Tesi: "Confocal imaging on living cells"

1

Bokhari, Ramiz Ahmed. "Confocal Imaging and Analysis of Quantum Dots on living Cells". Thesis, KTH, Tillämpad fysik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-129972.

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2

Bayard, Anaïs. "Study of the Physiological Response of NucS to Genotoxic Stress in Actinobacteria". Electronic Thesis or Diss., Institut polytechnique de Paris, 2024. http://www.theses.fr/2024IPPAX063.

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La précision de la réplication de l'ADN assure la fidélité de la transmission génétique. Les dommages causés par des facteurs externes ou internes menacent l'intégrité génomique. Les Actinobactéries, dépourvues des protéines majeur de la Réparation des Mésappariements (MMR) canonique, possèdent NucS (EndoMS), une enzyme qui répare ces erreurs indépendamment de l'ATP. Bien que l'activité de NucS dans ce mécanisme soit étudiée, sa fonction in vivo et dans d’autres systèmes de réparation de l'ADN conservent des zones d’ombre.Cette étude vise à caractériser la fonction de NucS dans la réparation des cassures double brin (DSB). Nos résultats montrent que mScarlet1-NucSD144A forme des foyers polaires en réponse aux dommages de l'ADN, particulièrement les DSB. Chez Corynebacterium glutamicum, les cellules CglΔnucS présentent une activation plus élevée de la Recombinaison Homologue (HR) et un nombre accru de DSB par rapport aux CglWT, indiquant un rôle de NucS dans l'efficacité et la sélectivité de la réparation des DSBs. Les bactéries CglΔnucS présentent un avantage de croissance en présence de stress génotoxiques, probablement en raison de mécanismes de réparation des DSB altérés. Nos analyses bioinformatiques prédisent l’interaction de NucS avec des enzymes clés de la RH et d'autres voies de réparation de l'ADN, ainsi que des gènes impliqués dans la réparation des dommages, le métabolisme et la régulation énergétique.NucS pourrait stabiliser les extrémités libres de l'ADN générées par les DSBs rapidement après leur formation, favorisant leur réparation par une voie alternative telle que la Jonction des Extrémités par Microhomologie (MMEJ). Les études futures devraient explorer les modifications post-traductionnelles et les conditions métaboliques régulant l'activité de NucS et son activité in vitro sur les DSB et les intermédiaires de HR
DNA replication accuracy ensures proper genetic transmission. Damage from external factors or internal events threatens genomic integrity. Actinobacteria, lacking canonical MMR proteins, possess NucS (EndoMS), an ATP-independent enzyme involved in a non-canonical mismatch repair pathway. While NucS's activity on mismatches is documented, its in vivo role and implications in DNA Damage Repair systems require further understanding.This study aims to characterise NucS's role in Double-Strand Break Repair (DSBR). Our findings show that mScarlet1-NucSD144A forms polar foci in response to DNA damage, especially DSBs and complex recruitment in apoptosis-like cells.Corynebacterium glutamicum, CglΔnucS bacteria exhibits higher homologous recombination (HR) activation and increased DSBs compared to CglWT, indicating NucS's role in DSBR efficiency and regulation. CglΔnucS bacteria have a growth advantage under genotoxic stress, likely due to altered DSBR mechanisms. Bioinformatic analyses predict NucS interactions with key enzymes of RH and other DNA repair pathways and metabolism and energy regulation.NucS may bind and stabilise free DNA ends generated by DSBs, balancing HR and participating in DSB repair through microhomology-mediated end joining (MMEJ). Future studies should explore post-translational modifications and metabolic conditions regulating NucS reponse and its in vitro activity on DSBs and HR intermediates

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3

Zoladek, Alina. "Confocal Raman imaging of live cells". Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/13338/.

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The objective of this thesis is to present the development of Raman microscopy for biochemical imaging of living cells. The main aim was to construct a Raman micro-spectrometer with the ability to perform time-course spectral measurements for the non-invasive study of biochemical processes in individual cells. The work can be divided into two parts: first, the development and characterization of the instrument; and second, completion of two experiments that demonstrate the suitability of Raman technique for studies of live cells. Instrumental development includes the design of optics and software for automated measurement. The experiments involve data collection and development of mathematical methods for analysis of the data. Chapter One provides an overview of techniques used in cell biology, with a special focus on Raman spectroscopy. It also highlights the importance of experiments on living cells, especially at the single cell level. Chapter Two explains the theoretical background of Raman spectroscopy. Furthermore, it presents the Raman spectroscopy techniques suitable for cell and biological studies. Chapter Three details the instrumentation and software development. The main parts of the confocal Raman micro-spectrometer, as designed for studying living cells, are: inverted microscope, 785 nm laser and high quality optics, environmental enclosure for maintaining physiological conditions during measurements of cells, and fluorescence wide-field microscopy facility for validation and confirmation of biochemical findings by Raman studies. Chapter Four focuses on the evaluation of the performance of the Raman setup and explains calibration and analysis methods applied to the data. Chapter Five and Six describe experiments performed on living cells. Chapter Five focuses on studies of the immunological synapse formed between primary dendritic and T cells indicating the polarisation of actin. Chapter Six describes time-course experiment performed on cancerous cells in the early phases of the apoptosis process, which enabled detection of the DNA condensation and accumulation of unsaturated lipids. Chapter Seven summarizes the work and gives concluding remarks.

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4

Zeskind, Benjamin J. "Quantitative imaging of living cells by deep ultraviolet microscopy". Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/38693.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.
Includes bibliographical references (p. 139-145).
Developments in light microscopy over the past three centuries have opened new windows into cell structure and function, yet many questions remain unanswered by current imaging approaches. Deep ultraviolet microscopy received attention in the 1950s as a way to generate image contrast from the strong absorbance of proteins and nucleic acids at wavelengths shorter than 300 nm. However, the lethal effects of these wavelengths limited their usefulness in studies of cell function, separating the contributions of protein and nucleic acid proved difficult, and scattering artifacts were a significant concern. We have used short exposures of deep-ultraviolet light synchronized with an ultraviolet-sensitive camera to observe mitosis and motility in living cells without causing necrosis, and quantified absorbance at 280 nm and 260 nm together with tryptophan native fluorescence in order to calculate maps of nucleic acid mass, protein mass, and quantum yield in unlabeled cells. We have also developed a method using images acquired at 320nm and 340nm, and an equation for Mie scattering, to determine a scattering correction factor for each pixel at 260nm and 280nm. These developments overcome the three main obstacles to previous deep UV microscopy efforts, creating a new approach to imaging unlabeled living cells that acquires quantitative information about protein and nucleic acid as a function of position and time.
by Benjamin J. Zeskind.
Ph.D.

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5

Chen, Wei. "Analysis of mass transport properties of plant cells by confocal microscopy and imaging techniques /". free to MU campus, to others for purchase, 1999. http://wwwlib.umi.com/cr/mo/fullcit?p9953850.

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6

Tabone, Roberta. "Sinthesys of heteroleptic zinc complexes for imaging in living cells". Master's thesis, Alma Mater Studiorum - Università di Bologna, 2019. http://amslaurea.unibo.it/19188/.

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This thesis arose from an interest in luminescence heteroleptic bis(dipyrrinato) Zn (II) complexes and their application in cell imaging, due to their attractive and fascinating characteristics. Among imaging technologies, near-infrared fluorescence imaging has been dedicated immense attention owing to its low absorption and autofluorescence from surrounding organism and tissues in this specific spectral region, which minimize background interference and improve tissue depth penetration. An ideal near-infrared probe should be equipped with excellence chemical and photophysical properties. The target of this work is the synthesis of new heteroleptic bis(dipyrrinato) Zn (II) complexes having two main features: the emission in the near-infrared region and water-solubility. In order to purse these intentions, the low-energy emission was achieved by expansion of π-conjugation of simple dipyrrins using Knoevenagel condensation106 and tri(ethylene)glycol chain was introduced to increase the water solubility of the final complex. Photophysical and luminescent properties of the new complexes were investigated. Finally, with a view to a potential biological use of these new complexes in biological environments, their biocompatibility was tested using a cell viability assay: (3-(4,5-dimethylthiazol-2-yl)-2’-5’-diphenyltetrazolium bromide (MTT) assay.

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7

Zou, Peng 1985. "Enzyme-based reporters for mapping proteome and imaging proteins in living cells". Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/79264.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
Each eukaryotic cell is exquisitely divided into organellar compartments whose functions are uniquely defined by the set of proteins they possess. For each individual protein, precise targeting to a specific sub-cellular location and trafficking between compartments are often key to its proper function. In fact, many human diseases are linked to mutations that cause mistargeting and/or defective trafficking. This thesis describes the development of enzyme-based reporters for measuring protein localization and trafficking. We employ two complementary approaches: a top-down approach, involving proteomics, to simultaneously acquire the subcellular localization information for hundreds of proteins; and a bottom-up approach, involving fluorescence imaging, to record detailed spatial information for proteins on an individual basis. This thesis is therefore divided into the following two parts. Part A describes a promiscuous protein labeling technique for proteomic mapping of organelles. This method capitalizes on peroxidase as a source of free radical generator. Compared to traditional sub-cellular fractionation methods, this novel approach obviates the need of organelle purification, thereby not only eliminating the potential artifacts associated with purification, but also greatly improving the temporal resolution of the proteomic mapping. Applying this technique to study the proteome of mitochondrial matrix and endoplasmic reticulum lumen has led to the discovery of several mitochondrial proteins whose localizations have previously been unknown or ambiguous. Part B discusses the development and application of site-specific protein labeling methods for studying receptor trafficking mechanisms. Building upon previous work in our lab, we utilized the Escherichia coli biotin ligase BirA and its acceptor peptide to site-specifically label the low-density lipoprotein receptor and studied its internalization and trafficking both at the ensemble imaging and single-molecule level. We discovered that this receptor internalizes as an oligomer into cells.
by Peng Zou.
Ph.D.

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8

Hammar, Petter. "lac of Time : Transcription Factor Kinetics in Living Cells". Doctoral thesis, Uppsala universitet, Beräknings- och systembiologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-198814.

