20-80.

[3] C. A. Balanis, Antenna Theory: Analysis and Design, 3rd ed, Wiley; 2005, pp. 30-51.

[4] P. Rusch and R. Harig. “3-D Reconstruction of Gas Clouds by Scanning Imaging IR Spectroscopy

and Tomography’’, IEEE sensors journal, Vol. 10, no. 3, Mar. 2010,pp. 71-80.

[5] Y. Ung, Lin, Bernd M “Dual-Mode Terahertz Time-Domain Spectroscopy System’’, IEEE

Transactions on terahertz and technology, vol. 3, March. 2012.

[6] K. Shimamura, K. Michigami, B. Wang and K. Komurasaki, “Photo in IN Precursor of laser

induced plasma by ultraviolet radiation’’ IEEE Plasma Science (ICOPS), May. 2011.

[7] S. M. Wentworth, Fundamentals of Electromagnetics with Engineering Applications. John Wiley

& Sons, July. 2006, pp. 45-68.

[8] C. Elachi and V. Zyl, Introduction to the physics and techniques of remote sensing, 2nd ed, John

Wiley & Sons, Apr. 2006, pp. 95-119.

[9] C. N. Banwell,Fundamentals of Molecular & Spectroscopy, Mcgraw-Hill Education (India) Pvt

Limited, Jan. 1994, pp.48-95.

[10] M. Thomas, Ultraviolet and visible spectroscopy, Analytical chemistry by open learning, 2nd ed,

Apr. 2000, pp. 214-249.

[11] B. C. Smith, Fundamentals of Fourier Transform Infrared Spectroscopy, CRC Press LLC,

May.1996, pp 128-170.

[12] F. Diedrich, J. C. Bergquist, and D. J. Wineland , “ Laser Cooling to the Zero-Point Energy of

Motion’’, The American Physical Society,Vol-4, Jan. 1989, pp. 406-406.

[13] B. Ferguson and X.C. Zhang, “Materials for terahertz science and technology’’, Nature

Materials, vol. 1, 2002, pp. 26-33.

[14] P. G. Coble, “Characterization of marine and terrestrial DOM in seawater using excitation-

emission matrix spectroscopy,’’ Marine Chemistry, Vol.45, Jan.1996, pp. 335–346.

[15] P. Grodzinski, M. Silver and L. K Molnar, “Nanotechnology for cancer diagnostics: promises and

challenges,’’ US National Library of Medicine, Vol. 6, May. 2006, pp. 307-318.

[16] Y. Liu, C. Sullivan, G. Fang, A. Cox and X. Li, “Molecular signaling involved in low doses of

arsenic-promoted cell proliferation’’, Oxford Journals, Life Sciences & Mathematics & Physical

Sciences,Vol. 42 1, Feb 2001, pp.19-66.

[17] H.Kang , A. C. Trondoli , G. Zhu , “Near-Infrared Light-Responsive Core–Shell Nanogels for

Targeted Drug Delivery’’, ACS Nano, Aug. 2011, pp. 94–99.


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[18] Helium-Neon (HeNe) Lasers, “Newport Corporation,USA’’, May. 2009, www.newport.com.

[19] He-Ne Laser Mount, “Holmarc Opto-Mechatronics (P) Ltd,UK’’, Jan. 2011, www.holmarc.com.

[20] Objective Lenses, “Newport Corporation, USA’’, Apr. 2011, www.newport.com.

[21] High-Precision XY Positioning System, “Piezo Nano Positioning’’, 2010, www.pi.ws

[22] Miniature Linear Actuator, “Zeber Techologies’’, May 2011, www.zaber.com.

[23] Silicon Pin Photodiode, “Vishay Techologies, Germany’’, May 1999, www.vishay.de

[24] Fluke 45 Bench Meter dual display, “Fluke Techologies, USA’’, 1995, www.fluke.se.

[25] LAMBDA 950, “PerkinElmer Inc., 1998, www.perkinelmer.com.

http://www.newport.com/

http://www.holmarc.com/

http://www.newport.com/

http://www.pi.ws/

http://www.zaber.com/

http://www.vishay.de/

http://www.fluke.se/

http://www.perkinelmer.com/


42

Appendix-A

Below are the the matlab codes to control the equipment , acquire data from multimeter

and imaging.

