Studying Grazing Behavior of Cafeteria Roenbergensis with Two-Photon Microscopy Faisal Abedin Abedin University of Texas at El Paso, [email protected]

Studying Grazing Behavior of Cafeteria Roenbergensis with Two-Photon Microscopy Faisal Abedin Abedin University of Texas at El Paso, Faisalabedin0@Gmail.Com

University of Texas at El Paso DigitalCommons@UTEP

Open Access Theses & Dissertations

2016-01-01 Studying Grazing Behavior of Cafeteria Roenbergensis with Two-Photon Microscopy Faisal Abedin Abedin University of Texas at El Paso, [email protected]

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Recommended Citation Abedin, Faisal Abedin, "Studying Grazing Behavior of Cafeteria Roenbergensis with Two-Photon Microscopy" (2016). Open Access Theses & Dissertations. 787. https://digitalcommons.utep.edu/open_etd/787

This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. STUDYING GRAZING BEHAVIOR OF CAFETERIA ROENBERGENSIS WITH TWO-PHOTON MICROSCOPY

FAISAL ABEDIN Master’s Program in Physics

Approved

Chunqiang Li, Ph.D., Chair

Cristian Botez, Ph.D.

Chuan Xiao, Ph.D.

Charles Ambler, Ph.D. Dean of the Graduate School

STUDYING GRAZING BEHAVIOR OF CAFETERIA ROENBERGENSIS WITH TWO-PHOTON MICROSCOPY

Faisal Abedin

THESIS

Presented to the Faculty of the Graduate School of

The University of Texas at El Paso

in Partial Fulfillment

of the Requirements

for the Degree of

MASTER OF SCIENCE

Department of Physics

THE UNIVERSITY OF TEXAS AT EL PASO

August 2016

Acknowledgement

At first, I would like to express my gratitude and thanks to my supervisor Dr. Chunqiang Li,

Assistant Professor, Department of Physics, University of Texas at El Paso. I am truly grateful to him, not only as a supervisor but also as a mentor. His constant guidance, valuable suggestions, encouragement, innovative ideas and new experimental techniques were the driving force behind the success of the research work. He has taught me the art of quality research and independent thinking. I thank him for everything he has done for me and helping me to complete this work.

It is my great pleasure to express my gratitude to Dr. Chuan Xiao, Assistant Professor,

Department of Chemistry, University of Texas at El Paso, not only for providing us the samples, but also because of offering much advice during our weekly meetings. I would like to thank him for his suggestions, and continuous encouragement in my research.

I would like to express my thanks to Dr. Cristian Botez, Chair , Department of Physics,

University of Texas at El Paso for allowing me to use the facilities of Physics Department and for being a member of my thesis committee .Also I am grateful to him for his support and help in

Physics department .

Finally, I would like to express my gratitude to my parents and sisters, who raised me and trusted me with their endless love.

iv Abstract

Major innovations in recent years have largely revolutionized the fluorescence imaging. Two- photon fluorescence microscopy is one of them. Two photon fluorescence microscopy has evolved as an alternative to conventional single-photon confocal microscopy and has been shown to provide several advantages. These include three-dimensionally resolved fluorescence imaging of living cells deep within thick, strongly scattering samples, and reduced phototoxicity, enabling long term imaging of photosensitive biological specimens. The inherent three-dimensional resolution of TPE microscopy has been exploited in a number of studies wherein spatial discrimination of fluorescence signals at the micrometer and submicrometer scale within thick biological specimens proved critical. In this study, we used our two-photon microscope to observe grazing behavior of fast moving marine organism Cafeteria roenbergensis with the interaction of bacteria.

v Table of Content

Chapter 1: Two-photon Microscopy……………………………………………………………..1

1.1 Introduction …………………………………………………………………………….……1

1.2 History of Two-Photon Microscopy……………………………………………..….……….2

1.3 Theory of Two-Photon Absorption…………………………………………………………..3

1.3.1 Intensity Square Dependence versus Power and Optical Sectioning………….…………3

1.3.2 Cross-section and Colocalization……………………………………………...…………8

1.4 Experimental Setup…………………………………………………………………….……10

1.4.1 Light Source…………………………………………………………….……………….10

1.4.2 Waveplate and Polarizer ………………………………………………..………………10

1.4.3 Scanning Platform………………………………………………………...……………..13

1.4.4 Dichroic Mirror and Filters……………………………………………………….……..14

1.4.5 Objective Lens…………………………………………………………………………..15

1.4.6 Photomultiplier Tube (PMT)…………………………………………………………….16

1.4.7 3D Stage……………………………………………………………………………...…..17

1.5 Advantages of Two-Photon Microscopy………………………………………………….....18

Chapter 2: Grazing Behavior of Cafeteria roenbergensis ………………………………………21

2.1 Introduction………………………………………………………………………..…………21

2.2 Cafeteria Roenbergensis…………………………………………………………..…………21

2.3 Bacteria………………………………………………………………………………………24

2.4 Microsphere ………………………………………………………………………...……….25

2.5 Samples Preparation…………………………………………………………….……………26

vi 2.5.1 Cafeteria roenbergensis Preparation………………………………………………….……26

2.5.2 Bacteria Preparation…………………………………………………………………….….27

2.5.3 Microsphere Preparation………………………………………………………………..….28

2.6 Two-Photon Fluorescence Microscopy Imaging…………………………………………….28

2.6.1 Cafeteria roenbergensis (Cro) Imaging…………………………………………………….28

2.6.2 Bacteria Imaging……………………………………………………………………...……30

2.6.3 Carboxylate-modified FluoSphere beads Imaging…………………………………..…….32

2.6.4 Imaging for Interaction between Cafeteria roenbergensis and Bacteria …………….……32

2.6.5 Imaging for Interaction between Cafeteria roenbergensis and Microsphere………………37

2.7 Statistical Analysis of Interactions …………………………………………………….…….40

2.8 Conclusion ………………………………………………………………………..…………51

Reference ………………………………………………………………………………..………52

Vita ………………………………………………………………………………………………55

vii List of Tables

1.1 Lines per image corresponding to each frame rate…………………………………………..13

2.1 Counting number of particles (Interaction of Cro and bacteria OD 1.2) by using ImageJ software ……………………………………………………………………………..…………..42

2.2 Counting number of particles (Interaction of Cro and bacteria OD 0.6) by using ImageJ software ……………………………………………………………………………...…………..43

2.3 Counting number of particles (Interaction of Cro and bacteria OD 0.3) by using ImageJ software ……………………………………………………………………………………...…..44

2.4 Counting number of particles (Interaction of Cro and microsphere) by using ImageJ software

……………………………………………………………………………………..……………..49

viii List of Figures

1.1 Simplified scheme of the energy transition occurring under TPE regime………….…………5

1.2 Schematic of the Two-Photon Laser Scanning Fluorescence Microscope developed in the

Biophotonics Laboratory of the Physics Department at UTEP……………………..……….12

1.3 One Photon Vs Two Photon Fluorescence Imaging …………………………………...……18

1.4 Comparison of Confocal Microscopy and Two-Photon Microscopy………………………..19

2.1 Cafeteria roenbergensis ……………………………………………………………………..23

2.2 Scanning electron mictoscope of Eschericha Coli (E.coli) …………………………………24

2.3 FluoSpheres Carboxylate-Modified Microscopes………………………..………………….26

2.4 Cafeteria roenbergenesis in NADH Autofluorescence ……………………………………..29

2.5 Cafeteria roenbergenesis in NADH Autofluorescence (after changing the color)…………..30

2.6 Bacteria (OD 1.2) signal with stained by SYBR Gold Stain ……………………………….31

2.7 Bacteria (OD 0.6) signal with stained by SYBR Gold Stain …………………….………….31

2.8 Bacteria (OD 0.3) signal with stained by SYBR Gold Stain …………………….………….31

2.9 Carboxylate-modified FluoSpheres beads in Red Channel………………………….………32

2.10 Carboxylate-modified FluoSpheres beads in Red Channel (after changing the color)……………………………………………………………………………………….……32

