Theses supervised by Prof. Dr. Alper Kiraz
16 theses · Koç University
Deep learning enhanced microscopy by training a convolutional neural network and a generative adversarial network with data collected using a line scanning confocal microscope
Eliminating out-of-focus background, confocal microscopy provides images with a higher resolution and signal-to-noise ratio in comparison with wide-field illumination microscopy. Besides the advances in confocal microscopy, new techniques like light-sheet microscopy, structured illumination microscopy, line-scanning confocal microscopy, or deep learning enhanced microscopy draw attention due to their advantages, including higher image acquisition speeds, better contrast, and resolution, and are more cost-effective. In this work, we first demonstrate the operation of a line-scanning confocal microscope developed using a digital light projector (DLP) and CMOS camera with a rolling shutter. In this method, a series of illumination lines are projected on a sample by a DLP with a focusing objective (50X, NA=0.55). The reflected light is imaged with a rolling shutter CMOS camera. The line-scanning confocal imaging is achieved by overlapping the illumination lines and the rolling shutter of the sensor. Significant improvements in image contrast and minimum feature size are obtained using this technique. Then by using this method, a dataset including 500 pairs of images obtained by imaging fibers of a paper tissue was prepared. This dataset is employed for training a Convolutional Neural Network (CNN) and a Generative Adversarial Network (GAN) for deep learning enhanced microscopy. For this, a data set containing 500 pairs of simple and line scanning mode images of this dataset is utilized. 95% of this amount is used for the training of the network, while the remaining 5% is used for the testing. Significant contrast improvement is obtained in the output of the CNN and GAN, comparable to those obtained with the ground-truth images.
Aerogel-based 3D printed optofluidic waveguided multi-channel photocatalytic microreactors
This thesis investigates the use of additive 3D printing technology for creating micro photoreactors within aerogels. The study focuses on developing a low-cost 3D printing system tailored for aerogels and optimizing an ink formulation suitable for 3D printing processes. The research begins with customizing an off-the-shelf 3D printer to fabricate a low-cost system specifically for aerogel-based structures. This customized printer integrates a 3D-printed syringe pump system and custom software for accurate calibration and printing of sub-millimeter lines. The ink formulation designed for 3D printing comprises a silica aerogel starter solution with additives and titanium dioxide nanoparticles. Post-printing steps including gelation and solvent exchange refine the aerogel's structures enhancing stability for micro photoreactors designed to interact with water. Rheological characterization studies inform ink optimization ensuring durability and usability over time. The optimized 3D printing system and ink formulations demonstrate advancements in printing aerogels with additives where applications promise impactful solutions and future advancements in materials engineering and environmental sciences.
Reconfigurable lightsheet microscopy using a superpixel-based digital micromirror device
In this thesis, a cost-effective and highly modular light sheet fluorescence microscope (LSFM) setup that can accommodate to different sample sizes is demonstrated. A digital micromirror device (DMD) is used in a Superpixel configuration conjugated with the sheet forming optics allowing independent control of both the amplitude and phase in creating the lightsheet. Arbitrary beam shaping enables a multitude of performance improvements such as chromatic correction, sheet optimization and tailoring, structured illumination microscopy (SIM) methods, fast mirrorless scanning and high adaptability to different imaging needs. Relative simplicity and lower cost aspects of the setup are also an advantage for research groups without optics experts with LSFM imaging needs. The Superpixel method extends the normally amplitude-only modulation capabilities of the DMD into arbitrary phase and amplitude modulation and is integrated into a conventional open-top setup. Non-diffracting Bessel and Airy beams requiring specific phase profiles are demonstrated with a scanning range of up to 180 um without additional scan optics along with the possibility of complex field control by using Zernike phase profiles. Simple switching between static and dynamic lightsheet modes ensure that modifications to the setup can be easily reverted without needing realignment. The demonstrated setup and design steps can be adjusted to demands of the sample, allow high-resolution imaging with fast dynamic control and can be combined with other techniques depending on the required performance metrics. In the spirit of the OpenSPIM project, the implementation and the material for constructing the setup along with used control software will be openly available online and free to use under the CC-BY-SA license.