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Gene regulation mediated by transcription factors (TFs) is essential for all organisms. The functionality of TFs can largely be described by the fraction of time they occupy their regulatory binding sites on the chromosome. DNA-binding proteins have been shown to find their targets through facilitated diffusion in vitro. In its simplest form this means that the protein combines a random 3D search in the cytoplasm with 1D sliding along DNA. This has been proposed to speed up target location. It is difficult to mimic the in vivo conditions for gene regulation in biochemistry experiments; i.e. the ionic strength, chromosomal structure, and the presence of other DNA-binding macromolecules. In this thesis single molecule imaging assays for live cell measurements were developed to study the kinetics of the Escherichia coli transcription factor LacI. The low copy number LacI, in fusion with a fluorescent protein (Venus) is detected as a localized near-diffraction limited spot when being DNA-bound for longer than the exposure time. An allosteric inducer is used to control binding and release. Using this method we can measure the time it takes for LacI to bind to different operator sequences. We then extend the assay and show that LacI slides in to and out from the operator site, and that it is obstructed by another DNA-binding protein positioned next to its target. We present a new model where LacI redundantly passes over the operator many times before binding. By combining experiments with molecular dynamics simulations we can characterize the details of non-specific DNA-binding. In particular, we validate long-standing assumptions that the non-specific association is diffusion-controlled. In addition it is seen that the non-specifically bound protein diffuses along DNA in a helical path. Using microfluidics we design a chase assay to measure in vivo dissociation rates for the LacI-Venus dimer. Based on the comparison of these rates with association rates and equilibrium binding data we suggest that there might be a short time following TF dissociation when transcription initiation is silenced. This implies that the fraction of time the operator is occupied is not enough to describe the regulatory range of the promoter.

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9

Tamura, Tomonori. "Endogenous protein imaging and analysis in living cells by selective chemical labeling methods". 京都大学 (Kyoto University), 2013. http://hdl.handle.net/2433/174965.

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10

Perez, Cota Fernando. "Opto-acoustic thin-film transducers for imaging of Brillouin oscillations on living cells". Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/32946/.

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In any given media, the speed of sound is considerably slower than speed of light, and the exploration of the acoustic regime in the GHz range gives access to very short acoustic wavelengths. Short acoustic wavelengths is an intriguing path for high resolution live-cell imaging. At low frequencies, ultrasound has proved to be a valuable tool for the mechanical characterisation and imaging of biological tissues. There is much interest in using high frequency ultrasound to investigate single cells due to its mechanical contrast mechanism. Mechanical characterisation of cells has been performed by a number of techniques, such as atomic force microscopy, acoustic microscopy or Brillouin microscopy. Recently, Brillouin oscillations measurements on vegetal and mammal cells have been demonstrated in the GHz range. In this thesis, a method to extend this technique, from the previously reported single point measurements and line scans, into a high resolution acoustic imaging tool is presented. A novel approach based around a three-layered metal-dielectric-metal film is used as a transducer to launch acoustic waves into the cell being studied. The design of this transducer and imaging system is optimised to overcome the vulnerability of a cell to the exposure of laser light and heat without sacrificing the signal to noise ratio. The transducer substrate shields the cell from the laser radiation by detecting in transmission rather than reflection. It also generates acoustic waves efficiently by a careful selection of materials and wavelengths. Facilitates optical detection in transmission due to simplicity of arrangement and aids to dissipate heat away from the cell. The design of the transducers and instrumentation is discussed and Brillouin frequency images (two and three dimensions) on phantom, fixed and living cells are presented.

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11

Silwal, Achut Prasad. "Raman Spectroscopic Imaging Analysis of Signaling Proteins and Protein Cofactors in Living Cells". Bowling Green State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1528721394633565.

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12

Umehara, Yui. "Synthesis and Evaluation of Nanoparticle-based Probes for Visualizing the Concentration and Fluctuation of Oxygen in Living Cells". Kyoto University, 2020. http://hdl.handle.net/2433/253296.

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13

Ma, Qijun [Verfasser]. "Protein interactions in living cells studied by multiparameter fluorescence imaging spectroscopy (MFIS) / Qijun Ma". Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2016. http://d-nb.info/108283713X/34.

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14

Tanhuanpää, Kimmo. "Introduction of hydrophobic fluorescent lipid analogues into living cultured cells and their imaging therein". Helsinki : University of Helsinki, 2001. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/tanhuanpaa/.

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15

Nitin, Nitin. "Optical and MR Molecular Imaging Probes and Peptide-based Cellular Delivery for RNA Detection in Living Cells". Diss., Available online, Georgia Institute of Technology, 2005, 2005. http://etd.gatech.edu/theses/available/etd-08102005-120350/.

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Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006.
Dr. X. Hu, Committee Member ; Dr. Al Merrill, Committee Member ; Dr. Niren Murthy, Committee Member ; Dr. Gang Bao, Committee Chair ; Dr. Nicholas Hud, Committee Member. Includes bibliographical references.

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16

Uttamapinant, Chayasith. "Cellular delivery and site-specific targeting of organic fluorophores for super-resolution imaging in living cells". Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/79263.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
Recent advances in super-resolution fluorescence microscopy have pushed the spatial resolution of biological imaging down to a few nanometers. The key element to the development of such imaging modality is synthetic organic fluorophores with suitable brightness and photostability. However, organic fluorophores are very difficult to use in live cells because of their chemical compositions. Many excellent fluorophores, such as cyanine and Alexa Fluor dyes, are highly charged with sulfonate groups and do not cross the plasma membrane. Even if the fluorophores get inside cells, there exist few methods that can be used to target these nongenetically encoded probes to specific cellular proteins with high specificity and minimal interference. We describe herein the development of new methods for cellular delivery and sitespecific targeting of organic fluorophores to proteins in living cells. Building on our lab's previous work on engineering new substrate specificity for E. coli lipoic acid ligase (LplA), we created a mutant ligase that catalyzes covalent conjugation of a 7-hydroxycoumarin fluorophore onto a 13-amino acid peptide substrate, called LAP. We showed that enzymatic fluorophore ligation is compatible with the living cell interior and is highly specific for LAP fusion proteins. To extend the repertoire of fluorophores targetable by LplA inside cells, we devised a two-step labeling approach based on enzymatic azide ligation, followed by chemoselective derivatization with any membrane-permeable fluorophore via strain-promoted cycloaddition. As an auxiliary tool for enzymatic probe ligation, we also developed a very efficient and biocompatible variant of copper-catalyzed azide-alkyne cycloaddition that can be used for modification of cell-surface proteins. To overcome the lack of membrane permeability of sulfonated fluorophores, we identified a chemical reaction that efficiently masks charged sulfonate groups as esterase-labile sulfonate esters. Such masked sulfonated fluorophores enter cells readily and can be sitespecifically targeted to intracellular proteins. Our efforts in developing protein labeling and fluorophore delivery methods culminated in their application to super-resolution imaging of cellular proteins in living cells.
by Chayasith Uttamapinant.
Ph.D.

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17

Basu, Srinjan. "Study of Chromatin Structure Using Stimulated Raman Scattering Microscopy in Living Mammalian Cells". Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10416.

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DNA is packaged into the nucleus of a mammalian cell as a nucleoprotein complex called chromatin. Changes in chromatin structure occur during processes that are critical to an understanding of mammalian cell biology such as cell division. Existing fixed-cell techniques have provided insight into chromatin organization in the mammalian nucleus. In addition, fluorescence microscopy techniques have allowed us to study changes in chromatin structure in living cells. However, most of these fluorescence techniques cannot be used for tissue imaging or long-term imaging due to photobleaching. In this thesis, we demonstrate that a label-free technique called Stimulated Raman Scattering (SRS) microscopy can be used to solve these problems and study chromatin structure in living mammalian cells both in culture and in tissue. SRS is a vibrational microscopy technique that takes advantage of intrinsic contrast arising from specific chemical bonds in a molecule. Nucleic acids have specifc phosphate and CH vibrations that can be used to determine their cellular distributions. Imaging at specific phosphate peaks using fingerprint SRS microscopy allows the detection of polytene chromosomes in Drosophila salivary gland cells and condensed chromatin in metaphase mammalian cells. In addition, we develop a technique called multicolor SRS microscopy, in which we image at several wavelengths across the CH vibrational band, and then use linear combination to simultaneously determine the nucleic acid, lipid and protein distributions in living mammalian cells. This technique achieves greater contrast than imaging at the phosphate vibrational peak due to the stronger SRS signal in the high wavenumber CH band and so allows us to determine chromatin structure in interphase mammalian cells. This technique also allows long-term imaging of living mammalian cells and the imaging of tissue such as mouse skin. The technique is used to monitor mammalian cell division in culture and paves the way for similar studies in living tissue. This technique will provide insight into cell division, differentiation and apoptosis during development and in disease models such as cancer.

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18

Ellis, Jonathan. "FRET analysis of splicing factors involved in exon and intron definition in living cells". Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/4397.

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I have analyzed the interactions between SR proteins and splicing components that are bound at the 5’ or 3’ splice site using fluorescence resonance energy transfer (FRET) microscopy. The SR proteins interact with the U1 snRNP-associated 70 kDa protein (U170K) at the 5’splice site and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) at the 3’ splice site. These interactions have been extensively characterized biochemically in the past, and are proposed to play roles in both intron and exon definition. We employed FRET acceptor photobleaching and fluorescence lifetime imaging microscopy (FLIM) to identify and spatially localise sites of direct interactions of SF2/ASF, and other SR proteins, with U2AF35 and U1-70K in live cell nuclei. These interactions were shown to occur more strongly in interchromatin granule clusters (IGCs). They also occur in the presence of the RNA polymerase II inhibitor, DRB, demonstrating that they are not exclusively co-transcriptional. FLIM data have also revealed a novel interaction between HCC1, a factor highly related to the large subunit of the U2AF splicing factor, with both subunits of U2AF that occur in discrete domains within the nucleoplasm but not within IGCs. These data demonstrate that the interactions defining intron and exon definition do occur in living cells in a transcription-independent manner.

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19

Li, Qifei. "In Vitro, Non-Invasive Imaging and Detection of Single Living Mammalian Cells Interacting with Bio-Nano-Interfaces". DigitalCommons@USU, 2015. https://digitalcommons.usu.edu/etd/4520.