2.11 Interaction between Cro and Bacteria in different time ……………………….………….34

2.12 Interaction between Cro and Bacteria in different time…………………………………...36

2.13 Interaction between Cro and Microsphere in different time………………………..……..38

2.14 Interaction between Cro and Microsphere in different time……………………….………39

2.15 Plot of percentage of overlapping particles (between cro and bacteria) with time (in middle surface)…………………………………………………………………………………….……..45

ix 2.16 Plot of percentage of overlapping particles (between cro and bacteria) with time (for 3 different experiments)……………………………………………………………………………46

2.17 Plot of percentage of overlapping particles (between cro and bacteria) with time (in top surface)……………………………………………………………………………..…………….47

2.18 Plot of percentage of overlapping particles (between cro and bacteria) with time (in both surfaces)…………………………………………………………………………………....…….48

2.19 Plot of percentage of overlapping particles (between cro and microsphere) with time (in both surfaces)…………………………………………………………………………….………50

x Chapter 1: Two- photon Microscopy (TPM)

1.1 Introduction

In light of non-linear optical wonders different laser-scanning techniques have been made in the latest a quarter century. Two-photon excited fluorescence microscopy (TPM), Second Harmonic

Generation (SHG) microscopy and Coherent Anti stokes Raman Scattering (CARS) microscopy are a portion of the intense imaging devices. The upsides of these creative system have empowered incredible advancements in biological imaging, particularly in thick tissue and live creature studies than the ordinary optical microscopy strategies. A standout amongst the most essential late innovations in biological imaging is two-photon fluorescence microscopy (TPM). This innovation empowers noninvasive investigation of organic specimens in three dimensions with submicrometer resolution. Two-photon fluorescence microscopy keeps on finding an expanding number of uses in science and medicine. Two photon excitation of fluorophores results from the simultaneous absorption of two photon [1].

Two-photon excitation is a fluorescence process in which a fluorophore (a molecule that fluoresces) is excited by the simultaneous absorption of two photons. The familiar one photon fluorescence process involves exciting a fluorophore from the electronic ground state to an excited state by a single photon. This process typically requires photons in the ultraviolet or blue/green spectral range. However, the same excitation process can be generated by the simultaneous absorption of two less energetic photons (typically in the infrared spectral range) under sufficiently intense laser illumination. This nonlinear process can occur if the sum of the energies of the two photons is greater than the energy gap between the molecule’s ground and excited states. Since 1 this process depends on the simultaneous absorption of two infrared photons, the probability of two-photon absorption by a fluorescent molecule is a quadratic function of the excitation radiance.

1.2 History of Two-Photon Microscopy

The potential for highly intense light to trigger nonlinear procedures has for quite some time been perceived. In 1931 Maria Göppert-Mayer anticipated multiphoton excitation forms in her doctoral dissertation on the hypothesis of two-photon quantum transtion in particle [2]. By focusing on second harmonic generation of light, in 1961 Franken and his gathering had started the trial work in nonlinear optics [3]. They demonstrated that ruby laser light, at wavelength λ, propagating through a quartz precious stone will produce light at the second harmonic generation with a wavelength of λ/2. After the publication of the paper by Franken and his gathering, In 1963

Kasier and Garret published the principal report on two-photon excitation (TPE) of CaF2:Eu2+ fluorescence [4]. Later they checked that TPE likewise can excite the fluorescence of natural colors. From that point forward, numerous case of TPE procedures in sub-atomic spectroscopy have been accounted for. To consider the electronic structure of the sub-atomic energized states, two-photon spectroscopy has turned into a vital instrument. Göppert-Mayer's hypothesis was at last affirmed 32 years after its detailing. Three-photon excitation spectroscopy has likewise been depicted by the correlation with the two-photon forms [5].

2 1.3 Theory of Two-Photon Absorption

1.3.1 Intensity Square Dependence versus Power and Optical Sectioning

The strategy for perturbation hypothesis can connected when the interaction amongst light and atoms is not very solid. As indicated by this approach, the full Hamiltonian has been part into a perturbation free, time independent 퐻0 and a time dependent perturbation V [6]:

퐻 = 퐻0 + 푉 (1.1)

Where 퐻 is very close to the unperturbed Hamiltonian 퐻0 . The impact of the electromagnetic collaboration can be seen at different order in V.

Take that the system have just two state, 풊 and 풇 , eigenstates of time independent

Hamiltonian 퐻0. The transition probability amongst 풊 and 풇 can be assessed as far as the time evolution operator U as:

2 푊𝑖→푓 = |< 푓|푈(푡)|𝑖 > | (1.2)

Where 푈 can be expressed as a function of 푉 (D-labelled operators) as:

1 푛 푡 푡 푡 푈 (푡, 0) = 퐼 + ∑∞ ( ) ∫ 푑푡 ∫ 푛 푑푡 … … ∫ 2 푑푡 푉 (푡 )푉 (푡 ) … 푉 (푡 ) (1.3) 퐷 푛=1 𝑖ℎ 0 푛 0 푛−1 0 1 퐷 푛 퐷 푛−1 퐷 1

퐻 푈 = 푈 푈 ; 푉 = 푈+푉푈 ; 푈 = exp {−𝑖 ( 0) 푡} (1.4) 0 퐷 퐷 0 0 0 ℏ

In this way the transition probability can be assessed at various order out of equations (1.3) and

(1.4). The zeroth order remedy prompts null probability of the transition amongst 풊 and 풇, attributable to the orthogonality property of eigenfunctions. Actually, it can be demonstrated that

3 the first order transition probability is relative to the matrix component:

2 2 |푉푓→𝑖| = |⟨푓|푉|𝑖⟩| (1.5)

This is associated to the single-photon absorption cross-section.

The decision of wavelengths in TPE applications is regularly performed to get the single photon cross-section irrelevant. This permits the calculation of a second order transition probability:

2 1 푡 푡′′ 푊2 = |∫ 푑푡′′ ∫ 푑푡′⟨푓|푈 (푡, 푡′′)푉(푡′′) × 푈 (푡′′, 푡′)푉(푡′)푈 (푡′)|𝑖⟩| (1.6) 𝑖→푓 ℏ4 0 0 0 0 0

It is advantageous to compose transition amplitude as far as the eigenstates of 퐻0 since the transitions are performed between them:

1 퐸 푡 푡′′ 퐸 −퐸 푊2 = |exp {−𝑖( 푓)푡} ∑ ∫ 푑푡′′ ∫ 푑푡′ exp {−𝑖( 푓 푚)푡′′} × 𝑖→푓 ℏ4 ℏ 푚 0 0 ℏ

퐸 −퐸 2 exp {−𝑖( 푓 푚)푡′}⟨푓|푉(푡′′)|푚⟩⟨푚|푉(푡′)|𝑖⟩| (1.7) ℏ

Where 푚 are the intermediate virtual state for which 푉푚𝑖 ≠ 0 and 푉푓푚 ≠ 0

Fig 1.1: Simplified scheme of the energy transition occurring under TPE regime

The perturbing electric field 푉(푡) felt by the fluorophores can be depicted by a constant operator

C and a time dependent element:

푉(푡) = 퐶푒−𝑖휔푡 (1.8)

Where 퐶 is the maximum amplitude, a vector that defines the polarization direction of light.

It is currently time to present the Bohr frequency and work under single intermediate level estimation (a and b are two states):

퐸 − 퐸 휔 = 푎 푏 푎푏 ℎ

Therefore, we can write form the equation (1.7)

2 |퐶 퐶 | 푡 2 푊2 = 푓푚 푚푖 × | 푑푡′{exp(𝑖휔 푡′) − exp (𝑖휔 푡′)}| (1.9) 𝑖→푓 4 2 ∫0 푛𝑖 푛푚 ℏ 휔푚푖

5 This speaks to a transition intervened by stand out virtual level 푚 . Two photons of suited energy need to interface at the same time with the fluorescent color to be absorbed and energize it, as per the rearranged Perrin-Jablonsky plan appeared in Figure 1.1.

Let 퐶푎푏 be the matrix elements of the constant perturbation operator. In the Coulomb gauge, they can be written as:

푒 푝⃑ 퐶 = 휀̂퐴 exp (𝑖푘⃗ . 푟 ) (1.10) 푐 푚 0

Where 퐴0 is the vector potential and 휀 is the polarization vector.

It is presently conceivable to determine the second order transition rate, presenting the harmonic temporal behavior of the perturbation and considering the transition rate for the symmetric middle level between the ground and excited state:

2 2 2휋 2 ′ 푊𝑖→푓 = lim 푊𝑖→푓 = |퐶푓푚퐶푚𝑖| 훿(휔 − 휔) (1.11) 푡→∞ ℏ4휔2

Where 훿 is the Kronecker’s delta function.