Süperhidrofobik yüzeylerde duran mikrodamlacıklarda yüksek Q-faktör ölçümü ve sürdürülebilir Raman lazeri
Optical microcavities are platforms in which the light-matter interactions are investigated and novel photonic and optoelectronic device concepts are introduced and tested. From the basic scientific point of view, they are important for quantum and nonlinear optical studies, whereas from a technological perspective, future computers and telecommunication components can benefit from microcavity based devices. The results of this thesis are presented in two parts; i) characterizing liquid microdroplets as optical microcavities and ii) sustainable microdroplet Raman laser, both with the help of recently demonstrated reversible photothermal tuning of a droplet.High Quality factors (Q-factors) are essential for studies in especially quantum and nonlinear optics. Previous attempts of Q-factor measurements were limited by the resolution (0.07 nm) of the monochromator present in the laboratory which corresponds to a Q-factor of about 8100 around 650 nm. In the first part of the thesis, we made use of reversible photothermal tuning to overcome this limitation. When heated, certain amount of water evaporates, thus changes the equilibrium size of the microdroplet. In order to measure the Q-factor, we continuously change the power of the IR laser in very small steps, which enables tuning of the size of the microdroplet accordingly. WGMs of a dye-doped microdroplet are simultaneously excited by a probe laser and dye emission is monitored with an avalanche photo diode (APD). A peak is observed in the APD signal when the resonance condition is satisfied. Q-factors up to 105 are measured with this method. We achieved a resolution around 0.004 nm, which implies that we have the ability to tune the droplet?s radius with a precision of ?1Å.In the second part of the thesis, we demonstrate sustained Raman lasing from NaCl-water microdroplets with time periods as long as 25 minutes. In principle, there is no upper limit and all the experiments were terminated after sufficient amount of data were gathered. The technique we present relies on two lasers: a cw IR laser for photothermal tuning, and a pulsed green laser for size stabilization and excitation of the Raman band. It was previously shown that resonant heating of a droplet can lead to size stabilization due to size-dependent absorption. In this study, we combine this effect with photothermal tuning to sustain Raman lasing in long time scales. By photothermal tuning, the size of the droplet is tuned to a specific size in resonance with the green laser. From then on, resonant heating of the green laser leads to size stabilization, which eliminates the previously observed on-off behavior. However, long term fluctuation in the chamber still needs to be suppressed and it is compensated by slight manual adjustments of IR laser power after Raman lasing is detected.
Yüzey destekli sıvı mikrodamlacıklarda ışımayan erk dönüşümlü optoakışkan mikrolazerler
Optofluidics is a recently established research field combining the microfluidic chip technology with optics. It allows for a broad range of applications aiming to develop new photonic devices whose functions are defined by the liquid or to build new integrated photonic devices in which light is used for excitation, analysis, or manipulation of the fluid and the particles in the fluid. In particular, optofluidic lasers hold great promise for developing biological sensors thanks to the high sensitivity of stimulated emission to small perturbations in the laser cavity and gain medium. In an optofluidic dye laser, the gain medium can be directly excited by tuning the pump laser into dye absorption band or non-radiative Förster resonance energy transfer (FRET) can be employed exciting donor molecules which transfer their energy efficiently to acceptor molecules. Optofluidic dye lasers based on FRET enable the extension of the lasing wavelength range and provide new ways of biological sensing thanks to the high sensitivity of FRET efficiency to the donor-acceptor pair separation. In this thesis, the behavior of energy-transfer dye lasers using a mixture of saturable absorber and laser dye as an active medium is investigated theoretically by using rate-equation model. The rate equation model describing the working principle of such dye laser consists of coupled differential equations for the population of energy levels of the dye and the absorber and photon density inside the cavity. Here, we study the temporal characteristics and power of the output lasing pulses as a function of the fluence of the pulsed excitation beam. In the second part, we demonstrate optofluidic microlasers using highly efficient non-radiative FRET for pumping of the gain medium placed within glycerol/water microdroplets situated on a superhydrophobic surface. These droplets can serve as natural microcavities hosting whispering gallery modes (WGMs). Droplets are doped with the FRET donor-acceptor dye pair Rhodamine 6G-Rhodamine 700. FRET lasing is characterized for different acceptor and donor concentrations, and low threshold pump fluences of acceptor lasing are demonstrated. We also verify the nature of the energy transfer (non-radiative vs. radiative) for the range of parameters studied in the experiments.