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Understanding of bio-nano-interfaces of living mammalian cells will benefit the identification of cellular alterations (e.g. nucleic acids, amino acids, biomechanics, etc.) due to external stimuli, the design of biomaterials (e.g. nanoparticles, nanotubes) and the investigation of the interaction between cells and bio-nano-interfaces (e.g. cell differentiation on 3D nanostructured materials). Analytical techniques can be applied to evaluate the chemical, physical, and mechanical properties of mammalian cells when exposed to such bio-nano-interfaces. In this study, non-invasive advanced spectroscopy techniques including atomic force microscopy (AFM) and Raman microscopy (RM), in conjunction with traditional biological methods are utilized to elucidate specific characteristic information for biological samples and how these property changes reflect the interaction with external stimuli. The focus of this dissertation is on the biophysical, biochemical and cytotoxic detection of mammalian cells interacting with bio-nano-interfaces, and this dissertation can be classified into three topics: biomechanics/cellular biopolymers measurement, bio-interfaces and nano-interfaces studies. For the topic of biomechanics/cellular biopolymers measurement, cellular biophysical and biomechanical properties could be used as differentiation markers to classify cellular differentiation. For the bio-interfaces part, it was observed that BRMS1 expression changed cellular biochemical and biomechanical properties, and the expressions of reactive oxidative species (ROS), apoptosis and cell viability of five types of cells displayed similar patterns over doxorubicin (DOX) incubation time. Secondly, A549 cells were treated with diesel exhaust particles (DEP) and resveratrol (RES) to study the effect of RES on the DEP-induced cells, and it was found that RES can alleviate DEP intervention on cellular structure and increase DEP-induced biomechanical and inflammatory changes. For the nano-interfaces topic, first we synthesized a hybrid nanoparticle with the multimodal properties of fluorescence imaging, Surface-enhanced Raman spectroscopy (SERS) detection and photothermal therapy (PTT) for single living cell analysis of epidermal growth factor receptor (EGFR) and specifically killed cancer cells with high EGFR expression. Additionally, to increase surface area, light-heat conversion efficiency and biocompatibility, we developed a silica coated nanoparticle conjugated with anti-human epidermal growth factor receptor 2 (HER2) antibody. Finally, three-dimensional TiO2 nanotubes with Au nanoparticles coating were synthesized and used to study trophoblast-derived stem-like cells growth on such 3D nanostructures.

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20

Wang, Renjie. "Quantitative analysis of chromatin dynamics and nuclear geometry in living yeast cells". Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30122/document.

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L'analyse de l'organisation à grande échelle des chromosomes, par des approches d'imagerie et de biologie moléculaire, constitue un enjeu important de la biologie. Il est maintenant établi que l'organisation structurelle du génome est un facteur déterminant dans tous les aspects des " transactions " génomiques: transcription, recombinaison, réplication et réparation de l'ADN. Bien que plusieurs modèles aient été proposés pour décrire l'arrangement spatial des chromosomes, les principes physiques qui sous-tendent l'organisation et la dynamique de la chromatine dans le noyau sont encore largement débattus. Le noyau est le compartiment de la cellule dans lequel l'ADN chromosomique est confiné. Cependant, la mesure quantitative de l'influence de la structure nucléaire sur l'organisation du génome est délicate, principalement du fait d'un manque d'outils pour déterminer précisément la taille et la forme du noyau. Cette thèse est organisée en deux parties: le premier axe de mon projet était d'étudier la dynamique et les propriétés physiques de la chromatine dans le noyau de la levure S. cerevisiae. Le deuxième axe visait à développer des techniques pour détecter et quantifier la forme et la taille du noyau avec une grande précision. Dans les cellules de levure en croissance exponentielle, j'ai étudié la dynamique et les propriétés physiques de la chromatine de deux régions génomiques distinctes: les régions codant les ARN ribosomiques regroupés au sein d'un domaine nucléaire, le nucléole, et la chromatine du nucléoplasme. Le mouvement de la chromatine nucléoplasmique peut être modélisé par une dynamique dite de " Rouse ". La dynamique de la chromatine nucléolaire est très différente et son déplacement caractérisé par une loi de puissance d'exposant ~ 0,7. En outre, nous avons comparé le changement de la dynamique de la chromatine nucléoplasmique dans une souche sauvage et une souche porteuse d'un allèle sensible à la température (ts) permettant une inactivation conditionnelle de la transcription par l'ARN polymérase II. Les mouvements chromatiniens sont beaucoup plus importants après inactivation transcriptionnelle que dans la souche témoin. Cependant, les mouvements de la chromatine restent caractérisés par une dynamique dite de " Rouse ". Nous proposons donc un modèle biophysique prenant en compte ces résultats : le modèle de polymère dit "branched-Rouse". Dans la deuxième partie, j'ai développé "NucQuant", une méthode d'analyse d'image permettant la localisation automatique de la position de l'enveloppe nucléaire du noyau de levures. Cet algorithme comprend une correction post-acquisition de l'erreur de mesure due à l'aberration sphérique le long de l'axe Z. "NucQuant" peut être utilisée pour déterminer la géométrie nucléaire dans de grandes populations cellulaires. En combinant " NucQuant " à la technologie microfluidique, nous avons pu estimer avec précision la forme et la taille des noyaux en trois dimensions (3D) au cours du cycle cellulaire. "NucQuant" a également été utilisé pour détecter la distribution des regroupements locaux de complexes de pore nucléaire (NPCs) dans des conditions différentes, et a révélé leur répartition non homogène le long de l'enveloppe nucléaire. En particulier, nous avons pu montrer une distribution particulière sur la région de l'enveloppe en contact avec le nucléole. En conclusion, nous avons étudié les propriétés biophysiques de la chromatine, et proposé un modèle dit "branched Rouse-polymer" pour rendre compte de ces propriétés. De plus, nous avons développé "NucQuant", un algorithme d'analyse d'image permettant de faciliter l'étude de la forme et la taille nucléaire. Ces deux travaux combinés vont permettre l'étude des liens entre la géométrie du noyau et la dynamique de la chromatine
Chromosome high-order architecture has been increasingly studied over the last decade thanks to technological breakthroughs in imaging and in molecular biology. It is now established that structural organization of the genome is a key determinant in all aspects of genomic transactions. Although several models have been proposed to describe the folding of chromosomes, the physical principles governing their organization are still largely debated. Nucleus is the cell’s compartment in which chromosomal DNA is confined. Geometrical constrains imposed by nuclear confinement are expected to affect high-order chromatin structure. However, the quantitative measurement of the influence of the nuclear structure on the genome organization is unknown, mostly because accurate nuclear shape and size determination is technically challenging. This thesis was organized along two axes: the first aim of my project was to study the dynamics and physical properties of chromatin in the S. cerevisiae yeast nucleus. The second objective I had was to develop techniques to detect and analyze the nuclear 3D geomtry with high accuracy. Ribosomal DNA (rDNA) is the repetitive sequences which clustered in the nucleolus in budding yeast cells. First, I studied the dynamics of non-rDNA and rDNA in exponentially growing yeast cells. The motion of the non-rDNA could be modeled as a two-regime Rouse model. The dynamics of rDNA was very different and could be fitted well with a power law of scaling exponent ~0.7. Furthermore, we compared the dynamics change of non-rDNA in WT strains and temperature sensitive (TS) strains before and after global transcription was actived. The fluctuations of non-rDNA genes after transcriptional inactivation were much higher than in the control strain. The motion of the chromatin was still consistent with the Rouse model. We propose that the chromatin in living cells is best modeled using an alternative Rouse model: the “branched Rouse polymer”. Second, we developed “NucQuant”, an automated fluorescent localization method which accurately interpolates the nuclear envelope (NE) position in a large cell population. This algorithm includes a post-acquisition correction of the measurement bias due to spherical aberration along Z-axis. “NucQuant” can be used to determine the nuclear geometry under different conditions. Combined with microfluidic technology, I could accurately estimate the shape and size of the nuclei in 3D along entire cell cycle. “NucQuant” was also utilized to detect the distribution of nuclear pore complexes (NPCs) clusters under different conditions, and revealed their non-homogeneous distribution. Upon reduction of the nucleolar volume, NPCs are concentrated in the NE flanking the nucleolus, suggesting a physical link between NPCs and the nucleolar content. In conclusion, we have further explored the biophysical properties of the chromatin, and proposed that chromatin in the nucleoplasm can be modeled as "branched Rouse polymers". Moreover, we have developed “NucQuant”, a set of computational tools to facilitate the study of the nuclear shape and size. Further analysis will be required to reveal the links between the nucleus geometry and the chromatin dynamics

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Zachar, Peter C. "A Comparative Study of Neuroepithelial Cells and O2 Sensitivity in the Gills of Goldfish (Carrasius auratus) and Zebrafish (Danio rerio)". Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/30343.

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Serotonin (5-HT)-containing neuroepithelial cells (NECs) of the gill filament are believed to be the primary O2 chemosensors in fish. In the mammalian carotid body (CB), 5-HT is one of many neurotransmitters believed to play a role in transduction of hypoxic stimuli, with acetylcholine (ACh) being the primary fast-acting excitatory neurotransmitter. Immunohistochemistry and confocal microscopy was used to observe the presence of the vesicular acetylcholine transporter (VAChT), a marker for the presence of ACh, and its associated innervation in the gills of zebrafish. VAChT-positive cells were observed primarily along the afferent side of the filament, with some cells receiving extrabranchial innervation. No VAChT-positive cells were observed in the gills of goldfish; however, certain key morphological differences in the innervation of goldfish gills was observed, as compared to zebrafish. In addition, in zebrafish NECs, whole-cell current is dominated by an O2-sensitive background K+ current; however, this is just one of several currents observed in the mammalian CB. In zebrafish NECs and the CB, membrane depolarization in response to hypoxia, mediated by inhibition of the background K+ (KB) channels, is believed to lead to activation of voltage-gated Ca2+ (CaV) channels and Ca2+-dependent neurosecretion. Using patch-clamp electrophysiology, I discovered several ion channel types not previously observed in the gill chemosensors, including Ca2+-activated K+ (KCa), voltage-dependent K+ (KV), and voltage-activated Ca2+ (CaV) channels. Under whole-cell patch-clamp conditions, the goldfish NECs did not respond to hypoxia (PO2 ~ 11 mmHg). Employing ratiometric calcium imaging and an activity-dependent fluorescent vital dye, I observed that intact goldfish NECs respond to hypoxia (PO2 ~ 11 mmHg) with an increase in intracellular Ca2+ ([Ca2+]i) and increased synaptic vesicle activity. The results of these experiments demonstrate that (1) ACh appears to play a role in the zebrafish, but not goldfish gill, (2) goldfish NECs likely signal hypoxic stimuli primarily via the central nervous system (CNS), (3) goldfish NECs express a broad range of ion channels as compared to the NECs of zebrafish, and (4) goldfish NECs rely on some cytosolic factor(s) when responding to hypoxia (PO2 ~ 11 mmHg). This thesis represents a further step in the study of neurochemical and physiological adaptations to tolerance of extreme hypoxia.