Then again, the vector potential can be identified with the radiation intensity by method for the

Poynting Vector (푁⃗⃗ ):

푐 휔2 퐼 = |푁⃗⃗ | = |퐸⃗ × 퐵⃗ | = 퐴2 (1.12) 4휋 2휋푐 0

Substituting equations (1.10) and (1.12) into (1.11) we obtain:

6 2 2 푊𝑖→푓 ∝ 퐼 (1.13)

Equation (1.13) demonstrates the proportionality between the second-order transition probability

(two-photon absorption) and the square of radiation intensity. The lasers utilized for microscopy fill in as a part of the TEM00 mode. The light power dispersion of a laser shaft orthogonal segment can be approximated as a Gaussian-Lorentian distribution having cylindrical shaped symmetry (Xu, 2002):

2 2푃(푡) 2휌 푧 2 퐼(푝, 푧, 푡) = 2 exp [− 2 ] ; 푤(푧) = 푤0√1 + ( ) (1.14) 휋푤 (푧) 푤 (푧) 푧푅

Where 휌 and 푧 are the radial and axial coordinates respectively referred to the origin in the in- focus spot; 푤0 is the laser beam diameter in the focus plane; and 푧푅 is the Rayleigh length.

Since the radiated fluorescence is the relative to the transition probability, equation (1.13) and

(1.14) demonstrate that the intensity of discharge falls along the optical axis as the forth power of the focal distance:

퐹 ∝ 푧−4 (1.15)

This is the principal vital result entirely associated with the ability of TPE optical sectioning.

7 1.3.2 Cross-section and Colocalization

The proportionality element in equation (1.13) can give further understandings about the two-photon cross-section. It is plausible to infer it under dipole approximation:

exp(𝑖. 푘⃗⃗⃗ . 푟 ) = 1 + 푘⃗⃗⃗ . 푟 + ⋯ ≈ 1

푝 1 = [푟 , 퐻 ] 푚 𝑖ℏ 0

8휋3푒4 푊2 ≈ 퐼2|⟨푓|휀̂. 푟 |푚⟩⟨푚|휀̂. 푟 |𝑖⟩|2훿(휔′ − 휔) (1.16) 𝑖→푓 ℏ4휔2푐2

2휋 The first order approximation is good one since 푘 = and 휆 ≫ 푟 . Indeed a fluorophore is 휆 very small compared to the wavelength of light, and the spatial variation of the electric field within the molecule can be ignored [7].

A few constants can be gathered together in the fine structure consistent and the radiation intensity partitioned by the single photon energy to get it as far as the quantity of photons:

퐼 푊2 ≈ 8훼2휋3|⟨푓|휀̂. 푟 |푚⟩⟨푚|휀̂. 푟 |𝑖⟩|2훿(휔′ − 휔)( )2 (1.17) 𝑖→푓 ℏ휔

Besides, the two-photon cross-section can be composed presenting again a total over all the conceivable excitable intermediate levels:

2 3 2 ′ 휎푇푃퐸 ≈ ∑푚 8훼 휋 |⟨푓|휀̂. 푟 |푚⟩⟨푚|휀̂. 푟 |𝑖⟩| 훿(휔 − 휔) (1.18)

On the other hand, the single-photon cross-section is expressed as (Sakurai, 1985):

2 2 ′ 휎푆푃퐸 ≈ 4훼휋 |⟨푓|휀̂. 푟 |𝑖⟩| 휔훿(휔 − 휔) (1.19)

8 In equation (1.18), the square of the matrix element gives the selection rules, the lifetime of the transition, and the delta-function is the ideal absorption spectra profile.

Looking at equations (1.18) and (1.19), we can layout the different selection rules and the same delta function profile at the same time, in the previous case, summed over an intermediate level set. Because of the different selection rules and to the total over the diverse state, the tentatively observed two-photon cross-section, if rescaled in a half-wavelength scale, are very more extensive or possibly equivalent to the single-photon one.

According to equations (1.18) and (1.19), TPE cross-section can also be written (Xu, 2002) as:

휎푇푃퐸 ≈ 휎푓푚휎푚𝑖휏푚 (1.20)

Where single-photon cross-sections for transition to and from an intermediate level 푚 are stresses together with its lifetime.

More extensive cross-sections let more fluorescent colors be all the while excited, so the decision of emission spectra whose overlap is insignificant permits three-dimensional distribution to be at the same time recuperated through TPE strategy.

9 1.4 Experimental Setup

1.4.1 Light Source

Taking into account a femtosecond titanium-sapphire (Ti:Sapphire) laser source the microscope is produced in our lab . This framework delivers pulses with duration on the order of

100 fs with a redundancy rate of 80 MHz. It conveys peak powers of more than 300 kW while the average power can reach above 2.5 W. This laser likewise has a wide tuning range from

690nm to 1040nm permitting specific excitation of a wide assortment of fluorophores. The most liberally utilized fluorophores have single photon excitation wavelength extending from 350nm to 690nm. Considering that for two photon absorption the excitation wavelength can be approximated as double the wavelength utilized for single photon, we are transported to the near infrared region of the spectrum. Another vital normal for the scope of wavelengths gave by our laser source is that the absorption coefficients of most organic examples are minimized in this extent. As it were, the way that there is low absorption and dispersing in this wavelength range represents more prominent infiltration depth in organic specimens.

1.4.2 Waveplate and Polarizer

The light leaving out of our laser source is straightly polarized in the horizontal direction.

Directly after the source there is a half waveplate connected to a revolving mounting that permits us to move the polarization direction by pivoting the quick and moderate axis of the waveplate.

A typical half waveplate presents a phase shift of π between the polarization parts. For directly polarized light this translate into a turn of 2θ, where θ is characterized as the angle framed by the polarization vector and the fast axis of the waveplate. Taking after the half waveplate there is a direct polarizer that parts the beam into two sections with various linear polarization. This sort of

10 polarizer is more reasonable for use with our femtosecond laser since it doesn't have to retain the high intensity light; rather it permits a specific polarization heading to experience parallel to the direction of the beam while all other polarization directions are avoided perpendicular to the beam path. We utilize this mix of waveplate and polarizer to viably control the average power of our laser beam. More often than not, the polarizer is kept in an altered position while the wave plate is pivoted to acquire the sought average power. This can be effectively comprehended from

Malus' law which depicts the intensity of a beam when an impeccable polarizer is set in its way and is given by

2 퐼 = 퐼0 (cos 훼)

Where 퐼0 and 퐼 are the intensities of the laser beam prior and then afterward the polarizer and α is the angle shaped between the polarization vector leaving the waveplate and the axis of the polarizer. In a perfect circumstance the full intensity is transmitted when 훼 = 00 while the light beam is blocked if 훼 = 900 .

11 Ti: Sapphire Laser

Half Waveplate

Polarizer

PMT

(Blue Channel) APD

PMT Polygonal (Red Channel) d Mirror e

c b f Z Objective Galvanometer Lens Mirror

650nm Y Laser Diode Bicell Photodiode PMT X 3D Stage (Green Channel)

Figure 1.2: Schematic of the Two-Photon Laser Scanning Fluorescence Microscope developed in the Biophotonics Laboratory of the Physics Department at UTEP.

12 1.4.3 Scanning Platform

The scanning instrument and its timing circuitry shape a key some portion of our two-photon framework. It gives the quick filtering capacity important to picture dynamic cell procedures and makes it reasonable for live creature thinks about. As of now it is conceivable to image a full frame of 320μm by 320μm at a rate of 30 frames for every second. Moreover, an electronic circuit permits tuning the frame rate to 60 and 120 frames for every second, which can be utilized to image considerably speedier procedures. The field of perspective is diminished in the vertical direction by one portion of its full frame measurement on account of 60 frames for each second and by one fourth for 120 frames for each second. The filtering stage is made out of a galvanometer mounted mirror and a turning polygonal mirror to deliver a unidirectional raster examine design. The galvanometer mounted mirror filters the vertical direction or axis while the horizontal direction or axis is examined by the turning polygonal mirror. The later comprises of a plate with 36 facets similarly disseminated along the edge; its steady rotational rate is 480 cycles for every second.