Düzlemsel genişlemeli akış içinde hidrodinamik tuzaklanmış mikrodamlaların ışıması ve çözülmesi
Dissolution of one phase into an immiscible phase is of great importance to food and drug industry. Dissolution process is mainly affected by diffusion coefficient, solubility, density and viscosity of the dissolving material in the immiscible phase. This thesis reports dye lasing in hydrodynamically manipulated microdroplets and proposes the hydrodynamic trap as a new method to observe dissolution of microdroplets. In the first part of this thesis, utilizing previously available hydrodynamic trapping method, we trap and manipulate the position of a dye doped liquid microdroplets in a microfluidic chip. By manipulating the position of the trapped droplets along the outlet channels of the microfluidic chip, we show lasing can be achieved for different positions of the droplet. When a trapped microdroplet is excited at a fixed position, lasing modes in the consecutive spectra of the droplet show a blue shift for all modes. The shift in modes is an indicator of the dissolution. Therefore, the next part focuses on dissolution of liquid microdroplets. Within a microfluidic chip, a trapped liquid microdroplet is exposed to planar extensional flow which increases the rate of the mass transfer out of the microdroplet. The static dissolution, that is diffusion, of droplets is modelled by Epstein and Plesset. However, the hydrodynamic trap induces flow around a trapped particle, therefore besides diffusion, the effect of convection should also be considered. The change in particle's radius as a function of time in the presence flow is numerically investigated by Zhang et al. To fit the experimental data we use the model provided by Zhang. The liquids used in the microdroplet dissolution experiments were n-octanol, n-decanol, undecanol and benzyl benzoate. Benzyl Benzoate and n-octanol showed good agreement with the Zhang model. In addition, experiments revealed that concentration of surfactant is an important parameter affecting dissolution rate which requires further investigation.
Mikrodamla lazerlerin olası opto-akışkan biyoalgılama uygulamaları
Dye-doped microdroplets can host lasing whispering gallery modes (WGMs). The sensitivity of laser emission wavelength, lasing threshold, lasing differential efficiency or even lasing beam spatial profile to the physical properties of microdroplets such as size, shape, relative refractive index of the droplet with respect to its surrounding medium, cavity Q-factor, or molecular binding/unbinding inside their constructive material can be used in sensing applications. To this end the necessity of control and manipulation of lasing microdroplets with minimum perturbation in their performance during the study suggests the utilization of advantages of optical forces as non-contact remote manipulation tools. In this thesis firstly we demonstrate lasing from optically manipulated dye-doped emulsion and aerosol droplets. We study their lasing properties such as lasing stability in time and dependence of the lasing WGMs on the physical properties of the microdroplets such as size, refractive index and dye concentration. Moreover, we use optical forces as deforming tools to stretch the microdroplet lasers for tuning their lasing emission. To do this a dual-beam trap is used to stretch the optically pumped dye-doped droplets of oil dispersed in water. Subsequently, resonant path lengths of WGMs propagating in the droplet are modified, leading to shifts in the microlaser emission wavelengths. Using this technique, we present all-optical, almost reversible spectral tuning of the lasing WGMs and show that the direction of tuning depends on the position of the pump beam focus on the droplet. In addition, we study the effects of temperature changes on the spectral position of lasing WGMs and demonstrate that droplet heating leads to red-tuning of the droplet lasing wavelength. In the biosensing side of this thesis, we study microdroplet biolasers that exploit active liquid optical resonators formed by surface-supported aqueous microdroplets containing purified yellow fluorescent protein or a suspension of live E. coli bacterial cells expressing the fluorescent protein. We first demonstrate lasing in fluorescent protein solutions at concentrations as low as 49 µM. Subsequently, we show that a single fluorescent bacterial cell of micrometre size confined in a droplet-based cavity can serve as a laser gain medium. Aqueous droplet microcavities allow the maintenance of the bacterial cells under conditions compatible with unimpeded growth. Therefore, our results also suggest a direct route to microscopic sources of laser light with self-regenerating gain media. Fluorescence resonance energy transfer (FRET) from a donor to an acceptor chromophore is used as an atomic scale ruler to measure the conformational changes in biomolecules. Incorporating FRET into a laser cavity can increase the sensitivity of FRET-based biochemical sensors due to the nonlinear dependence of the lasing output on the FRET parameters. In order to obtain a fundamental understanding of the sensing capabilities by FRET lasing in microdroplets we carry out a comprehensive theoretical analysis of optofluidic FRET lasers on a simpler counterparts of microdroplet lasers called Fabry-Perot microcavity using a rate equation model. We compare conceptually distinct cases of donor and acceptor molecules diffusing freely in bulk solution versus molecules connected by a fixed-length linker and show that the latter arrangement is especially well-suited for sensing of low-concentration analytes. By comparing FRET lasing-based sensors with conventional FRET sensors, we show that for optimal pump fluence and FRET-pair concentration, FRET lasing can lead to more than 100-fold enhancement in detection sensitivities of conformational changes in the Forster radius range. We study the dependence of the sensitivity enhancement on the cavity Q-factor. We show that the highest enhancements can be obtained for Q-factors between 10^4-10^6, and enhancement values decrease for Q-factors above 10^6 due to the radiative energy transfer in the cavity. Finally, we demonstrate FRET lasing from self-assembled tetrahedral DNA complexes labeled with Cy3 and Cy5 dyes and suspended as a gain medium in aqueous microdroplet cavities deposited on a superhydrophobic surface. Threshold fluence and differential efficiency are characterized for DNA complexes containing 1Cy3-3Cy5 and 3Cy3-1Cy5. We demonstrate that at a constant Cy5 concentration, average threshold fluence is reduced 3 to 8 times and average differential efficiency is enhanced 6 to 30 times for 3Cy3-1Cy5 as compared to 1Cy3-3Cy5. Using 3Cy3-1Cy5 nanostructures, FRET lasing is observed at very low concentrations down to 1 µM. This work shows that optofluidic microlasers based on droplet resonators can be combined with DNA nanotechnology to explore applications in biochemical sensing and novel photonic devices.