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Anastos, Thèo. "Studying the Efficacy of an Injectable 3-Dimensional Fibrin Extracellular Matrix to Characterize the Effects of Antitumor Agents on SW620 Cells in A Microfluidic Device". DigitalCommons@CalPoly, 2021. https://digitalcommons.calpoly.edu/theses/2277.

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Colorectal cancer is the third most common cancer in the United States and there is currently a lot of research going into new antitumor agents to kill the cancer. One method for replicating the tumor response to a drug in vivo is by creating an in vitro drug testing model to replicate the in vivo condition. This research project was conducted to determine the efficacy of testing tumor cultures in a microfluidic device as a way to provide accurate drug responses in vitro instead of using in vivo subjects in clinical trials. A total of four experiments were conducted with each experiment increasing the complexity of the culture model. The first experiment was a 2-dimensional tumor culture that was seeded in a well plate to study how 5-fluorouracil treatments affected the tumor cell viability. The second experiment was a 2-dimensional tumor culture that was seeded on top of a fibrin extracellular matrix (ECM) gel to determine how the tumor cells would respond to the 5-luorouracil treatments while growing on the fibrin. The third experiment was to create a 3-dimensional tumor culture that was seeded inside the fibrin ECM gel. This experiment was conducted to determine if tumor cells cultured within the fibrin gel could receive nutrients from the medium diffusing through the gel. Once the tumors responded as expected in the fibrin gel, the gel could be injected into a microfluidic device for the fourth experiment. The fourth experiment was a proof of concept to determine if the tumor cells could survive in the microfluidic device and be properly treated with 5-fluorouracil. The experiment with the cells seeded in the well plates showed that an increase in 5-fluorouracil concentration caused a significant decrease in cell viability. Both fibrin gel experiments showed that the average tumor size, total tumor area, and tumor count decreased as the 5-fluorouracil concentration increased. The tumor cells were successfully able to be cultured in the microfluidic device and the average tumor size decreased significantly when the culture was exposed to the 5-fluorouracil treatment.

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23

Salehi, Hamideh. "L'étude des cellules vivantes et la dentine humaine par microscopie confocale Raman". Thesis, Montpellier 1, 2013. http://www.theses.fr/2013MON12201/document.

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"L'étude des cellules vivantes et la dentine humaine par microscopie confocale Raman" La microscopie confocale Raman est utilisée pour suivre des médicaments et des nanoparticules dans les cellules et dans les tissus durs. La microscopie Raman est non-invasive, ne nécessite aucun marqueur et permet une imagerie à haute résolution. Dans la première partie de l'étude cette méthode est utilisée pour suivre un médicament anticancéreux, le paclitaxel, au sein d'une lignée de cellules cancéreuses vivantes Michigan Cancer Foundation-7 (MCF-7). Les images Raman ont été traitées par un algorithme de partitionnement des données par k-moyennes pour détecter le paclitaxel dans les cellules. La distribution du paclitaxel dans les cellules est vérifiée par le calcul du coefficient de corrélation de Pearson entre le spectre de référence du traitement et les spectres de l'image entière. Le temps progressif de diffusion du paclitaxel dans toute la cellule est observé. Cette observation demande une étude complémentaire sur l'action pharmaceutique du produit, basé sur la liaison rapide de la tubuline libre au paclitaxel cristallisé. L'apoptose dans les cellules a été suivies par partitionnement de données et par corrélation. Le partitionnement de données a été utilisé pour déterminer la position de mitochondries dans les cellules ; le cytochrome C de distribution à l'intérieur des cellules est basé sur l'analyse de corrélation. L'apoptose des cellules est défini par le cytochrome C dans le cytoplasme de diffusion. Le cytochrome C agit comme un déclencheur pour l'activation en cascade des caspases, et sa libération par les mitochondries est un signe d'apoptose. La Co-localisation de cytochrome C est effectuée après incubation de cellules avec une concentration différente de paclitaxel. L'autre produit étudié est le dioxyde de titane. Le titane est largement utilisé pour les matériaux orthopédiques et dentaires implantés dans le corps humain. Il est inévitable que le sang prenne contact avec la surface de l'implant et des nanoparticules. Les nanoparticules de dioxyde de titane ont été suivies en intracellulaire dans les cellules MCF-7 et TERT épithéliales humaines (lignée orale cellulaire de kératinocytes OKF6/TERT-2). La détection des nanoparticules et leur toxicité ont été étudiées en utilisant deux méthodes d'analyse. La microscopie confocale Raman a également été utilisée pour réaliser l'analyse structurale et chimique de la jonction émail-dentine-résine et de la carie dentaire, grâce à une analyse précise des constituants minéraux et organiques. La microscopie Raman associée à des méthodes d'analyse de données ouvre de nouvelles portes pour la recherche en biologie-santé et en particulier en odontologie
"The Study of living cells and human dentin by confocal Raman microscopy"Confocal Raman microscopy is employed to trace drugs and nanoparticles intracellular and in hard tissues. Raman spectroscopy a non-invasive, label-free and high spatial resolution imaging technique in first part of the study is being used to trace the anticancer drug paclitaxel in living Michigan Cancer Foundation-7 (MCF-7) cells. An analytical method was developed and applied to Raman data acquired. The Raman images were treated by K-mean cluster analysis to detect the drug in cells. Distribution of paclitaxel in cells is verified by calculating the Pearson correlation coefficient between the reference spectrum of the drug and the whole Raman image spectra. A time dependent gradual diffusion of paclitaxel all over the cell is observed suggesting a complementary picture of the pharmaceutical action of this drug based on rapid binding of free tubulin to crystallized paclitaxel. The apoptosis in the cells were followed by post-measurement analysis including K-mean clustering and Pearson correlation coefficient. K-mean clustering was used to determine mitochondria position in cells and cytochrome c distribution inside the cells was based on correlation analysis. Cell apoptosis is defined as cytochrome c diffusion in cytoplasm. Cytochrome c acts as a trigger for the activation of the caspase cascade, and its release from mitochondria is a sign of apoptosis. Co-localization of cytochrome c is done after cell incubation with different concentration of paclitaxel. The other product used was titanium dioxide. Titanium has been widely used for orthopedic and dental implant materials. When biomaterial is implanted into the human body, it is unavoidable that blood will contact the implant surface and nanoparticles. The question is: do these nanoparticles cause toxicity? Titanium dioxide nanoparticles were followed intracellular in MCF-7 cells and TERT epithelial human oral keratinocyte cell line (OKF6/TERT-2). Detection of nanoparticles and their toxicity were studied using two analytical methods. Confocal Raman microscopy were also used to obtain Structural analysis and chemical profile of Enamel – Dentine- Resin and Raman map of decay and sound dentin samples, through accurate analysis of the mineral and organic components. The Raman spectroscopy combined with this novel method developed in this study, will provide accurate finger prints of chemical composition and by post-measurement analysis of the data acquired more information would be obtained, which might open new gates in pharmaceutical and dentistry researches

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24

Klein, Katharina [Verfasser], Jürgen [Akademischer Betreuer] Schlegel, Vasilis [Akademischer Betreuer] Ntziachristos e Bernhard [Akademischer Betreuer] Hemmer. "Label-free microscopic bioimaging by means of confocal Raman spectroscopy on living glioblastoma cells / Katharina Klein. Gutachter: Vasilis Ntziachristos ; Bernhard Hemmer ; Jürgen Schlegel. Betreuer: Jürgen Schlegel". München : Universitätsbibliothek der TU München, 2013. http://d-nb.info/1036494993/34.

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25

Sabaté, Thomas. "Modeling, visualizing and quantifying chromatin loop extrusion dynamics in living human cells". Electronic Thesis or Diss., Sorbonne université, 2023. http://www.theses.fr/2023SORUS304.