Every feature relates to a line in the image parallel to the x axis. It is straight forward to compute the line scanning rate for the polygonal mirror, which is altered to 17280 Hz. Table 1.1 outlines the amount of lines per image figured for every frame rate.

Table 1.1: Lines per image corresponding to each frame rate

Frame Rate (Hz) Lines per image

30 576

60 288

120 144

13 The way that the quantity of lines per images are whole number various of the quantity of facets

(36) ensures that the same facet scans the same line on each progressive frame maintaining a strategic distance from vertical looking over impacts. Dissimilar to the polygonal mirror, whose scanning rate is altered, the galvanometer-mounted mirror can be scanned in the vertical dimension at 30 Hz, 60 Hz or 120 Hz, consequently setting the scanning rate of the microscope.

So as to synchronize the galvanometer and polygonal mirrors a bicell photodiode is utilized to check the quantity of facets (lines) and create an electronic sign that is utilized by the hardware to drive the galvanometer at a particular frequency. The photodiode can create the synchronization signal by means for a 650nm laser diode that sparkles on the polygonal scanner in a way that its refection scans over the bicell photodiode.

1.4.4 Dichroic Mirrors and Filters

A dichroic mirror can be utilized to separate laser beams with various wavelengths. They have the property of transmitting a specific scope of wavelengths while reflecting others. As should be obvious in figure 1.2, our microscope has three dichroic mirrors named as a, b and c. The dichroic mirrors in our set up are situated in a way that the angle shaped between the incident light and the mirror is near 45 degrees. Dichroic a transmits every one of the wavelengths above

660nm while reflecting wavelengths underneath it. This permits the excitation light achieves the objective lens while the fluorescence radiated by the specimen is reflected in a perpendicular direction to the underlying beam path. The following two dichroic, b and c, isolate the fluorescent sign, diverting particular range of wavelengths to every detector (PMT). For instance, dichroic b reflects wavelengths beneath 495nm, permitting the blue range of wavelengths to come to the PMT assigned for the blue channel. The transmitted fluorescence proceeds with a straight way until it comes to the dichroic c, where it is isolated once again; wavelengths beneath

14 580nm, containing the green part of the spectrum, are reflected towards the green channel detector while the staying fluorescent sign is transmitted towards the red channel detector. With the reason for narrowing down the range of wavelengths and specifically recognize fluorescence from the specimen comparing to the blue, green and red portion of the visible spectrum, there are a few band pass channels before every detector. They are represented to in figure 1.2 and named as d, e and f.

1.4.5 Objective Lens

In our research facility we count with two Olympus objective lenses contrasting predominantly in the numerical aperture (NA) and the working distance. The NA is a unit less number that describes the range of angles over which the objective lens can acknowledge or transmit light. It is characterized as

푁퐴 = 푛 푠𝑖푛훽

Where n is the refractive index of the objective submersion fluid and β is the half angle of the most extreme cone of light that can enter or leave the lens. The working distance, then again, is characterized as the separation from the front lens component of the goal to the nearest surface of the coverslip when the example is in sharp core interest. When in doubt, the objective working distance diminishes as the amplification and NA both expansions.

Our two objective lenses have a magnification factor of 60X and a NA of 1 and 1.2. .They are both water immersion objective lenses (n=1.33 for pure water).

The qualities of the objective lens assume a vital part in deciding the resolution of an imaging framework. As it was clarified some time recently, optical sectioning is an inherent property of a two-photon system, giving it the capacity of 3D imaging.

15 The lateral resolution of our microscope can be approximated using Abbe’s equation

휆 휆 푅 = 0.6 ≈ 푁퐴 2푁퐴

Where R is the minimum distance between recognizable objects in a picture and is the wavelength of the light utilized for enlightening the sample. This formula has its starting point in the diffraction of light and the limited opening of the optical components; it speaks as far as possible for the determination of an optical system.

1.4.6 Photomultiplier Tube (PMT)

The photomultiplier tubes are one of the imperative segments of our system. They have a photosensitive surface that catches incident photons and creates electronic charges that are detected and intensified. The yield of a PMT is a present relative to the quantity of photons striking the photosensitive surface. The quantum proficiency (QE) of these gadgets, which is the rate of photons that are identified, is a component of the light wavelength and tried and true on the chemical composition of the surface. The qualities for QE may run between 20% and 40%.

PMTs can react to changes in the info photon flux inside a couple of nanoseconds which makes them reasonable for identification and recordings of to a great degree quick occasions. The dynamic scope of these devices is likewise extensively wide yet the electrical yield current precisely mirrors the incident photon flux.

The sign to noise (SN) proportion is another estimation that serves to depict the execution of electronic imaging sensors. On account of PMTs the SN proportion is high on the grounds that the dim current, which emerges in electronic devices without light, is extensively low.

In our framework, each PMT is combined with a Hamamatsu C7950 attachment which changes over the little current high-impedance output of the PMT into a low-impedance voltage output with a transformation element of 0.3 V/μA. Moreover, each C7950 is associated with a +/ - 15

16 and a variable force supply (0 V to +3.6 V) for high voltage alterations. The last is utilized to physically control the increase of the enhancement circuit inside each C7950. The extensive zone of the photocathode or photosensitive surface is another favorable position of PMTs in light of the fact that it permits effective collection of fluorescence. In our set up this fluorescence originates from a point in the example at once and each PMT can deal with a most extreme count tally rate of around 1 MHz.

1.4.7 3D Stage

As clarified some time recently, the mix of the polygonal mirror and mounted galvanometer mirror delivers a two dimensional image at a given z-position. With a specific end goal to make a three dimensional remaking of the sample it is important to get images at various focuses in the axial direction. Our 3D mechanized stage (Shutter Instrument, model MP-285) gives this capacity. It can travel one inch on each of the three axes and gives a low resolution of 0.2

μm/step and a high resolution of 0.04 μm/step. Ordinarily in our lab a pile of images is brought with a partition of 1 μm/step between planes. As far as pace the most extreme worth indicated by the producer for this stage is 2.9 μm/step which adds to a little procurement time.

17 1.5 Advantages of Two-Photon Microscopy

The probability per pulse that a fluorophore will experience excitation by two-photon absorption when it is enlightened by a train of pulses through a focusing lens is given by

2 2 2 훿2푃푎푣푒 푁퐴 푛푎 ∝ 2 ( ) (1.21) 휏푝푓푝 2ℏ푐휆

Where 훿2 is the two-photon cross-section of the fluorophore at wavelength λ, Pave the laser beam average power, NA the numerical aperture of the focusing lens, ℏ is the decreased Plank constant, and c the speed of light [8]. In a normal analysis, the laser normal power and pulse width are set precisely keeping in mind the end goal to maximize signal generation. It is imperative to maintain a strategic distance from immersion (푛푎 ≈ 1) generally the image will lose the optical resolution characterized by the numerical gap of the focusing lens and the excitation wavelength.

Figure 1.3: One Photon Vs Two Photon Fluorescence Imaging [9]

The fluorescence signal delivered in TPM is relative to the probability of two-photon absorption.

Since this event scales with the laser intensity squared, two-photon excited fluorescence can be spatially restricted by focusing the excitation light.

18 The mix of two-photon absorption probability over sections of the enlightening cone is biggest at the focal plane and ceases to exist quickly in the regions in front and behind it. The restriction of excitation and accordingly signal generation in a little volume inside samples is absolutely the key component of TPM. It is shown in Figure 1.3 by contrasting the impact of one-and two- photon absorption on signal generation.

Another preferred standpoint of TPM is the capacity to image thick specimens, notwithstanding when they are very heterogeneous. This is because of the utilization of longer wavelengths (close

IR) in respect to shorter ones (Unmistakable and UV) utilized as a part of ordinary one-photon fluorescence microscopy. Since light dissipating is relative to 휆−4 , TPM photons can achieve further into samples and not be lost. With suitable goals, TPM has been utilized to picture organic occasions up to a few hundred microns inside thick tissues [10]. The high entrance profundity of TPM is further improved in light of the fact that most biological samples show minimal linear absorption somewhere around 700 and 1000 nm.