Laser taramalı konfokal mikroskobu denetleyicisinin açık tasarım yaklaşımı ile donanımsal/yazılımsal olarak tasarlanması ve uygulanması
Laser scanning confocal microscope (LSCM) is a powerful electro-optic instrument in biological imaging and material science, compared to traditional wide-field microscopy methods. This stems from the fact that confocal microscopy enables optical imaging with a better spatial resolution. However, high cost and complexity of commercially available confocal systems hinder their wider usage. In this thesis, a new LSCM hardware/software is studied and developed using an open design approach. This device is made more convenient by adapting low cost design techniques, widely adapted components and well supported open source software. For this reason, a CDAQ (Control and Data Acquisition) unit is designed and implemented to perform essential electrical input/output (I/O) operations. This unit consists of a 2-channel 16-bit ultralow glitch Digital to Analog Converter (DAC) for driving the galvanometer scanning mirrors, and interference reduction circuitry for a 3-channel on-board 12-bit Analog to Digital Converter (ADC) which is used for sampling photomultiplier tube and motor position feedback signals. These I/O peripherals are controlled by an ARM CortexM based microcontroller which runs a multithreaded firmware to accomplish given tasks. Components of the CDAQ are all pluggable I/O interfaces and they are open to modification for the desired application. Along with these operations, CDAQ provides power supply and gain control units. CDAQ unit is controlled as a device from the user interface named "Konfokal" which is developed using Python programming language along with PyQt framework. "Konfokal" is an open source software which aims at increasing the productivity of researchers by providing environment where LSCM image acquisition and modification can be done under one single program. Also, the opportunity to tailor or modify the source for the application is possible. Capabilities of this complete working device are shown with acquired images of test samples and it is offered as a more convenient and modern device for use of researchers. "Konfokal" program was also extended in order to incorporate a Digital Micromirror Device (DMD) as a scanning unit in place of the galvanometer scanning mirrors.
Optodijital konfokal mikroskopun opto-mekanik tasarım ve geliştirmesi
Laser scanning confocal microscopy (LSCM) technique allows for obtaining high resolution, clear images of thin sections in a sample. The power of a LSCM arises from exciting a very small region of the sample at a time and using a spatial filter, usually a pinhole, in front of the detector cancelling the out of focus light. Then, 2D digital images can be constructed by scanning the desired field of view of the sample. Combining these images at the end, high resolution 3D images can be obtained. Although, LSCM systems are widely used in biology, genetics, physics and other sciences, their high cost often requires central research facilities that hinders their wide use. The aim of this thesis was to develop a cost-efficient, compact and computer controlled LSCM using a Digital Micromirror Device (DMD) that eliminates the need for a scan lens and galvanometer. DMD chip is composed of micro mirrors, each of them functioning as a pinhole due to their rotation to two positions. High speed parallel scanning can be achieved by applying different patterns on the DMD chip that is located at the plane conjugate to the focal plane of the objective. Subsequently, by using a detector at a confocal point, digital images can be produced. In this thesis, first a galvanometer mirror based LSCM was constructed. A program was developed to control the mirrors and gather data to form the images. Then a DMD-based optodigital LSCM was developed by using sequential patterns to scan the specimen. Optical and mechanical designs of the optical setups, optical alignment, optical simulation with Zemax , 3D CAD design with Solidworks and sampling with the program were accomplished.