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L’étude de l’organisation 3D du génome a révélé l’existence de boucles de chromatine et des Topologically Associating Domains (TADs) de l’ordre de plusieurs centaines de kilobases, créés par l’anneau de cohésine par le processus d’extrusion de boucle d’ADN. Cependant, ces structures ont été caractérisées presque exclusivement par des techniques de génomique et d’imagerie de cellules fixées, leur dynamique temporelle reste donc peu comprise. Par exemple, la durée des contacts créés par extrusion de boucles n’est pas définie et des paramètres majeurs de ce processus comme la durée de vie des contacts ancre-ancre et la vitesse d’extrusion in vivo sont toujours inconnus. Pour répondre à cette lacune, j’ai quantifié la dynamique de l’extrusion de boucle cohésine-dépendante en visualisant et suivant dans le temps plusieurs paires d’ancres de boucles dans des cellules humaines vivantes. Il est attendu que l’extrusion de boucle soit identifiée par une diminution progressive de la distance ancre-ancre. Cependant, cette signature est occultée par la dynamique stochastique de la chromatine, les ancres de boucles pouvant entrer en contact même sans extrusion. De plus, mesurer la distance ancre-ancre à partir d’images fluorescentes est rendu difficile par plusieurs sources d’erreurs comme les erreurs aléatoires liées à la localisation de points fluorescents. Pour estimer les conditions expérimentales qui permettent de détecter et quantifier l’extrusion de boucles malgré ces complications, j’ai utilisé des simulations de polymères et modélisé le processus d’extrusion de boucle in silico. De plus, j’ai testé et validé de nouvelles méthodes d’analyse pour quantifier les boucles de chromatine à partir d’images statiques (e. g. à partir d’images d’ancres de boucles acquises par DNA FISH), estimer la fraction, fréquence et durée de vie des contacts ancre-ancre, ainsi qu’estimer la vitesse d’extrusion effective in vivo à partir d’images dynamiques. En se basant sur les résultats des simulations de polymères, j’ai taggué par fluorescence de multiples ancres de boucles et TADs dans des cellules vivantes par le système CRISPR/Cas9. Nous avons conclu que les contacts entre les ancres étaient peu fréquents et de courte durée, par rapport à la durée du cycle cellulaire. Cependant, les boucles sont presque constamment soumises à l’extrusion par la cohésine. En comparant les résultats de modélisation et les expériences, nous avons pu estimer des paramètres biophysiques généraux de la dynamique d’extrusion de boucles. Ces résultats suggèrent que l’extrusion de boucles de chromatine cohésine-dépendante est un processus hautement dynamique, qui crée des interactions à longue portée transitoires plutôt que des contacts stables. Mes résultats aideront à comprendre quantitativement des processus biologiques fondamentaux qui utilisent les contacts transitoires mais à longue distance créés par l’extrusion de boucles, comme la réparation de l’ADN et la régulation de l’expression des gènes
Studies of spatial genome organization have revealed the existence of chromatin loops and Topologically Associating Domains (TADs) of several hundred kilobases in size, which are created by the cohesin ring complex through a process of DNA loop extrusion. However, these structures have been characterized almost exclusively by genomic techniques and fixed cell imaging, thus their temporal dynamics are still poorly understood. For example, it is not clear whether loop extrusion creates stable or transient contacts at loop anchors and key parameters of this process, including loop lifetime and extrusion speed, remain unknown. To address this gap, my thesis aims to quantify the dynamics of cohesin-dependent loop extrusion by visualizing and tracking in time pairs of anchors at several loops and TADs in living human cells. Extrusion is expected to manifest itself as a progressive decrease in anchor-anchor distances. However, this signature is obscured by stochastic motions of the chromatin, whereby anchors can occasionally come into contact even without extrusion. Furthermore, measuring the anchor-anchor distance from fluorescent images is complicated by several sources of uncertainties, such as unavoidable random errors in the computational localization of fluorescent spots. To evaluate the experimental conditions under which one can expect to detect and quantify loop extrusion despite such complications, I first performed an analysis in silico using polymer simulations that account for loop extrusion. Using these simulations, I also tested and validated novel analysis methods to quantify chromatin loop dynamics from static imaging (e. g. from DNA-FISH images of loop anchors), and to estimate the lifetime and frequency of anchor contacts, as well as the effective loop extrusion speed from dynamic imaging in vivo. Using the simulation results as guidelines, we fluorescently labelled multiple loop and TAD anchors in human cells using the CRISPR/Cas9 system and tracked the loop anchors by live-cell imaging. Based on our analysis of the imaging data, we found that contacts between the two loop anchors are infrequent and short-lived as compared to the cell cycle duration. However, loops were found to be almost constantly extruded by cohesin. By comparing simulations and experimental data, we could estimate key biophysical parameters of loop extrusion dynamics including loop lifetimes and extrusion speed. Our results suggest that cohesin-dependent loop extrusion is a highly dynamic process, which creates transient long-range interactions rather than stable contact s. Our findings will help to quantitatively understand biological processes that involve short-lived but long-range contacts created by loop extrusion, including mechanisms of DNA repair and gene regulation

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26

Lin, Chi-Wang. "Methodology development for imaging histone modifications and for site-specific protein labeling in vitro and on the surface of living cells". Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/37605.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references (leaves 187-207).
A new methodology for monitoring post-translational modifications of histone H3 in living cells was developed using genetically encoded fluorescent reporters. These reporters were constructed for sensing histone phosphorylation and methylation by fluorescence resonance energy transfer (FRET). These reporters are four-part chimeric proteins with the domains from N- to C-terminus as follows: a cyan fluorescent protein (CFP), a phosphoserine recognition domain (14-3-3 tau) or a methyllysine recognition domain (HP1 or Pc chromodomain), a peptide whose sequence corresponds to the Nterminus of histone H3, and a yellow fluorescent protein. The histone phosphorylation reporter exhibited a 25 % YFP/CFP emission ratio increase upon phosphorylation in vitro by Msk-1 kinase. Site-directed mutagenesis studies suggested that Ser28 phosphorylation gave rise to the reporter FRET response. When tested in living HeLa cells, the reporter exhibited a rapid increase in the emission ratio 5-15 min prior to the nuclear membrane breakdown and the FRET peaked during cell division. Another reporter, in which the 14-3-3 tau and the H3 peptide were swapped, exhibited the FRET response to both Ser10 and Ser28 phosphorylation. Two methylation reporters, K9 and K27, were constructed for sensing H3-lysine9 and H3-lysine27 methylation.
(cont.) The reporters gave 60 % (K9) and 28 % (K27) emission ratio changes after in vitro methylation, catalyzed by the histone methyltransferase vSET. Applying the K9 reporter in cells showed different levels of reporter FRET in fibroblasts either expressing or lacking methyltransferases Suv39h1 and Suv39h2. Site-specific incorporation of biophysical probes onto cell surface proteins is critical for the study of protein trafficking. One general solution to achieve labeling specificity is the use of enzymes for the ligation of probes to a substrate peptide tagged onto a protein of interest. Transglutaminase incorporates amine probes to cell surface proteins expressing a 6- or 7-amino acid consensus sequence (Q-tag). A variety of probes such as biotin cadaverine and fluorescein cadaverine were incorporated to Q-tag-CFP and Q-tag-EGF receptor constructs expressed on the surface of living HeLa and HEK cells. The NF?B p50 transcription factor fused to a Q-tag was labeled with a benzophenone photo-affinity probe in vitro. Upon UV irradiation, elevated levels of p50 homodimerization were observed in the presence of DNA or the interacting protein myotrophin.
by Chi-Wang Lin.
Ph.D.

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27

Agrawal, Amit. "Nanoparticle Probes for Ultrasensitive Biological Detection and Motor Protein Tracking inside Living Cells". Diss., Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/19798.

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Semiconductor quantum dots (QDs) have emerged as a new class of fluorescent probes and labeling agents for biological samples. QDs are bright, highly photostable and allow simultaneous excitation of multiple emissions. Owing to these properties, QDs hold exceptional promise in enabling intracellular biochemical studies and diagnosis with unprecedented sensitivity and accuracy. However, use of QD probes inside living cells remains a challenge due to difficulties in delivery of nanoparticles without causing aggregation and imaging single nanoparticles inside living cells. In this dissertation, a systematic approach to deliver, image and locate single QDs inside living cells is presented and the properties of molecular motor protein driven QD transport are studied. First, spectroscopic and imaging methods capable of differentiating single nanoparticles from the aggregates were developed. These technologies were validated by differentiating surface protein expression on viral particles and by enabling rapid counting of single biomolecules. Second, controlled delivery of single QDs into living cells is demonstrated. A surprising finding is that single QDs associate non-specifically with the dynein motor protein complex and are transported to the microtubule organizing center. Accurate localization and tracking of QDs inside cell cytoplasm revealed multiple dynein motor protein attachment resulting in increased velocity of the QDs. Further, spectrin molecule which is known to recruit dynein motor protein complex to phospholipid micelles was found to associate with the QDs. These results may serve as a benchmark for developing new QD surface coatings suitable for intracellular applications. Since, nanoparticles are similar in size to viral pathogens; better understanding of nanoparticle-cell interactions should also help engineer nanoparticle models to study virus-host cell interactions. (Contains AVI format multimedia files)

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Hammadi, Shumoos T. H. "Novel medical imaging technologies for processing epithelium and endothelium layers in corneal confocal images. Developing automated segmentation and quantification algorithms for processing sub-basal epithelium nerves and endothelial cells for early diagnosis of diabetic neuropathy in corneal confocal microscope images". Thesis, University of Bradford, 2018. http://hdl.handle.net/10454/16924.

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Diabetic Peripheral Neuropathy (DPN) is one of the most common types of diabetes that can affect the cornea. An accurate analysis of the corneal epithelium nerve structures and the corneal endothelial cell can assist early diagnosis of this disease and other corneal diseases, which can lead to visual impairment and then to blindness. In this thesis, fully-automated segmentation and quantification algorithms for processing and analysing sub-basal epithelium nerves and endothelial cells are proposed for early diagnosis of diabetic neuropathy in Corneal Confocal Microscopy (CCM) images. Firstly, a fully automatic nerve segmentation system for corneal confocal microscope images is proposed. The performance of the proposed system is evaluated against manually traced images with an execution time of the prototype is 13 seconds. Secondly, an automatic corneal nerve registration system is proposed. The main aim of this system is to produce a new informative corneal image that contains structural and functional information. Thirdly, an automated real-time system, termed the Corneal Endothelium Analysis System (CEAS) is developed and applied for the segmentation of endothelial cells in images of human cornea obtained by In Vivo CCM. The performance of the proposed CEAS system was tested against manually traced images with an execution time of only 6 seconds per image. Finally, the results obtained from all the proposed approaches have been evaluated and validated by an expert advisory board from two institutes, they are the Division of Medicine, Weill Cornell Medicine-Qatar, Doha, Qatar and the Manchester Royal Eye Hospital, Centre for Endocrinology and Diabetes, UK.

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Conic, Sascha. "Analysis of transcription factor and histone modification dynamics in the nucleus of single living cells using a novel antibody-based imaging approach". Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ081.