Figure 1.4: Comparison of Confocal Microscopy and Two-Photon Microscopy

19 Customary light microscopy is a vital device, yet its capacity to determine infinitesimal structures in optically thick examples is constrained on the grounds that the image at the focal plane is obscured by out-of-center noise. The creation of confocal microscopy and two-photon microscopy has begun to address 3D imaging needs. Confocal microscopy is a strategy fundamentally the same as two-photon microscopy. Confocal microscopy accomplishes 3D determination utilizing an arrangement of conjugate apertures, one for enlightenment and one for recognition of the scattered or bright light [11]. These conjugate pinholes, working as spatial channels, guarantee that the microscope will enlighten and distinguish light from the same volume inside the sample.

20 Chapter 2: Grazing behavior of Cafeteria roenbergensis

2.1 Introduction Grazing behavior of free living heterotrophic nanoflagellates (HNF) on microorganisms are vital perspectives managing microbial biology. In most oceanic biological systems, heterotrophic nanoflagellates (HNF) are the most vital bacterivorous life forms [12]. Research center and field investigations of communications between heterotrophic nanoflagellates (HNF) and microorganisms recommend that heterotrophic nanoflagellates (HNF) are the principle microbial consumers of microscopic organisms and can standardize bacterial densities in oceanic biological system [13]. The flagellate Cafeteria roenbergensis, a typical marine bacterivorous flagellate, is a reasonable species for definite perceptions of the grazing behavoir of heterotrophic nanoflagellates (HNF). C.roenbergensis is a eukaryotic living being which has been found in all sea water, particularly in costal water [14]. In any case, C.roenbergensis swims quickly, likewise, the connection speed amongst C.roenbergensis and bacteria and microsphere are additionally fast, along these lines, there are numerous troubles and difficulties for customary imaging systems to see their communication. Two-photon fluorescence microscopy is produced to track quick moving Cafeteria roenbergensis originating before on microorganisms and microsphere. Our exploration gives a technique on concentrate fast interaction between microbial living beings.

2.2 Cafeteria Roenbergensis

Cafeteria roenbergensis was discovered by Danish marine ecologist Tom Fenchel and taxonomist David Patterson in 1988. It is found primarily in coastal waters where there are high concentrations of bacteria on which it grazes. Its voracious appetite plays a significant role in regulating bacteria populations [15]. Tom Fenchel, is credited with having joked about the 21 Chromalveolata’s name: "We found a new species of ciliate during a marine field course in

Rønbjerg and named it Cafeteria roenbergensis because of its voracious and indiscriminate appetite after many dinner discussions in the local cafeteria." —Tom Fenchel [16]. Cafeteria roenbergensis is a small bacterivorous marine flagellate. Bacterivores are free-living organisms, exclusively microscopic, which obtain energy and nutrients primarily or entirely from the consumption of bacteria. Similar to other flagellates it has whip-like organelles called flagella which are used for gathering food. Cafeteria roenbergensis is a slightly flattened, kidney- shaped bicosoecid (small group of unicellular flagellates). Its cell typically measures between 3 and 10 µm and it has a volume of around 20 µm3 [17]. It is colorless and has two unequally sized flagella. Cafeteria is a eukaryotic organism, so it contains the typical organelles such as mitochondria and nuclei [17]. Cafeteria roenbergensis is a suspension feeder, meaning it feeds by filtering suspended bacteria, its primary food source, and other particulate matter from the water [18]. Its two flagella facilitate feeding, locomotion and attachment to substrates. The anterior flagellum is responsible for locomotion and feeding. It propels the cell in a swift spiral movement. During feeding, it beats at about 40 times per second to create a current of water that moves about 100 micrometers/second. This current brings bacteria to its mouthparts. The food is ingested below the base of the flagella, which is referred to as the ventral side [19].

Figure 2.1: Cafeteria roenbergensis

Bacterivorous nanoflagellates, the general group to which C. roenbergensis belongs, make up a significant portion of the oceans’ protozoan communities, as well as those in freshwater, soils and other habitats. They are reported to be the primary consumer of bacteria in many habitats, controlling bacterial populations as they "graze"[20].

Since they are anything but difficult to grow in culture, Cafeteria roenbergensis has been liable to a differing qualities of more definite concentrates, for example, genomic and environmental studies that have uncovered that this species has the most practically reduced DNA amongst eukaryotes [5]. While in society, Cafeteria are nourished Vibrio microscopic organisms. In a test directed by Park and Simpson in 2010, it was found that Cafeteria cells develop best in salinities of 3 ppm to 100 ppm, however can't make due at concentrations any higher [21].

23 2.3 Bacteria

Bacteria constitute a large domain of prokaryotic microorganisms. Typically a few micrometers in length, bacteria have a number of shapes, ranging from spheres to rods and spirals. Bacteria were among the first life forms to appear on Earth, and are present in most of its habitats. It can only be seen with the use of microscope. The Dutch microscopist Antonie van

Leeuwenhoek in 1676 first observed bacteria by using his own designed single-lens microscope

[22]. There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a milliliter of fresh water. There are approximately 5×1030 bacteria on Earth [23], forming a biomass which exceeds that of all plants and animals. The ancestors of modern bacteria were unicellular microorganisms that were the first forms of life to appear on Earth, about 4 billion years ago. For about 3 billion years, most organisms were microscopic, and bacteria and archaea were the dominant forms of life [24].

Figure 2.2: Scanning electron micrograph of Eschericha Coli (E. coli)

In our experiment we used Eschericha Coli (E. coli) as the sample of bacteria. . E. coli was first discovered in 1885 by Theodor Escherich, a German bacteriologist. E. coli has since been

24 commonly used for biological lab experiment and research. E. coli is a facultative (aerobic and anaerobic growth) gram-negative, rod shaped bacteria that can be commonly found in animal feces, lower intestines of mammals, and even on the edge of hot springs. E. coli is a Gram- negative rod-shaped bacteria, which possesses adhesive fimbriae and a cell wall that consists of an outer membrane containing lipopolysaccharides, a periplasmic space with a peptidoglycan layer, and an inner, cytoplasmic membrane. Some strains are piliated and capable of accepting and transferring plasmid to and from other bacteria. Such property enables E. coli under bad/stress conditions to survive. [25] Even though it has extremely simple cell structure, with only one chromosomal DNA and a plasmid, it can perform complicated metabolism to maintain its cell growth and cell division.

2.4 Microsphere

Microspheres (also called latex beads or latex particles) are spherical particles in the colloidal size range that are formed from an amorphous polymer such as polystyrene. The molecular probes, fluospheres beads are manufactured using high-quality, ultraclean polystyrene and are loaded with a variety of proprietary dyes to create intensely fluorescent beads that typically show little or no photo bleaching, even when excited with the intense illumination required for fluorescence microscopy. Different surface modifications are available to facilitate the coupling of various molecules and proteins to the surface of the bead. [26]

Figure 2.3: FluoSpheres® Carboxylate-Modified Microspheres [26]

2.5 Samples Preparation

2.5.1 Cafeteria roenbergensis Preparation

Preparation of Cafeteria roenbergensis for imaging requires evacuation of 15 mL of Cro culture from substantial stock, contained in a 125 mL cup, and situation of the sum inside a tube for a rotator of 20 minutes with a revolution velocity of 2600xg. In the wake of centrifuging is finished the15 mL has all supernatant taken out. At that point resuspension must happen with 1 mL of 25 ppt ofF/2 media. Another rotator cycle is accomplished for 20 minutes at 3000xg pivot speed with this 1 mL resuspended Cro. After the second axis cycle the pellet inside the tube is to be inspected and relying upon size, resuspension is done either with 10-20 μL (10-little pellet,

20-major pellet) of 25 ppt F/2 media. Stain Cro and check utilizing hemocytometer. Dilute to necessary concentration in view of number.