Optik mikroçınlaçlar ile opto-akışkan algılamaları
In this thesis, detection of various fluids using optical microresonators is described. As optical microresonators, different geometries are used such as microdisks, microrings, microspheres, and microcylinders. The sensing mechanism relies on the change of optical properties, when there is a change in the amount of quantity of interest. Modes of optical microresonators, also known as whispering gallery modes (WGMs), are analyzed for the changes, and their spectral positions or quality factors are tracked, and employed as sensitive indicators of the changes. Sensing of air humidity and hydrogen gas, liquid refractometry, and contamination detection in liquid microspheres are demonstrated in the context of this thesis. In humidity sensing work, air humidity is sensitively detected using SU-8 polymer microdisk resonators. These resonators are produced with a single-step fabrication, and they provide a repeatable and long-term-stable sensing platform. When these SU-8 microresonators are coated with Pd, they can also operate as hydrogen sensors. Using this idea, hydrogen gas sensing using Pd-coated SU-8 microdisk resonators is presented in the hydrogen concentrations well below its flammable limit of 4%. Apart from gas sensing, detection in a liquid environment is also studied in two studies. In the first study, the refractive index of the liquid environment is detected using optical fiber resonators (OFRs). These OFRs are easily fabricated from standard optical fibers, and they provide refractive index sensitivity in the order of 10-5 RIU. Refractive index detection results, as well as the analytical calculations of the expected response of OFR sensors, are discussed. In the second study, contamination of the oil droplets is investigated for sunflower seed oil-olive oil mixtures. In addition, benzyl benzoate (BB) droplets are investigated. WGM shifts due to dissolution of the BB droplets, as well as incoming and outgoing small particles are discussed. The experiments performed in microfluidic chips and the droplets are held in position using optical trapping. Based on the Q-factors of the droplets, the material contained inside the droplets can be predicted. Finally, hydrogen gas sensing using polymer and Pd-coated OFRs is presented. These OFRs present even higher sensitivity than the Pd-coated SU-8 microdisk resonators. OFR sensors are easier to prepare and more flexible than the SU-8 microdisk sensors. Hydrogen concentrations down to 1000 ppm are successfully detected, with a potential detection limit less than 100 ppm. For all these sensing applications, detection results and underlying sensing mechanisms are presented, and they are compared with the state-of-the-art WGM-based sensors.
Yüzey destekli akışkan kontrol sistemlerinin geliştirilmesi
In this thesis, controlled motion of various polar liquids along patterned tracks defined over certain polymer surfaces is demonstrated. The liquid motion along the tracks is maintained due to their relatively hydrophilic (water attractive) nature as compared to the surrounding hydrophobic (water repellent) region. As a result, liquid, either in the from of droplets or bulk, is driven along the tracks in the direction of external pressure flow. Such polymer microfluidic devices are fabricated using polydimethyl siloxane (PDMS) which allows easy handling, artificial roughness addition, reconfigurability, non toxic nature and sensitivity to certain external stimulus. Fabrication of reconfigurable polymer surfaces are demonstrated which switch their wettability from superhydrophobic to superhydrophilic upon exposure to oxygen plasma and return to their original state after appropriate thermal treatment for many cycles. This is a purely chemical process taking place due to the migration of functional groups from surface to the bulk and vice versa which causes dramatic transition of wetting state and also allows absolute recovery of surface hydrophobicity. Wetting properties of these surfaces are determined by measuring their static, advancing and receding water contact angle. A specific PDMS mask with narrow openings is developed which allows selective exposure to the surface to make hydrophilic channels. Over these channels, water filaments are produced due to surface tension driven transport phenomenon from one side called input reservoir to the other side, output reservoir. Those patterns are absolutely erasable by thermal treatment, so that, new patterns can be made in the same way for many times. Hydrophilic tracks can also be defined by exposing PDMS–coated glass slides via laser ablation which is an irreversible process and only changes the topography. Laser ablation is a process of exposure of a high power extremely focused laser beam to remove PDMS coating, thus, uncovering the glass substrate. Since the wettability contrast is not too much, instead of surface driven transport, this protocol is adopted to manipulate droplets of nanoliter volume immersed in oil in a closed microfluidic environment. The difference in wettability of glass and PDMS surfaces together with the shallow step-like transverse topographical profile of the ablated tracks allow polar droplets wetting preferentially the glass surface to follow the track. Droplets with smaller wettability are found to be unguided even in the presence of topographic step. Based on this phenomenon, passive sorting of microdroplets of different chemistry injected in the same microfluidic chip is also explored. A comprehensive experimental and theoretical study is done with different droplet liquids, flow speeds and geometry of guiding tracks to gain deep and detailed understanding of the system. Liquid filaments over patterned surface are of paramount importance in photonics for fabricating fluidic optical waveguides. Guiding of light through these liquid waveguides can benefit bio-sensing, bio-lasing mostly within the visible spectrum in a highly efficient way. The theoretical implementation of an optofluidic rhodamine B dye laser based on holey fiber is demonstrated in the last chapter of the thesis. Conventional dye laser is also modeled for a comparison with the fiber laser. It is concluded that a specific fiber based variant of dye laser is superior to the conventional dye laser for having smaller lasing threshold and higher slope efficiency. These types of fluidic lasers can also be established by fluidic waveguiding over reconfigurable patterned surfaces.