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Dans les cellules des eucaryotes, la transcription des gènes est contrôlée par une pléthore de complexes protéiniques. Cependant, la plupart de nos connaissances fondamentales sur la régulation de la transcription viennent des expériences biochimiques ou des expériences d’immunofluorescences utilisant des cellules fixées. Par conséquent, beaucoup d’efforts ont été consacré récemment pour obtenir des informations sur les mouvements dynamiques ou sur l’assemblage des facteurs de transcription directement dans des cellules vivantes. Nous avons développé une stratégie de marquage, appelé « versatile antibody-based imaging approach » (VANIMA), dans laquelle des anticorps marqués avec un fluorochrome sont introduit dans des cellules vivantes pour visualiser spécifiquement des protéines endogènes ou des modifications post-traductionnelle. Nous avons pu montrer que VANIMA peut être utilisé pour étudier des processus dynamique des mécanismes fondamental de la biologie y compris les facteurs de la machinerie de transcription ainsi que les modifications des histones dans des cellules vivantes de cancer humaine en utilisant la microscopie conventionnelle ou à super-résolution. Dans l’avenir VANIMA va servir comme un outil valable pour révéler les dynamiques des processus endogènes en biologie y compris la transcription directement dans des cellules vivantes individuelles
In eukaryotic cells, gene transcription is controlled by a plethora of protein complexes. However, most of our basic knowledge about transcription regulation originate from biochemical experiments or immunofluorescence experiments using fixed cells. Consequently, many efforts have been devoted recently to obtain information about the dynamic movements or assembly of transcription factors directly from living cells. Therefore, we developed a labeling strategy, named versatile antibody-based imaging approach (VANIMA), in which fluorescently labeled antibodies are introduced into living cells to image specific endogenous proteins or posttranslational modifications. We were able to show that VANIMA can be used to study dynamical processes of fundamental biological mechanisms including factors of the transcription machinery as well as histone modifications in living human cancer cells using conventional or super-resolution microscopy. Hence, in the future VANIMA will serve as a valuable tool to uncover the dynamics of endogenous biological processes including transcription directly in single living cells

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Fu, Yubin, Feng Qiu, Fan Zhang, Yiyong Mai, Yingchao Wang, Shibo Fu, Ruizhi Tang, Xiaodong Zhuanga e Xinliang Feng. "A dual-boron-cored luminogen capable of sensing and imaging". Royal Society of Chemistry, 2015. https://tud.qucosa.de/id/qucosa%3A36054.

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A new dual-boron-cored luminogen ligated with a nitrogen-containing multidentate ligand and four bulky phenyl rings was readily synthesized. The unique molecular structure endows this BN-containing luminogen with rich photophysical properties in either solution or in the solid state, including a large Stokes shift, aggregation induced emission activity and reversible piezochromism. Furthermore, this BN-containing luminogen exhibits good capabilities for imaging living cells and sensing of fluoride anions.

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Neuhaus, Jochen, Birgit Schröppel, Martin Dass, Hans Zimmermann, Hartwig Wolburg, Petra Fallier-Becker, Thomas Gevaert, Claus J. Burkhardt, Do Hoang Minh e Jens-Uwe Stolzenburg. "3D-electron microscopic characterization of interstitial cells in the human bladder upper lamina propria". Universitätsklinikum Leipzig, 2017. https://ul.qucosa.de/id/qucosa%3A15544.

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1) Aims To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. 2) Methods Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. 3) Results 3View-SEM image stacks as large as 59µm x 59µm x 17µm (xyz) at a resolution of 16nm x 16nm x 50 nm and high resolution (5nm x 5nm x 10nm) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium sheet-like morphology. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. 4) Conclusions Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.

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Sharifian, Gh Mohammad. "Adsorption and Transport of Drug-Like Molecules at the Membrane of Living Cells Studied by Time-Resolved Second-Harmonic Light Scattering". Diss., Temple University Libraries, 2018. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/524558.

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Chemistry
Ph.D.
Understanding molecular interactions at the surfaces of cellular membranes, including adsorption and transport, is of fundamental importance in both biological and pharmaceutical studies. At present, particularly with respect to small and medium size (drug-like) molecules, it is desirable to gain an understanding of the mechanisms that govern membrane adsorption and transport. To characterize drug-membrane interactions and mechanisms governing the process of molecular uptake at cellular membranes in living organisms, we need to develop effective experimental techniques to reach quantitative and time-resolved analysis of molecules at the membrane surfaces. Also, we preferably want to develop label-free optical techniques suited for single-cell and live cell analysis. Here, I discuss the nonlinear optical technique, second-harmonic light scattering (SHS), for studying molecule-membrane interactions and transport of molecules at the membrane of living cells with real-time resolution and membrane surface-specificity. Time-resolved SHS can quantify adsorption and transport of molecules, with specific nonlinear optical properties, at living organisms without imposing any mechanical stress onto the membrane. This label-free and surface-sensitive technique can even differentiate molecular transport at individual membranes within a multi-membrane cell (e.g., bacteria). In this dissertation, I present our current research and accomplishments in extending the capabilities of the SHS technique to study molecular uptake kinetics at the membranes of living cells, to monitor bacteria membrane integrity, to characterize the antibacterial mechanism-of-action of antibiotic compounds, to update the molecular mechanism of the Gram-stain protocol, to pixel-wise mapping of the membrane viscosity of the living cells, and to probe drug-induced activation of bacterial mechanosensitive channels in vitro.
Temple University--Theses

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Heinlein, Thomas. "Development of methods for structure and function determination in living and fixated cells on the single-molecule level based on coincidence analysis and spectrally-resolved fluorescence lifetime imaging microscopy [SFLIM]". [S.l. : s.n.], 2005. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB11611929.

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34

Vodičková, Marie. "Měření indexu lomu a morfometrie živých buněk pomocí koherencí řízeného holografického mikroskopu". Master's thesis, Vysoké učení technické v Brně. Fakulta strojního inženýrství, 2018. http://www.nusl.cz/ntk/nusl-392854.

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This master’s thesis deals with the design of methodology for measurement of refractive index and thickness of living cells by coherence-controlled holographic microscope. The theoretical part summarises the holographic microscopy and its development at IPE FME BUT in Brno. The thesis focuses on the multimodal holographic microscope, its description, the principle, the procedure of work and data processing. Confocal microscopy is also described, which serves to compare the acquired values with the proposed methodology. The last part of the theoretical part deals with the testing of statistical hypotheses, which is needed for the processing of measured data. Experiments were designed for the verification of methodology for determination of the refractive index and cell thickness. The experimental part of the thesis deals with the sample preparation and measurement. The procedure and results of the proposed experiments and their evaluation follows.

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35

Hoque, Apu E. (Ehsanul). "Migration and invasion pattern analysis of oral cancer cells in vitro". Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526220239.

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Abstract Desmoglein 3 (Dsg3) is an adhesion receptor in desmosomes, but relatively little is known about its role in cancer. In this study, the function of Dsg3 was investigated in oral squamous cell carcinoma (SCC) cell lines in vitro using locally established human leiomyoma tumor microenvironment (TME) matrices. Since Dsg3 has been identified as a key regulator in cell adhesion, we hypothesized that it may play a role in oral SCC cells adhesion and motility. Thus, one aim of the study was to explore this hypothesis by both gain and loss of function methods in four human buccal mucosa SCC SqCC/Y1 cell lines: transduction of vector control (Ct), full-length (FL) or two different C-terminally truncated Dsg3 mutants (Δ238 and Δ560). Live cell imaging was performed for 2D migration and 3D sandwich, alongside other assays. In 3D sandwich, we tested the effects of the monoclonal antibody, AK23, targeting the extracellular domain of Dsg3 in SqCC/Y1 cells. Our results showed that loss of Dsg3 disrupted cell adhesion and protein expression. In 2D assays, FL and Dsg3 mutants migrated faster with higher accumulated distances than Ct. In contrast with 2D, mutants showed accelerated invasion over the Ct in 3D models. The AK23 antibody inhibited only the invasion of FL cells. The TME in vivo consists of cellular and matrix elements playing a leading role in carcinoma progression. To study carcinoma cells invasion in vitro, mouse Matrigel® and rat type 1 collagen are the most commonly used matrices in 3D models. Since they are non-human in origin, they do not perfectly mimic human TME. To address this, we have developed a solid organotypic myoma disc model derived from human uterus leiomyoma tumor. Here, we introduce a novel Myogel, prepared from leiomyoma similar to Matrigel®. We validated Myogel for cell-TME interactions in 3D models, using SqCC/Y1 and HSC-3 cell lines. Compared with Matrigel® and type I collagen, oral SCC cell lines invaded more efficiently in Myogel containing matrices. This study describes promising 3D models using human TME mimicking Myogel which is suitable to analyze oral SCC cells both in carcinoma monocultures and in co-cultures, such as with TME fibroblasts. We also introduce a possible novel therapeutic target against Dsg3 to suppress cancer cell invasion
Tiivistelmä Desmogleiini 3 (Dsg3) on desmosomien adheesioreseptori, jonka merkityksestä syövässä tiedetään vähän. Koska Dsg3 on tärkeä epiteelisolujen välisissä liitoksissa, oletimme sillä olevan vaikutusta myös suun karsinoomasolujen tarttumisessa ja niiden liikkuvuudessa. Testasimme hypoteesiamme muuttamalla Dsg3:n toimintaa ihmisen posken karsinoomasolulinjassa SqCC/Y1, josta oli aiemmin valmistettu neljä erilaista muunnosta: tyhjän vektorin sisältävä kontrollisolulinja (Ct), kokopitkää Dsg3 tuottava solulinja (FL), sekä kaksi Dsg3 C-päästä lyhennettyä mutanttisolulinjaa (Δ238 ja Δ560). Immunofluoresenssi-menetelmää käyttäen analysoimme solulinjoissamme solujen välisiä liitoksia. Lisäksi mittasimme solujen liikkeitä 2D-migraatio- ja 3D-sandwich-kokeissa. Testasimme myös Dsg3:n solunulkoista osaa tunnistavan monoklonaalisen vasta-aineen (AK23) vaikutusta solujen invaasioon. Osoitimme, että Dsg3:n rakenteen muuttaminen ja toiminnan estyminen häiritsi solujen tarttumista. 2D-kokeissa sekä FL että mutanttilinjat (Δ238 ja Δ560) migroivat kontrollisoluja nopeammin ja pidemmälle, mutta 3D-kokeissa vain mutanttilinjat invasoituivat kontrollisoluja tehokkaammin. AK23-vasta-aine esti vain FL-solujen invaasiota. Syöpäsolujen 3D-invaasiota mittaavissa kokeissa käytetään yleensä hiiren kasvaimesta valmistettua kaupallista Matrigeeliä® tai rotan kudoksista eristettyä tyypin I kollageenia. Tutkimusryhmämme on jo aiemmin kehittänyt organotyyppisen myoomamallin, jossa valmistamme myoomakudosnapit ihmisen kohdun leiomyoomakasvaimista. Tässä työssä valmistimme leiomyoomasta Myogeelia, vertasimme sitä Matrigeeliin®, sekä tutkimme tarkemmin Myogeeli-valmisteen soveltuvuutta 3D-tutkimuksiin. Totesimme, että kielen (HSC-3) ja posken (SqCC/Y1) karsinoomasolut invasoituivat tehokkaimmin Myogeeli-pitoisissa matrikseissa kuin Matrigeeliä® tai kollageeniä sisältävissä kasvatusalustoissa. Tutkimustulostemme perusteella Myogeeli-pohjaiset 3D-mallit soveltuvat hyvin sekä syöpäsolulinjojen invaasiotutkimuksiin että yhteisviljelmiin, joissa syöpäsoluja viljellään yhdessä syöpäkasvaimen ympärillä olevien solujen, kuten fibroblastien, kanssa

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Surampudi, Vasudha. "POLYSACCHARIDE-BASED SHEAR THINNING HYDROGELS FOR THREE-DIMENSIONAL CELL CULTURE". VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3872.