26 2.5.2 Bacteria Preparation

Start by expelling 15 mL of E. coli from principle stock then rotator this sum at 1000xg turn speed for one moment to evacuate any cell garbage or garbage in the media. Next, exchange the main 8.5 mL of supernatant to a perfect 15 mL tube and check the optical thickness with a spectrophotometer. Dilute the microbes to an optical density (OD) of 1.5 with no less than 800

μL of total bacteria volume. Once that is done, play out a 1:200 dilution of SYBR gold stain utilizing 25 ppt F/2 media or 40% ethanol getting a last 400 μL volume. At that point blend both the 400 μL of 1.5 OD E. Coli with the 400 μL 1:200 diluted SYBR gold stain to get a final concentration of 0.75 OD of E. Coli and 1:400 SYBR gold stain. Next, let the solution sit for 30 minutes in the dark. While the mixture is sitting in the dark, take a different tube and dilute the

1.5 OD E. coli to 0.75 utilizing 25 ppt F/2 media achieving a final volume of 800 μL. At that point take this 0.75 OD E. coli and let it sit for the remaining time with the gold recolored E. coli. After time for resting oblivious has slipped by take both specimens and rotator them at

2000xg for a term of ten minutes. Expel both specimens from the rotator then take out 700 μL of supernatant from the two examples. After expulsion of supernatant include 700 μL of 25 ppt F/2 media and vortex from that point. Repeat centrifuge at 2000xg for ten minutes, evacuation of same volume of supernatant, expansion of 25 ppt F/2 media of same volume and vortexing of both specimens. Presently, read the OD of the unstained specimen and taking into account the

OD centrifuge down the recolored sample for five minutes at a rotation velocity of 2000xg and resuspend for a final 1.2 OD reading.

27 2.5.3 Microsphere preparation

Our laboratory purchased the specific microsphere from the manufactured company. The label of the beads was red. The excitation and emission wavelength of the beads are 580nm and 605nm respectively. The diameter of the microsphere we chose to do experiment was 1.0 µm. the coupling surface of the beads was Carboxylate. Carboxylate-modified FluoSpheres beads have a high density of pendent carboxylic acids on their surface, making them suitable for covalent coupling of proteins and other amine-containing biomolecules using water-soluble carbodiimide reagents such as EDAC. It also contained 2% of solid.

2.6 Two-photon Fluorescence Microscopy Imaging

2.6.1 Cafeteria roenbergensis

Before we put Cafeteria roenbergensis sample on a slide glass, we used tape on a slide glass to make a gap between the cover glass and that slide glass. After dropped 2 μl sample on the slide glass, we secured that with cover glass. At that point we putted the slide under the two-photon microscopy and began recording images.

Figure 2.4: Cafeteria roenbergensis in NADH Autofluorescence

Figure 2.4 shows the NADH autoflurescence signal from C.roenbergensis in the blue channel, the excitation wavelength was 710nm, the laser power was about 200mW on the sample and the voltage of PMT was 3.36 V. We change the image color from blue to red to see better in our eyes.

Figure 2.5: Cafeteria roenbergensis in NADH Autofluorescence (after changing the color)

In figure 2.4 and 2.5, we can see only one frame captured at time 0.03s. We took videos for fast moving Cafeteria roenbergensis and each time we took more than 600 frames. In the video, we are able to see the fast moving C. roenbergensis and their interaction with E. coli bacteria.

2.6.2 Bacteria Imaging

To get image bacteria, same method were utilized as Cro imaging. To make little gap between slide glass and cover glass, we utilized tape on slide glass. At that point we dropped 2μl E.coli sample on slide glass and secured it with cover glass. Figures 2.6 to 2.8 demonstrate the Green signal from bacteria with recoloring SYBR Gold stain from the Green channel, the excitation wavelength was 710nm, the laser power was around 200mW on the specimen and the voltage of

PMT was 1.6 V.

30 Bacteria signal was stronger than NADH autoflurescence signal from C.roenbergensis so that is the reason, PMT pick up for Green Channel was lower than PMT pick up for Blue channel. In the picture, now and then we can see bacteria makes little cluster, else they are extremely uniform in the entire edge.

Figure 2.6: Bacteria (OD 1.2) signal with stained by SYBR Gold Stain

Figure 2.7: Bacteria (OD 0.6) signal with Figure 2.8: Bacteria (OD 0.3) signal with Stained by SYBR Gold Stain Stained by SYBR Gold Stain

31 2.6.3 Carboxylate-modified FluoSpheres beads Imaging

To get image microsphere, same procedure were used as Cro and bacteria imaging. To make little gap between slide glass and cover glass, we used tape on slide glass. Then we dropped 2μl beads sample on slide glass and covered it with cover glass. Figure 2.9 shows the Red signal from microspheres from the Red channel, the excitation wavelength was 710nm, the laser power was about 200mW on the sample and the voltage of PMT was 1.5 V. To maintain the color of the prey similar, we change the microsphere color red to green. Figure 2.10 shows the microspheres after changing the color.

Figure 2.9: Carboxylate-modified Figure 2.10: Carboxylate-modified FluoSpheres beads in Red Channel FluoSpheres beads in Red Channel (After Change the color)

2.6.4 Imaging for Interaction between Cafeteria roenbergensis and Bacteria

To get the image interaction in middle surface, we dropped 1.5μL C.roenbergensis sample on the slide glass and with for a minute or two to stable. Then we injected 1.5μL E.coli sample into the

32 Cafeteria roenbergensis sample and covered the slide glass with cover glass. Then we putted the slide under our two- photon microscopy and started recording their interaction. Before that we started recording time from when we mixed them up. We used to get our sample ready for imaging around 2 min after mixing up. The interaction process between C. roenbergensis and bacteria is shown in figure 2.11, the laser power was about 200mW on the sample and the voltage of PMT in green channel was 1.6V and blue channel was 3.36 V. Here we merged blue to red color to see better in our eyes.

33 (a) (b)

Figure 2.11: (a) interaction between Cro and Bacteria in 2 min after mixing up; (b) interaction between Cro and Bacteria in 20 min after mixing up; (c) interaction between Cro and Bacteria in 40 min after mixing up; (d) interaction between Cro and Bacteria in 60 min after mixing up

To get the image interaction in top cover surface, we dropped 1.5μL C. roenbergensis sample on the cover glass and with for a minute or two to stable. Then we injected 1.5μL E. coli sample 34 into the cro sample and flipped the cover glass on the slide glass. Then we putted the slide under our two- photon microscopy and started recording their interaction. Before that we started recording time from when we mixed them up. We used to get our sample ready for imaging around 2 min after mixing up. The interaction process between C. roenbergensis and bacteria is shown in figure 2.12, the laser power was about 200mW on the sample and the voltage of PMT in green channel was 1.6V and blue channel was 3.36 V. Here we merged blue to red color to see better in our eyes.

(a) (b)

35 (c) (d)

Figure 2.12: (a) interaction between Cro and Bacteria in 2 min after mixing up; (b) interaction between Cro and Bacteria in 20 min after mixing up; (c) interaction between Cro and Bacteria in 40 min after mixing up; (d) interaction between Cro and Bacteria in 60 min after mixing up

We can see in the frames that number of overlapping particles (C. roenbergensis and bacteria together) is increasing with time .This specifies that C. roenbergensis are eating bacteria and we are losing bacteria with time. We can see in our frame that overlapping particles look like little yellowish and C. roenbergensis looks red before eating bacteria. That’s how we can confirm that

C. roenbergensis are eating bacteria. In figure 2.11 (a) - (d) and 2.12 (a) - (d), all of the frames have been take from different time duration’s video. We used to take around 35s video in different times after mixing C. roenbergensis and Bacteria.

36 2.6.5 Imaging for Interaction between Cafeteria roenbergensis and Microsphere

To get the image interaction in middle surface, we follow the similar procedure that we did for the interaction of C. roenbergensis and bacteria .we dropped 1.5μL C.roenbergensis sample on the slide glass and with for a minute or two to stable. Then we injected 1.5μL microsphere sample into the Cafeteria roenbergensis sample and covered the slide glass with cover glass. The interaction process between C. roenbergensis and microsphere is shown in figure 2.13, the laser power was about 200mW on the sample and the voltage of PMT in red channel was 1.5V and green channel was 1.9 V. To maintain the color of predator and prey, we change the color of C. roenbergensis from green to red and the color of microsphere red to green.

(a) (b)

37 (c) (d)

Figure 2.13: (a) interaction between Cro and Microsphere in 2 min after mixing up; (b) interaction between Cro and Microsphere in 10 min after mixing up; (c) interaction between Cro and Microsphere in 16 min after mixing up; (d) interaction between Cro and Microsphere in 30 min after mixing up

To get the image interaction in top cover surface, we dropped 1.5μL C. roenbergensis sample on the cover glass and with for a minute or two to stable. Then we injected 1.5μL microsphere sample into the Cafeteria roenbergensis sample and flipped the cover glass on the slide glass.