Opto-sayısal konfokal mikroskop enstrümantasyonu ve sayısal mikroayna temelli görüntüleme düzeneği kurulumu
Confocal microscopy has become a vital technique for life sciences due to higher lateral and axial resolution it provides compared to standard epifluorescence microscopy. Improved axial resolution increases sectioning capabilities of the microscope, allowing for the observation of living specimens in three dimensions using image reconstruction. Despite these, accessibility to confocal microscopes did not escalate in proportion to its usage around the globe. Therefore, in this thesis, we present a home-built laser scanning confocal microscope (LSCM) setup using commercially available equipment. The design of the setup was realized using a 488 nm laser beam, an inverted microscope, a photon multiplier tube (PMT) and optical/opto-mechanical parts including mirrors, lenses, beamsplitter, scan lens. In addition, X-Y axis galvanometer scanner was employed for beam steering. Instrumentation was made using a DAQ card and LabVIEW based software. For characterization purposes, confocal images of calibration samples were obtained. As an alternative approach to standard LSCM, digital micromirror device (DMD) chip was implemented to confocal configuration for scanning the laser beam on specimen. Being cost-efficient and digital makes DMD-based scanners a promising candidate to be used in optical microscopy. Two different scanning algorithms were developed to modulate array of mirrors on DMD chip and results were compared accordingly.
Lazer işleme ile geliştirilen mikroakışkan çip içerisinde su damlalarının boyutlarına bağlı sınıflandırılması ve anjiyojenez çalışmalarına yönelik mikroakışkan çip tasarımı
We demonstrate an autonomous, high throughput and rigorous mechanism for sorting of droplets with different dimensions over inclined (10 degree), shallow (700 nm) and narrow (22 and 30 µm) guiding tracks defined by laser micromachining. We fabricated a microfluidic device containing two independent T-junctions and inlets for droplet generation allowing broad range of size and speed tuning as well as droplet merging before entering the Hele-Shaw channel hosting the guiding track. In the first part, we investigate partial guiding of different sizes of droplets under same host liquid flow conditions based on the three forces acting on the droplets namely drag force, frictional force and confinement force. For a bigger droplet, drag force being a quadratic function of droplet diameter dominates the confinement force causing the droplets leaving the inclined track earlier covering less vertical distance and guided partially. Secondly, as the speed of the droplet is increased by increasing the channel flow rate while keeping its size constant, higher drag force is exerted on the droplet once again causing partial guiding. Finally, we demonstrate sorting of smaller guided droplets coflowing with bigger unguided droplets as a result of merging from two inlets in the common tapered region just before entering the Hele-Shaw channel. For all the experiments, we have considered two chip designs with different guiding track widths (22 µm and 30 µm) and found that the droplets undergo stronger guiding for the case of wider track because of the higher confinement force. All experimental results are correlated with analytical model results incorporating droplet size, speed, interfacial tension, contact angle and realistic droplet shape by finite volume method. Microfluidic technology combined with tissue engineering has significantly increased the progress in cell biology and helped in understanding the physiological and pathophysiological transitions. PDMS due to its bio-compatibility, optical transparency and elasticity is widely used in fabrication of microfluidic devices for rapid prototyping to understand the 3D physiological microenvironment. We designed a novel microfluidic chip that incorporates a collagen hydrogel scaffold for 3D cell growth and enables controlled diffusion of medium through it. Thus, it is a convenient design that incorporates a hydrogel that mimicks the cellular microenvironment. Our design consists three parallel microchannels in contact with each other. By the unique height design, the hydrogel solution stays in the defined region and takes the shape of the collagen loading channel due to capillary action and surface tension effect. Angiogenesis; the formation of new capillaries from existing ones is mediated by endothelial cells. The hydrogel provides a porous 3D support which mediates the exchange of O2 and nutrients. We studied the response of endothelial cells by stimulating them with vascular endothelial growth factor (VEGF) that diffuses through the 3D hydrogel scaffold from biochemical channel to the cell channel inside a microfluidic chip. Our novel design provides a favorable microenvironment for Endothelial cells to grow sprouts under the