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The recreation of the complicated tissue microenvironment is essential to reduce the gap between in vitro and in vivo research. Polysaccharide-based hydrogels form excellent scaffolds to allow for three-dimensional cell culture owing to the favorable properties such as capability to absorb large amount of water when immersed in biological fluids, ability to form “smart hydrogels” by being shear-thinning and thixotropic, and eliciting minimum immunological response from the host. In this study, the biodegradable shear-thinning polysaccharide, gellan-gum based hydrogel was investigated for the conditions and concentrations in which it can be applied for the adhesion, propagation and assembly of different mammalian cell types in an unmodified state, at physiological conditions of temperature. Cell studies, to show successful propagation and assembly into three-dimensional structures, were performed in the range of hydrogels which were deemed to be optimum for cell culture and the cell types were chosen to represent each embryonic germ layer, i.e., human neural stem cells for ectoderm, human brain microvasculature cells for mesoderm, and murine β-cells for endoderm, along with a pluripotent cell line of human induced pluripotent stem cells, derived from human foreskin fibroblasts. Three-dimensional cell organoid models, to allow for gellan gum based bioprinting, were also developed using human induced pluripotent stem cells and human neural stem cells.

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Cheng, Sheng. "ATP induced intracellular calcium response and purinergic signalling in cultured suburothelial myofibroblasts of the human bladder". Doctoral thesis, Universitätsbibliothek Leipzig, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-88695.

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Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.

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38

Amin, Md Asif. "Time Resolved Spectroscopy and Microscopy: Application to Live Cells and Related Systems". Thesis, 2019. http://hdl.handle.net/10821/8243.

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In this thesis, we have applied time resolved confocal microscopy to five different processes, - intermittent oscillation of lipid droplets in live cells14, in situ imaging of cancer cell by fluorescent gold nanoclusters, selective killing of breast cancer cells, delivery of cytochrome c and hence imaging of live cell and effect of binary solvent mixture on structure and dynamics of a protein. Intermittent oscillation in live lung cell is observed using time resolved confocal microscopy and we have observed significant differences between normal and cancer cell. Imaging of cancer cell was done using in situ generated gold nanoclusters. We have shown how doxorubicin loaded gold nanoclusters (AuNC) selectively killed breast cancer cell. We have delivered cytochrome c, inside a cell by gold nanoclusters. We have studied the structure and dynamics of cytochrome c in water-ethanol binary mixture using FCS.
Research was conducted under the supervision of Prof. Kankan Bhattacharyya of the Physical Chemistry division under SCS [School of Chemical Sciences]
Research was carried out under DST grant and CSIR fellowship

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Rasmi, C. K. "Rapid light sheet fluorescence microscopy for dynamic imaging of living organisms". Thesis, 2018. https://etd.iisc.ac.in/handle/2005/4517.

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The primary goal of this thesis is to develop a light sheet based microscopy system that provides non-invasive images having high spatial and temporal resolution. The fluorescence microscopy has become an indispensable tool for biologists to understand the underlying mechanisms of various biological processes. The phenomenon of fluorescence offers non-invasive imaging with high speci ficity and single molecule sensitivity which is of interest in the life sciences research. The confocal microscopy has emerged as a potential technique which enables optical sectioning by employing a pinhole in the detector side to eliminate out of focus light. The confocal microscopy has been combined with other techniques such as multi-photon excitation microscopy to tackle scattering and to improve the penetration depth. The super resolution 4Pi microscopy is also combined with confocal detection to improve axial resolution. Past decade has seen the evolution of a large number of super resolution techniques such as STED, fPALM, PALM, STORM, SSIM, RESOLFT and GSDIM. This was a major breakthrough in fluorescence microscopy enabling single molecule resolution. Imaging of dynamical processes such as embryo development is an important and challenging problem in developmental biology. Long period monitoring is required to capture these processes which demand the sample to be kept in its natural environment with minimal interference from the probing light. The photobleaching and photodamage are the real issues when the sample is exposed to probing light for a long time. Imaging at high spatial and temporal resolution with minimal photodamage is a real challenge even for confocal and two photon excitation microscopy. Light sheet microscopy offers multitude of possibilities to the photobleaching problem and has found applications in various domains during the last decade. This thesis introduces an improvised light sheet microscopy technique where the number of images needed to construct 3D volume is greatly reduced by choosing an alternate acquisition strategy. The statistical image reconstruction techniques such as maximum likelihood (ML) approach is used for post-processing and has been shown to improve the image quality for the applications presented in the thesis. The introduction of light sheet illumination on a micro fluidic platform is also studied to enable 3D imaging of uni-cellular and multi-cellular organisms. A brief summary of the work is given below. Chapter 1 gives a brief outline of the developments in the field of fluorescence microscopy. The quest for improved resolution, contrast, penetration depth, speed and minimal damage to the sample has motivated researchers to come up with different microscopy designs which can tackle the aforementioned aspects. The emergence of light sheet microscopy is explained in detail as a promising tool in scenarios where the conventional techniques like confocal and multi-photon excitation microscopy are inadequate to overcome the challenges in various life science research areas. Chapter 2 is dedicated to explain the fundamentals of fluorescence. A brief outline is given to introduce various contrasts existing in optical microscopy, highlighting the advantages of fluorescence. Resolution of an imaging system is discussed in detail and the idea of the point spread function is introduced as it determines the performance of an optical system in terms of resolution. Some of the widely used fluorescence microscopy techniques are explained to provide an overview of developments happening in the fi eld of fluorescence microscopy. Image degradations due to blurring, noise and aberrations are inescapable in an imaging system. In general most of the microscopic samples are 3D objects. But what we acquire is a 2D image and can have ambiguities due to its 3D nature, like presence of out of focus features in the image. Hence the 2D image is a false representation of the 3D object. In order to tackle these issues image deconvolution techniques are usually employed. In chapter 3 we discuss about the state-of-the-art statistical image reconstruction technique maximum likelihood (ML) approach for image deconvolution. Due to its iterative nature ML algorithm is inherently slow. In order to process and view images in real-time, ML algorithm has to be accelerated. A step in this direction has been the incorporation of Biggs-Andrews algorithm into ML framework to accelerate ML. BA approach is based on vector extrapolation method which is a simple method without any derivative calculation and inherits automatic acceleration. We have tested the performance of the algorithm on microscopy images obtained from three techniques wide field, confocal and super-resolution 4Pi microscopy. The convergence is improved by a factor of two for all the tested images. Developmental biology is one of the promising areas which demands fast and high resolution imaging to understand various biological processes during embryo development. Light sheet florescence microscopy was introduced to tackle this highly challenging problem because of its inherent optical sectioning capability which helps in eliminating out of focus illumination. This reduces photobleaching and phototoxicity. We have developed limited view light sheet microscopy (LVLSM) in which the volume of zebra sh embryo is constructed from a very few angular views. Chapter 4 deals with LVLSM. The rotation and translation involved in multi-view microscopy is time consuming and hundreds of images are acquired to construct a 3D volume. In this work we have developed a scheme that uses only rotation to acquire the data. In addition, we have used ML algorithm to improve the contrast of the image and to remove the noise. We have reconstructed a ve day old zebra sh embryo using 18 views which is an order of magnitude less compared to the number of images used in multi-view light sheet microscopy. We study the effect of limited number of views in the 3D image reconstruction in the next chapter in the light of speeding up the imaging process as well as for reducing of photobleaching further. We have done the time-lapse imaging of a ve day old zebra sh embryo to study the effect of photobleaching. The uorescence decay curve is fitted with mono-exponential decay and the parameters are obtained. We have constructed 3D volume from 18, 9 and 6 angular views at 10o; 20o and 30o angular separation respectively and checked the performance in terms of contrast. The image quality with 18 and 9 views were found to be almost the same. However, there is a reduction in contrast for reconstruction with only 6 views. But even with 6 views, it is found that the structural details are retained in volume reconstruction. Intensity line plots are employed to quantitatively check the reconstruction with 18, 9 and 6 views and it is found that there is a good agreement between reconstructions with 18 and 9 views. In chapter 6 we have explored the light sheet illumination for the 3D imaging of organisms during ow on micro fluidic platform. Light sheet illumination can provide optical sectioning and hence it is possible to get 2D cross-sections of the sample during the fllow. The micro fluidic channel is kept at an angle with respect to the illumination axis and the illumination and detection arms are orthogonal. Optimization of the light sheet dimensions, ow parameters and camera settings facilitates 3D imaging without any translation of the sample. The performance of the system is checked by imaging samples of two different sizes, HeLa cells and C: elegans worms. In order to tackle the problem of motion blur, maximum likelihood algorithm is employed in which we have used experimentally measured PSF to deblur the image. PSF is estimated by owing nanobeads through the channel at the same ow rate as that of the sample. The reconstructed images show better contrast and less noise compared to raw images. The proposed system is promising for 3D imaging ow cytometry as well as for 3D imaging of live model organisms for high throughput screening. The conclusion for the thesis is given in chapter 7. Some of the prospects of the work is given as future scope.

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Leung, Tony Kin Shun. "A Contour Grouping Algorithm for 3D Reconstruction of Biological Cells". Thesis, 2009. http://hdl.handle.net/10012/4569.