Then we putted the slide under our two- photon microscopy and started recording their interaction. Before that we started recording time from when we mixed them up. We used to get our sample ready for imaging around 2 min after mixing up. The interaction process between C. roenbergensis and microsphere is shown in figure 2.14, the laser power was about 200mW on the sample and the voltage of PMT in red channel was 1.5V and green channel was 1.9 V. To maintain the color of predator and prey, we change the color of C. roenbergensis from green to red and the color of microsphere red to green.

38 (a) (b)

Figure 2.14: (a) interaction between Cro and Microsphere in 2 min after mixing up; (b) interaction between Cro and Microsphere in 10 min after mixing up; (c) interaction between Cro and Microsphere in 16 min after mixing up; (d) interaction between Cro and Microsphere in 30 min after mixing up

We can see in the frames the number of overlapping particles (C. roenbergensis and microsphere together) are very random with time increasing. This indicate that the C. roenbergensis try to eat

39 microsphere but cannot digest. So it slit out the microsphere. That’s why the number of overlapping particles was increased one time and decreased next. In figure 2.13 (a) - (d) and 2.14

(a) - (d), all of the frames have been take from different time duration’s video. We used to take around 12s video in different times after mixing C. roenbergensis and Microsphere.

2.7 Statistical Analysis of Interactions

We utilized a few programming for the statistical analysis of interaction amongst C. roenbergensis and bacteria and amongst C. roenbergensis and microsphere. ImageJ and Minitab were for the most part utilized as a part of this case. In our TPFM images, there were foundation noise and to see the interaction we needed to minimize that noise from the recordings. Image preparing with regular noise diminishment channels would dispose of a portion of the critical information in our recordings. To conquer this we built up another strategy [27] to analysis our recordings in ImageJ. Likewise we needed to do diverse examination for microsphere and microscopic organisms and C. roenbergensis, on the grounds that we had solid signal in green channel from bacteria and frail signal in blue channel from C. roenbergensis autofluorescence and less solid signal in red channel from microsphere. That is the reason, our blue and red channel recordings were noisy. We have done following algorithms for our statistical analysis:

For interaction between C. roenbergensis and bacteria:

Run (“Color”, “split channels”);

Run (“Blue channel”, “Noise”, “Despeckle”);

Run (“Math “, “Subtract = 40” );

Run (“Find edges”);

Run (“Gaussian Blur = 1.5”);

40 Run (“Green channel”);

Run (“Math”, “Subtract = 25”);

Run (“Find edges”);

Run (“Gaussian Blur =1.0);

Run (“Image Calculator”, “Green”, “AND”, “Blue”);

Run (“Convert to Mask”);

Run (“Analyze Particles”);

Run (“Blue channel”, “Convert to Mask”);

Run (“Analyze Particles”);

Run (“Green channel”, “Convert to Mask”);

Run (“Analyze Particles”);

For interaction between C. roenbergensis and microsphere:

Run (“Color”, “split channels”);

Run (“Red channel”, “Noise”, “Despeckle”);

Run (“Math “, “Subtract = 35” );

Run (“Find edges”);

Run (“Gaussian Blur = 1.0”);

Run (“Green channel”);

Run (“Math”, “Subtract = 35”);

Run (“Find edges”);

Run (“Gaussian Blur =1.5);

Run (“Image Calculator”, “Green”, “AND”, “Red”);

Run (“Convert to Mask”);

41 Run (“Analyze Particles”);

Run (“Red channel”, “Convert to Mask”);

Run (“Analyze Particles”);

Run (“Green channel”, “Convert to Mask”);

Run (“Analyze Particles”);

From these algorithm we were able to find the numerical data for all interactions and input to tables.

Table 2.1: Counting number of particles (Interaction of Cro and bacteria OD 1.2) by using ImageJ software

Time Frame: 1-200 Frame: 201-400 Frame: 401-600 Bacteria (Min) Perce Perce (OD 1.2) nt of Over nt of Percent Over. Over Cro Over Parti Cro Over Cro of Over Particles Particles Partic cles Particl Particles les es 2 560 1198 46.74 481 1061 45.33 470 1023 45.94 36851 4 511 1056 48.39 452 951 47.52 461 948 48.62 30158 6 520 842 61.75 510 835 61.07 500 820 60.97 27956 8 340 531 64.03 362 562 64.41 355 558 63.62 24946 10 457 685 66.71 452 684 66.08 448 680 65.88 24998 12 862 1265 68.14 882 1301 67.79 873 1285 67.93 25698 14 578 826 69.97 558 798 69.92 546 780 70 21498 16 751 1065 70.51 701 984 71.23 642 899 71.41 21645 18 566 745 75.97 579 768 75.39 574 756 75.92 20646 20 875 1088 80.42 860 1048 82.06 808 998 80.96 18464 25 828 985 84.06 835 990 84.34 821 973 84.37 19846 30 1315 1563 84.13 1258 1485 84.71 1205 1427 84.44 18473 35 863 1016 84.94 846 995 85.02 798 951 83.91 17965 40 1008 1186 85.01 892 1058 84.31 859 1017 84.46 14479 50 1262 1496 84.35 1155 1375 84.0 1082 1289 83.94 13568 60 1058 1254 84.37 995 1185 83.96 963 1134 84.92 12859

Table 2.2: Counting number of particles (Interaction of Cro and bacteria OD 0.6) by using ImageJ software

Time Frame: 1-200 Frame: 201-400 Frame: 401-600 Bacteria

(Min) Perce Perce (OD 0.6) nt of Over nt of Percent Over. Over Cro Over Parti Cro Over Cro of Over Particles Particles Partic cles Particl Particles les es 2 266 895 29.72 265 884 29.97 262 876 29.90 19874 4 226 642 35.20 220 635 34.64 210 601 34.94 17956 6 166 436 38.07 159 431 36.89 160 435 36.78 18424 8 368 798 46.11 354 782 45.26 347 772 44.94 15897 10 212 452 46.90 210 448 46.87 208 441 47.16 16014 12 372 743 50.06 371 750 49.46 361 738 48.91 14998 14 229 450 50.88 222 436 50.91 214 421 50.83 13652 16 342 658 51.97 342 655 52.21 335 643 52.09 14895 18 290 501 57.84 276 493 55.98 275 488 56.35 13654 20 197 319 61.75 185 305 60.65 183 301 60.79 11448 25 418 654 63.91 401 647 61.97 393 635 61.88 12014 30 305 465 65.59 295 452 65.26 296 455 65.05 11542 35 341 526 64.82 332 511 64.97 324 506 64.03 11654 40 196 298 65.77 189 288 65.61 181 279 64.87 9847 50 339 515 65.82 325 501 64.84 316 487 64.88 9984 60 215 326 65.95 200 308 64.93 197 304 64.80 9145

Table 2.3: Counting number of particles (Interaction of Cro and bacteria OD 0.3) by using ImageJ software

Time Frame: 1-200 Frame: 201-400 Frame: 401-600 Bacteria

(Min) Perce Perce (OD 0.3) nt of Over nt of Percent Over. Over Cro Over Parti Cro Over Cro of Over Particles Particles Partic cles Particl Particles les es 2 144 689 20.89 141 672 20.98 131 658 19.90 5894 4 133 534 24.90 128 513 24.95 119 498 23.89 6049 6 124 365 33.97 112 342 32.74 108 339 31.85 5949 8 173 482 35.89 171 489 34.96 148 437 33.86 5246 10 127 336 37.79 112 305 36.72 102 286 35.66 4982 12 211 542 38.92 204 539 37.84 191 517 36.94 4266 14 272 648 41.97 239 599 39.89 227 583 38.93 4668 16 238 530 44.90 231 525 44 224 511 43.83 3859 18 218 456 47.80 190 406 46.79 190 415 45.78 3498 20 148 285 51.92 145 286 50.69 138 276 50 3255 25 336 635 52.91 334 643 51.94 326 641 50.85 2897 30 166 315 52.69 148 286 51.74 148 291 50.85 2449 35 154 298 51.67 143 276 51.81 135 265 50.94 1989 40 377 726 51.92 357 688 51.88 330 649 50.84 1459 50 183 346 52.89 179 345 51.88 168 324 51.85 1299 60 190 364 52.19 190 367 51.77 157 309 50.80 1068

After getting numerical data from ImageJ software, we used Minitab for our statistical analysis.