influence of stimuli by having a wide contact area between the collagen loading channel and the side channels. We found that our design works for studying sprouting angiogenesis and can be used for mimicking 3D microenvironments. We also designed another microfluidic chip that will be used as an indicator of the angiogenic potential of a tumor. It incorporates cells trapping wells in one of the side channels for trapping tumor cells and can be grown to become tumor spheroids in a chip. The side channel enables the formation of spheroids from injected tumor cells on the chip. This design is different from the previous design in a sense that no stimulus in introduced directly in the chip rather the growth factors and molecules secreted by tumor spheroids in its microenvironment will trigger endothelial sprouts. Different type of tumors secrete different growth factors and molecules. By using our designed microfluidic chip the angiogenic tendency of different tumors spheroids can be studied.
Manyetik alanın photolyase ve flavin içeren çözeltilerdeki etkisinin floresan tabanlı tespit edilmesi
It has been known that bird's navigation mechanism relies on the geomagnetic fi eld; however, the primary sensor is still a matter of debate. One of the leading hypotheses is based on the magnetic sensitivity of the radical pairs. According to this model, inside the photoactivated proteins such as cryptochrome and photolyase, flavin-tryptophan radical pairs are formed by photon absorption and electron transfer, and they can undergo coherent singlet-triplet interconversion which is affected by an external magnetic field. Absorption spectroscopy has been predominantly employed to monitor such magnetic field effects. This technique requires relatively high sample concentrations. Moreover, magnetic field sensitivity of individual molecules with different orientations is lost and only an average magnetoreception behavior can be observed. Recently, fluorescence-based approach has emerged as an alternative experimental strategy which is generally easier to perform and more sensitive. Despite this, up to now fluorescence-based experiments only revealed the magnetic fi eld effect in solutions containing flavin and tryptophan, but not actual photoactivated proteins. In this thesis, the magnetic fi eld effect on flavin containing solutions and photolyases is detected by utilizing fluorescence-based experiments. In the presence of around 28 mT magnetic fi eld, up to -5.55% magnetic field effect is observed for the solution of 1 µM flavin adenine dinucleotide (FAD) and 300 µM Trp. The roles of laser intensity, pH of the buffer, and magnetic fi eld strength in the magnetic fi eld sensitivity are investigated for the FAD and Trp containing solutions. The fluorescence based experiments are also conducted with photolyases. -1.34% and -1.01% magnetic field effects are measured for the solutions of 170 µM Vibrio cholera photolyase (VcPHR) and 166 µM Escherichia coli photolyase (EcPHR) respectively. Moreover, simulations to understand quantum coherence in the radical pair mechanism are performed, and the fluorescence based observation of coherent interconversion is discussed. Finally, we suggest conjugating the photoexcited proteins or flavin with dye molecules which serve as acceptors in Förster resonance energy transfer (FRET). In this method, the decay rate of the singlet and triplet states is further enhanced, revealing higher sensitivity of magnetic fi eld effect.
Şeffaflaştırılmış doku görüntülemesi için ışık tabakası floresans mikroskobu geliştirilmesi
Light sheet fluorescence microscopy has become a popular technique for life sciences such as biomedical studies due to its advantages compared to confocal and epifluorescence microscopy. LSFM is a non-destructive technique using a narrow line of light; instead of a focused spot, for the illumination and it allows fast imaging with large field of view and reduced photobleaching effect. In this study, we present development of a home-built LSFM for cleared tissue imaging set-up with commercially available equipment. LSFM involves formation of a thin sheet of laser light using a cylindrical lens. The sample is scanned along the light sheet; thus the sample planes are selectively illuminated. The selective illumination of the sample allows for selective fluorescent excitation at desired focal planes. Besides three-dimensional image of the sample is acquired by the reconstruction of selected planes. The LSFM has been built utilizing Open Access platform for Selective Plane Illumination Microscopy. The sample was a stained cleared tissue with fluorescent tagged secondary antibodies. In order to illuminate the sample, several laser sources with a wavelength of 525 nm was used for example a Coherent Chameleon tunable laser with an SHG and a diode laser. Three-dimensional images were reconstructed from the imaged planes using the Fiji ImageJ software.