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Advances in computational modelling offer unprecedented potential for obtaining insights into the mechanics of cell-cell interactions. With the aid of such models, cell-level phenomena such as cell sorting and tissue self-organization are now being understood in terms of forces generated by specific sub-cellular structural components. Three-dimensional systems can behave differently from two-dimensional ones and since models cannot be validated without corresponding data, it is crucial to build accurate three-dimensional models of real cell aggregates. The lack of automated methods to determine which cell outlines in successive images of a confocal stack or time-lapse image set belong to the same cell is an important unsolved problem in the reconstruction process. This thesis addresses this problem through a contour grouping algorithm (CGA) designed to lead to unsupervised three-dimensional reconstructions of biological cells. The CGA associates contours obtained from fluorescently-labeled cell membranes in individual confocal slices using concepts from the fields of machine learning and combinatorics. The feature extraction step results in a set of association metrics. The algorithm then uses a probabilistic grouping step and a greedy-cost optimization step to produce grouped sets of contours. Groupings are representative of imaged cells and are manually evaluated for accuracy. The CGA presented here is able to produce accuracies greater than 96% when properly tuned. Parameter studies show that the algorithm is robust. That is, acceptable results are obtained under moderately varied probabilistic constraints and reasonable cost weightings. Image properties – such as slicing distance, image quality – affect the results. Sources of error are identified and enhancements based on fuzzy-logic and other optimization methods are considered. The successful grouping of cell contours, as realized here, is an important step toward the development of realistic, three-dimensional, cell-based finite element models.

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Lord, Samuel Joseph Moerner W. E. Pande Vijay Zare Richard N. "Fluorophores for single-molecule imaging in living cells characterizing and optimizing DCDHF photophysics /". 2010. http://purl.stanford.edu/ct011cv6501.

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42

Ling, Jian. "The development of Raman imaging microscopy to visualize drug actions in living cells". Thesis, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3037519.

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43

Χρηστάκου, Αθανασία. "Cell culture and confocal fluorescence imaging of natural killer‐target cell interactions in multi‐well microdevices". Thesis, 2009. http://nemertes.lis.upatras.gr/jspui/handle/10889/4207.

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Abstract (sommario):

The ability of culturing cells in vitro has given many advantages in biological research and has become a standard methodology in drug discovery and toxicology. However traditional culturing methods give limited possibilities comparing to microfluidic systems. In order to understand the cellular mechanisms of Natural killers against virus infected cells and tumors, we developed a method for observing in parallel, high numbers of individual Natural killer-target cell conjugates in confined regions. An important advantage of this method is that it gives the possibility to keep track of large numbers of specific conjugates in a time scale of several days. Thus live cell imaging of NK-Target cell interactions in multi-well microstructures, can offer valid statistical information about NK cells processes that can lead to a better understanding of the function and regulation of the immune system.
Ανοσολογία είναι ο επιστημονικός κλάδος που διερευνά τους σύνθετους μηχανισμούς με τους οποίους το ανθρώπινο σώμα αντιδρά και καταπολεμά μολύνσεις ή δυσλειτουργίες που προέρχονται είτε από παθογόνα ή από μεταλλάξεις των κυττάρων του ίδιου του οργανισμού. Οι αντιδράσεις του ανοσοποιητικού συστήματος διαχωρίζονται σε εγγενείς και προσαρμοσμένες άνοσες αντιδράσεις ανάλογα με την ταχύτητα και την εξειδίκευση των αντιδράσεων αυτών ενάντια στα παθογόνα. Το εγγενές ανοσοποιητικό σύστημα αντιδρά άμεσα και συνήθως είναι αρκετά αποτελεσματικό ώστε να εξοντώσει το παθογόνο πριν προκαλέσει αρρώστια. Σε περιπτώσεις όπου η δραστικότητα το εγγενούς δεν είναι επαρκής, το προσαρμοσμένο ανοσοποιητικό σύστημα ενεργοποιείται με την βοήθεια του εγγενούς και χρησιμοποιώντας πολύ συγκεκριμένους μηχανισμούς με τη βοήθεια των οποίων παύει η διαδικασία της μόλυνσης. Τα φυσικά κύτταρα δολοφόνοι (Natural killer cells-NK) ανήκουν στο εγγενές ανοσοποιητικό σύστημα και παίζουν σημαντικό ρόλο στην προστασία του οργανισμού και την ρύθμιση του ανοσοποιητικού συστήματος. Βασικός στόχος της διπλωματικής εργασίας είναι η διερεύνηση των αλληλεπιδράσεων μεταξύ φυσικών κυττάρων δολοφόνων και κυττάρων στόχων. Τα κύτταρα στόχοι είναι είτε κύτταρα μολυσμένα με ιούς ή καρκινικά κύτταρα. Η αρχική υπόθεση ήταν ότι οι πληροφορίες σχετικά με τις λειτουργίες των NK κυττάρων είναι ευκολότερο να καταγραφούν και να αναλυθούν εκτενέστερα, αν μεγάλος αριθμός μεμονωμένων ζευγών ΝΚ-στόχων παρατηρηθούν ξεχωριστά σε περιορισμένο μικρόχωρο. Για την επίτευξη του σκοπού αυτού χρησιμοποιήθηκαν μικροσυσκευές πολυκυψελών εντός των οποίων καλλιεργήθηκαν ξεχωριστά για αρκετές μέρες κύτταρα στόχοι και κύτταρα δολοφόνοι, έτσι ώστε να ελεγχθεί η ζωτικότητα και η λειτουργικότητα των κυττάρων μέσα στους μικρόχωρους. Πιο συγκεκριμένα, για τον έλεγχο αυτό τα κύτταρα τοποθετήθηκαν στις κυψέλες και καλλιεργήθηκαν για 3-4 ημέρες. Κάθε μέρα μία συγκεκριμένη περιοχή της μικροσυσκευής παρατηρήθηκε σε απλό οπτικό μικροσκόπιο και τα κύτταρα μέσα στις κυψέλες μετρήθηκαν. Τα δεδομένα καταγράφηκαν σε μορφή πινάκων και επεξεργάστηκαν στο MatLab. Τα ιστογράμματα που κατασκευάστηκαν έδειξαν ότι η κατανομή των κυττάρων μέσα στις κυψέλες μεταβάλεται και ο συνολικός αριθμός τους αυξάνεται. Τα πειράματα σχετικά με τον έλεγχο του πολλαπλασιασμού των κυττάρων πραγματοποιήθηκαν για 3 διαφορετικούς τύπους, 221Cw6, Nishi και NKL. Εφόσον πρώτα έγινε ο έλεγχος βίο-συμβατότητας των κυττάρων στις μικροκυψέλες, στη συνέχεια κύτταρα δολοφόνοι και κύτταρα στόχοι επεξεργάστηκαν με ειδικές φθορίζουσες βαφές, τοποθετήθηκαν στις μικροσυσκευές και παρατηρήθηκαν με τη χρήση συνεστιακού φθορίζοντος μικροσκοπίου. Με χρήση ειδικής λειτουργίας του μικροσκοπίου, εικόνες συλλέχθηκαν κάθε1-3 λεπτά για 6-12 ώρες. Με τη χρήση της λειτουργίας αυτής ήταν δυνατή η παρακολούθηση των κινήσεων των κυττάρων μέσα στις κυψέλες και η καταγραφή της συμπεριφοράς τους και των γεγονότων κατά την διάρκεια του πειράματος. Έχοντας μεγάλο αριθμό κυψελών (60-100) σε κάθε πείραμα, υπήρξε η δυνατότητα παρατήρησης μεγάλου αριθμού γεγονότων εκ των οποίων κάποια ήταν εξαιρετικά σπάνια η ακόμα και μοναδικά. Λεπτομέρειες σχετικά με την μεθοδολογία των πειραμάτων, την καταγραφή και ανάλυση των αποτελεσμάτων, αναγράφονται αναλυτικά και επεξηγούνται στην παρούσα εργασία.

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44

Siao, Siou-Huei, e 蕭琇卉. "A Ratiometric Fluorescent Probe for Detecting Alkaline Phosphatase Activity and Imaging in Living Cells". Thesis, 2019. http://ndltd.ncl.edu.tw/handle/3c3462.

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碩士
國立交通大學
應用化學系碩博士班
108
Alkaline phosphatase (ALP) is a kind of hydrolase that can remove phosphate from biological molecules such as proteins, nucleic acids, and carbohydrates. It is widespread in human tissues including bone, liver, kidney, intestine, and placenta. Therefore, ALP is an important diagnostic indicator for some diseases such as hepatitis and osteoblastic bone cancer. We developed a ratiometric fluorescent probe DCP to detect ALP in living cells for in vivo ratiometric cell imaging. The probe DCP consists of a fluorophore DC containing conjugating double coumarins and a phosphate group as a recognizing group. In the presence of ALP, the emission of the probe DCP showed a bathochromic shift from 470 nm to 546 nm in the fluorescence spectra with an isosbestic point at 520 nm. The presence of ALP caused the hydrolysis of the phosphate group from the probe DCP that changed the intramolecular charge transfer (ICT) effect and resulted in a ratiometric fluorescence change. The ratiometric fluorescence response enables quantification of the concentration of ALP activity in a range of 0 mU/mL to 150 mU/mL with a detection limit of 0.051 mU/mL. The probe DCP also has good selectivity and low cell cytotoxicity. Furthermore, the probe DCP successfully applied to image endogenous ALP activity in living cells such as human hepatocellular carcinoma cells (HepG2) and zebrafish.

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45

Lo, Jocelyn. "Homo-FRET Imaging of CEACAM1 in Living Cells using Total Internal Reflection Fluorescence Polarization Microscopy". Thesis, 2012. http://hdl.handle.net/1807/33307.

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Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.

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46

Chang, Ming-Hsuan, e 張明軒. "Intracellular Imaging of Living Cells by Split Ring Resonators Microscopy in Near-Infrared (NIR) Region". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/92903265257628820971.

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47

Mao, Shu. "Design and development of new optical probes and imaging systems for fluorescence microscopy of living cells". 2008. http://www.library.wisc.edu/databases/connect/dissertations.html.

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48

Chen, Hsiang-Jung, e 陳相榮. "Near-Infrared Fluorescence Switchable Merocyanine Dye for Organelle-Specific Protein Labeling and Imaging in Living Cells". Thesis, 2017. http://ndltd.ncl.edu.tw/handle/u5s8h4.

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49

Ramadass, Radhan [Verfasser]. "The behavior of DASPMI in living cells : spectrally and spatially resolved fluorescence lifetime imaging / von Radhan Ramadass". 2008. http://d-nb.info/990862011/34.

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50

Bacher, Christian Peter [Verfasser]. "Computational imaging of dynamic nuclear processes in living somatic and germ line cells / presented by Christian Peter Bacher". 2005. http://d-nb.info/977560732/34.

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Tesi sul tema "Confocal imaging on living cells"

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