By using Minitab we were able to plot percentage of overlapping particles (Cro +

Bacteria) vs time.

44 Percentages (%) VS Time (Min)

90 Y = aX + c Y = bX+d Total number of Cro = 48419 b1 =1.4 Total number of Bacteria = 350050 80

70 Total number of Cro = 25586

)

( Total number of Bacteria = 220998

s 60 b2 = 1.2

g Total number of Cro = 21831 a a1 = 3.3

n 50

e Total number of Bacteria = 58827

e b3 =1.1 P 40 a2 = 2.4

a3 = 2.5 20

0 10 20 30 40 50 60 Time (Min)

Figure 2.15: Plot of percentage of overlapping particles (between C. roenbergensis and bacteria) with time (In middle surface)

To ensure our perceptions we have done this examination a few times in various optical density of bacteria. For each slide, we took recordings in various time length. We have watched that number of overlapping particles expanded with time. In figure 2.15, we can see overlapping particles are expanding with time, however after certain time they are making saturation curve.

Overlapping particles are saturated around 25 min furthermore we can see little bump in around

10 min. To be affirm about this bump we have done a few analysis and each time we are getting same bump around 10 min.

Then we compare the current data with previous data done by other graduate researcher [27] and plot them in same graph to see how much it well-matched to that data.

45 Percentages (%) Vs Time (Min)

)

(

s 60

n 50

P 40

0 10 20 30 40 50 60 70 Time (Min)

Figure 2.16: Plot of percentage of overlapping particles (between C. roenbergensis and bacteria) with time (for 3 different experiment)

Figure 2.16 indicates that the current experimental data is compatible to the previous data taken from different time. Each time overlapping particles are increasing with time, but after certain time they are making saturation curve. Overlapping Particles are saturated around 25 min and also we can see little bump in around 10 min.

We also used the same algorethm to count the percentage of overlapping particles in top coverglass. Then we plot the graph in minitab software how it is increased with time.

46 Percentages VS Time

90 Y = aX+c Y = bX+d Total number of Cro = 433188

b1 = 1.6 Total number of Bacteria = 3939933 80

70 Total number of Cro = 339517 Total number of Bacteria = 2700315

) a1 = 3.27

% 60 b2 = 1.4

(

a 50 Total number of Cro = 387089

c a2 = 2.7 Total number of Bacteria = 836891 r 40 b3 = 1.1

30 a3 = 1.6

10 0 10 20 30 40 50 60 70 Time (Min)

Figure 2.17: Plot of percentage of overlapping particles (between C. roenbergensis and bacteria) with time (In top surface)

In fiugre 2.17 we see the increment of the percentage of overlapping particle in top surface of the slide is quite similar to middle surface of the slide. After 25 min it also get saturated and get first bump in the curve after 10 min . then we plot the data we collect from middle surface and the top in same graph.

47 Percentages VS Time

)

% 60

(

a 50

c r 40

10 0 10 20 30 40 50 60 70 Time (Min)

Figure 2.18: Plot of percentage of overlapping particles (between C. roenbergensis and bacteria) with time (In both surfaces)

In figure 2.18 the curves (1,3,5 from the top) are the curves for the interaction three different optical density of bacteria with cro from top coverglass. And the curves (2,4,6 from the top) are the curves from the middle surface of the slide glass. The figure indicates that the percentages of overlapping particles in top surface is higher than that in middle surface for a specific time. From that we can predict that the number of food can effect the eating behavoir of the predator . the speed of consuming prey get faster if the number of prey get higher. And that’s why the percentage of overlapping particles is higher in the top surface than that in middle surface.

We can see in figures 2.15 to 2.18, slopes for first 4 points for different optical density of bacteria are around identical. Also we calculated slope from points on 15min to 25 min and we got around same slope for different optical density of bacteria. So from the all bi-phase curves 48 we can say that the first fast increasing phase (0-9 minutes) where the slope is higher than 2 are most likely to be the capture and ingestion stages, digestion and egestion stages are in the first saturation phase (9-15 minutes), and the second increasing phase (15 -25 minutes) where slope is lower than 2, is another ingestion phase.

Then we apply another algorithm to find the data for interaction between C. roenbergensis and microspheres from middle surface and top surface.

Table 2.4: Counting number of particles (Interaction of Cro and microsphere) by using ImageJ software

Time Top Surface: Frames(1-300) Middle Surface: Frames(1-300)

(Min) Percent Percent Over. Microsphere Over. Microsphere Cro of Over Cro of Over Particles Particles Particles Particles 2 7213 52388 13.76 38115 165 1464 11.27 4389 4 8113 52456 15.46 44025 229 2046 11.19 3737 6 7741 48223 16.05 50658 154 2665 5.77 2545 8 5610 38574 14.54 48451 157 2511 6.25 1680 10 8925 52068 17.14 56119 427 2564 16.65 2490 12 13742 60180 22.83 80847 103 1781 5.78 1799 14 14532 59064 24.60 83757 52 1456 3.57 2446 16 14478 59363 24.38 85573 94 979 9.60 2128 18 9720 48358 20.10 71771 265 2372 11.17 1978 20 13461 62558 21.51 78356 193 1535 12.57 2322 25 9919 44474 22.30 70873 57 746 7.64 1452 30 9078 46305 19.60 74451 298 1945 15.32 1957

After the counting , we plot the percentage of overlapping particle for both top surface and middle surface in a same graph using Minitab software.

49 Percentages Vs Time

30 Total number of Cro = 624011

) Total number of Microsphere = 782996

(

e 20

P 10

Total number of Cro = 22064 Total number of Microsphere = 28923 0

0 5 10 15 20 25 30 35 Time (Min)

Figure 2.19: Plot of percentage of overlapping particles (between C. roenbergensis and Microsphere) with time (In both surfaces)

In figure 2.19, we can see that the percentages of overlapping particle is random with the increase of time for both cases. When cro try to capture microsphere the percentage of overlapping particle get incrase but cro can not digest the food so they egest the microsphere.

That’s why at certain time the percentage get decrease. Then cro again try to capture the microsphere and failed to digest again. By doing so, the caputuring and egesting happened continuously with time increase.

50 2.8 Conclusion

By utilizing two-photon fluorescence microscopy, we are capable observe the grazing behavior of moving Cafeteria roenbergensis. This high resolution video magnifying instrument permits us to observe the interaction between C. roenbergensis and bacteria furthermore between C. roenbergensis and microsphere in both middle surface and top surface.

For the interaction amongst C.roenbergensis and bacteria ,the C.roenbergensis was imaged by exciting and distinguishing the NADH autofluorescence signal and the bacteria was recognized by recoloring with SYBR gold stain, both C.roenbergensis and bacteria able to image with

710nm excitation wavelength and laser power 200 mW. We utilized E. coli microscopic organisms as a model bacteria for our investigation, since it's most dependable microbes to develop in research facility.

What's more, for the interaction amongst C. roenbergensis and microsphere, the C. roenbergensis was identified by recoloring with SYBR gold stain and the microsphere was distinguished by recoloring with Red stain, both C. roenbergensis and microsphere were likewise able to image with 710nm excitation wavelength and laser power 200 mW. We utilized

Carboxylate-adjusted FluoSpheres globules as a model microsphere for our examination

To be affirm about the grazing behavior of C. roenbergensis, we did our trial a few times and we get around same plot for the greater part of our investigation. We do have little noise in our two- photon microscopy, to expel this commotion we built up new algothrim which minimized information misfortune from our recordings. This strategy is valuable when the size of the obejct is in the same order of background noise.

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54 Vita

Faisal Abedin was born and raised in Bangladesh. In 2012, he obtained a B.S. in Physics from

University of Dhaka, Dhaka, Bangladesh. Then he came at University of Texas at El Paso to pursue higher study at USA, led him to pursue a M.S. in Physics at the University of Texas at El

Paso, Texas, USA. While completing the courses of the master's program, Faisal Abedin got involved in research activities at the Biophotonics Laboratory of the Physics Department. There he worked in interdisciplinary projects mainly focused in the applications of a Two-Photon Laser

Scanning Fluorescence Microscope to study interesting biological problems. He recently got accepted into the Physics Program for doctoral students at the University of Central Florida where he expects to continue performing research in the field of physics and optics.