Evre kaydırmalı çınlaç boşalım izgegözlem yöntemi ve sündürülmüş optik lif temelli doğrusal çınlaç algılayıcısı
This dissertation presents the linear optical cavity sensor based on fiber Bragg grat- ings and tapered optical fiber. The sensing and detection approach used in this thesis is phase shift cavity ring down spectroscopy. It is because of linear nature of the cavity and detection mechanism applied, that the whole study is named as "linear cavity tapered fiber sensor using phase shift cavity ring down spectroscopy". First part of the thesis is dedicated to the study of experimental investigations that are available in the literature for the purpose of optical sensing. Among the reviewed techniques are the surface plasmon resonance (SPR), resonant wavelength shift, direct absorption spectroscopy, cavity ring down spectroscopy (CRDS) and its vari- ants like cavity enhanced absorption spectroscopy (CEAS). Then these techniques are sorted out depending upon the advantages that they have on each other and limitations that put constraint on their performance. For example, surface plasmon resonance is a single pass approach, which is its major constraint. But multi-pass method dubbed as resonant wavelength shift, although it is sensitive enough but suf-fers from laser intensity changes. On the other hand, cavity ring down spectroscopy, is free from such errors but it is expensive and complicated technique. The sensing technique called phase shift cavity ring down spectroscopy not only combines the advantages of CRDS and resonant wavelength shift but also provide solutions to their short comings. That is, it is economically feasible and not affected by the noise related to laser intensity. In the manuscript under study, mathematical foundation of the phase shift cavity ring down spectroscopy is also given due attention. In addition to this, fabrication of fiber Bragg gratings, their character- ization, tapered optical fiber and its evanescent field are the topics which are also discussed in the same thesis.In the second part of the thesis, an experimental investigation has been reported by using tapered fiber based cavity. Here we employed a tapered fiber-based linear fiber cavity for the demonstration of a highly sensitive sensor for sucrose concentration in water using PS-CRDS. This, linear fiber cavity has a small cavity length (∼1.25 m) that enables the observation and tracking of individual cavity modes in transmission and phase spectra recorded during laser sweeps. Hence, it eliminates the need for Pound–Drever–Hall locking of the laser source to the cavity resonance and thus provide a simpler experimental scheme. The analysis of the recorded data sets was performed to track the changes in phase shifts observed only when the laser wavelength is in resonance with the cavity modes. Such a mode-tracking PS-CRDS approach reveals limit of detection values less than around 400 µM, better than the performance of previously demonstrated PS-CRDS sucrose concentration sensors employing fiber loop resonators. The sensitivity of our sensor critically depends on the fiber taper diameter and can reach up to around 6◦/1mM Suc. for 3.2 µm taper diameter at 6 MHz modulation frequency using fiber Bragg gratings (FBGs) with reflectivities around 86%. This value can be further increased by employing FBGs with higher reflectivities or fiber tapers with smaller diameters provided that the cavity loss due to water absorption is compensated with an amplifier. It is well known fact common sources of optical losses are scattering, optical components, splicing or water absorption (for liquid phase measurements). These losses become more prominent at small taper waist diameters. So, by com- pensating these losses, working at the taper diameter lesser than 2 µm is possible. Last section of the thesis explains the working of the system for above said idea. Basically, to compensate for the optical losses, a booster optical amplifier (BOA) was added in to the previously designed simple PS-CRDS experimental scheme. Af- ter carefully adjusting the gain, this optical amplifier, provides enhanced ring down time and more round trips of light into the cavity. Which ultimately manifests itself in the form of improved sensitivity and detection limit. Some exemplary measurements performed with the amplified PS-CRDS showed the sensitivity of 4.84◦/1mM Suc. for 2 µm taper diameter at 2 MHz modulation fre- quency. Higher sensitivity can also be achieved by adjusting booster optical amplifier at higher gains and using even more thinner tapers. The thesis ends with the con- cluding remarks and future directions. It highlights the potential applications of PS-CRDS and suitable candidates to replace tapered optical fiber as a sensing head. Implementation of these changes further enhance the performance of sensing device.