**************************

clear all, close all,

load matrix_04-07-2012_4.mat

image(64*matrix/max(max(matrix)));

colormap(jet);

threshold = graythresh(matrix);

bw = im2bw(matrix,threshold);

image(64*bw);

colormap(gray);

%imshow(bw)

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

%only steper forward and backwrd.

clear all; clc;

m=32; n=36; %image will be m x n pixels

% m rows, n columns

matrix=zeros(m,n);

%evenrow=0;

actual_value=0;

s=initialize_serial();

t=initialize_serial_move();

% for stepper

step_length_mm = .5;

data = step_length_mm*10000;

data_N=data;

temp_6 = uint32(data/256^3);

data = data-256^3*temp_6





temp_3 = data

home_all = [ 0 1 0 0 0 0 ];

move_1 = [1 21 temp_3 temp_4 temp_5 temp_6];

move_2f = [2 21 temp_3 temp_4 temp_5 temp_6];

move_2b = [2 21 0 236 255 255];

home_1 = [1 1 0 0 0 0];

home_2 = [2 1 0 0 0 0];

for x=1:m

for y=1:n

fwrite (t, move_2f);

pause(0.3);

actual_value=get_value(s);

matrix(x,y)=actual_value;

end

fwrite (t,move_1);

for y=n:-1:1

fwrite (t, move_2b);

pause(0.3);


43

actual_value=get_value(s);

matrix(x,y)=actual_value;

end

fwrite (t,move_1);

end

close_serial_port;

image(64*matrix/max(max(matrix)));

colormap(jet);

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

function [v] = get_value(s)

%s = serial('COM6');

%fopen(s);

% v=rand(1);

%CMD='VAL1?';

%CMD='*RST; VDC; VAL1?';

CMD='VAL1?';

fprintf(s,CMD);

%pause(3);

eco=fscanf(s);

out=fscanf(s);

cmd_chk=fscanf(s);

%pause(2)

out=out(1:end-2);

%pause(3);

% out = fscanf(s);

% pause(3);

% out = fscanf(s);

%v=out;

%fclose (s);

%delete(s);

v=str2num(out);

end

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

function s=initialize_serial()

s = serial('COM6');

fopen(s);

CMD='*RST; VDC; RANGE 3; RATE F; TRIGGER 1';

fprintf(s,CMD);

%pause(1);

eco=fscanf(s);

check=fscanf(s);

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

subplot (2,1,1), plot(LA (1:end,1),LA(1:end,2)),xlabel(

'Wavelength(\lambda),nm.') ylabel(

'Absorbance(A)'), subplot (2,1,2), plot(LT (1:end,1),LT(1:end,2)),xlabel(

'Wavelength(\lambda),nm') ylabel(

'Transmittance(T)'),

%%%%%%%%%%%%%%%%%%%%%%%%

subplot (2,1,1), plot(Auv (1:end,1),Auv(1:end,2)),xlabel(


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'Absorbance(A)'), subplot (2,1,2), plot(Tuv (1:end,1),Tuv(1:end,2)),xlabel(


'Transmittance(T)'), %%%%%%%%%%%%%%%%%%%%%%

subplot (2,1,1), plot(Avis (1:end,1),Avis(1:end,2)),xlabel(


'Absorbance(A)'), subplot (2,1,2), plot(Tvis (1:end,1),Tvis(1:end,2)),xlabel(


'Transmittance(T)'),

%%%%%%%%%%%%%%%%

subplot(3,2,1), plot(AirA (1:end,1),AirA(1:end,2)),xlabel(


'Absorbance(A)'), subplot (3,2,2), plot(TirA (1:end,1),TirA(1:end,2)),xlabel(


'Transmittance(T)'), subplot (3,2,3), plot(AirB (1:end,1),AirB(1:end,2)),xlabel(


'Absorbance(A)'), subplot (3,2,4), plot(TirB (1:end,1),TirB(1:end,2)),xlabel(


'Transmittance(T)'), subplot (3,2,5), plot(AirC (1:end,1),AirC(1:end,2)),xlabel(


'Absorbance(A)'), subplot (3,2,6), plot(TirC (1:end,1),TirC(1:end,2)),xlabel(



45

Appendix-B

Used Equipment at Laser Spectoscopy

1. HeNe lasers Newport. [18]

specification of

laser.

Specifications

Model R-31007

Wavelength 632.8 nm Output Power 1 mW

Beam Diameter (1/e²) 0.58 mm Polarization 500:1

Noise 1.2 % Spatial Mode TEMoo

Longitudinal Mode 1084 MHz Beam Divergence Full Angle 1.41 mrad

Power Requirements 120/240 VAC, 50/60 Hz Suggested Laser Mount ULM or ULM-TILT

Laser Head 7.00 L x 1.75 D in. Beam Drift <0.05 mrad

2. Laser holder[19]

3. Breadboard Table


46

4. x-y sample scanar [21]

5. 5” Stages for Linear

Actuators[22]

6. Valumax objective lens

By Newport[20]

7. Digital multimeter by FLUK[24]

8. Lamda 950 by

PerkingElmer [25]


47

9. Manual stepper

10. Liquid

holder

11. pin diode

holder

Multipoint spectroscopic analyzing & imaging method

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