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Jaritz, Fritz Simon. "Single Cell Expansion Microscopy". Thesis, KTH, Tillämpad fysik, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-279445.
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Raabe, Isabel. "Visualization of cell-to-cell communication by advanced microscopy techniques". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-178404.
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In order to maintain a multicellular organism cells need to interact and communicate with each other. Signalling cascades such as the Bone Morphogenic Protein (BMP) and Hedgehog (Hh) signalling pathways therefore play essential roles in development and disease. Intercellular signalling also underlies the function of stem cell niches, signalling microenvironments that regulate behaviour of associated stem cells. Range and intensity of the niche signal controls stem cell proliferation and differentation and must therefore be strictly regulated. The testis and ovary of the fruit fly Drosophila melanogaster are established models of stem cell niche biology.
In the apical tip of the testis, germ line stem cell (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. While it is clear which signals regulate GSC maintenance it is unclear how these signals are spatially regulated. Here I show that BMP signalling is specifically activated at the interface of niche and stem cells. This local activation is possible because the transport of signalling and adhesion molecules is coupled and directed towards contact sites between niche and stem cells. I further show that the generation of the BMP signal in the wing disc follows the same mechanism.
Hh signalling controls somatic stem cell populations in the Drosophila ovary and the mammalian testis. However, it was unknown what role Hh might play in the fly testis, where the components of this signalling cascade are also expressed. Here I show that overactivation of Hh signalling leads to an increased proliferation and an expansion of the cyst stem cell compartment.
Finally, while the major components of the Hh signalling pathway are known, detailed knowledge of how signal transduction is implemented at the cell biological level is still lacking. Here, I show that localisation of the key signal transducer Smo to the plasma membrane is sufficient for phosphorylation of its cytoplasmic tail and downstream pathway activation. Using advanced, microscopy based biophysical methods I further demonstrate that Smo clustering is, in contrast to the textbook model, independent of phosphorylation.
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Ronteix, Gustave. "Inferring cell-cell interactions from quantitative analysis of microscopy images". Thesis, Institut polytechnique de Paris, 2021. http://www.theses.fr/2021IPPAX111.
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Les systèmes biologiques sont bien plus que la somme de leurs constituants. En effet, ils sont souvent caractérisés par des comportements macroscopiques complexes résultant de boucles d'interactions et de rétroactions. Par exemple, la régulation et le rejet éventuel des tumeurs par le système immunitaire est le résultat de multiples réseaux de régulation, influençant à la fois le comportement des cellules cancéreuses et immunitaires. Pour simuler ces effets complexes in-vitro, j'ai conçu une puce microfluidique permettant de confronter des sphéroïdes de mélanome à de multiples cellules T et d'observer les interactions qui en résultent avec une haute résolution spatio-temporelle et sur de longues périodes de temps. En utilisant de l'analyse d'images avancée, combinée à des modèles mathématiques, je démontre qu'une boucle de rétroaction positive conduit l'accumulation de cellules T sur la tumeur, ayant pour conséquence une fragmentation accrue des sphéroïdes. Cette étude met en lumière l'initiation de la réponse immunitaire à l'échelle de la cellule unique : elle montre que même le tout premier contact entre une cellule T et un sphéroïde tumoral augmente la probabilité que la cellule T suivante arrive sur la tumeur. Elle montre également qu'il est possible de récapituler des comportements antagonistes complexes in-vitro, ce qui ouvre la voie à l'élaboration de protocoles plus sophistiqués, impliquant par exemple un micro-environnement tumoral plus complexe.De nombreux processus biologiques sont le résultat d'interactions entre de multiples types de cellules, en particulier au cours du développement. Le foie fœtal est le lieu de la maturation et de l'expansion du système hématopoïétique, mais on sait peu de choses sur sa structure et son organisation. De nouveaux protocoles expérimentaux ont été récemment mis au point pour imager cet organe et j'ai développé des outils pour interpréter et quantifier ces données, permettant la construction d'un "réseau jumeau" de chaque foie fœtal. Cette méthode permet de combiner les échelles unicellulaire et de l'organe dans une seule analyse, révélant l'accumulation de cellules myéloïdes autour des vaisseaux sanguins irriguant le foie fœtal aux derniers stades du développement de l'organe. À l'avenir, cette technique permettra d'analyser précisément les environnements de cellules d'intérêt de manière quantitative. Ceci pourrait à son tour nous aider à comprendre les étapes du développement de types cellulaires cruciaux tels que les cellules souches hématopoïétiques.Les interactions entre les bactéries et leur environnement sont essentielles pour comprendre l'émergence de comportements collectifs complexes tels que la formation de biofilms. Un mécanisme d'intérêt est celui de la rhéotaxie, par lequel le mouvement bactérien est entraîné par les gradients de la contrainte de cisaillement du fluide dans lequel les cellules se déplacent. J'ai développé une méthode pour calculer les équations semi-analytiques guidant le mouvement des bactéries dans la contrainte de cisaillement. Ces équations prédisent des comportements qui ne sont pas observés expérimentalement, mais la divergence est résolue une fois que la diffusion rotationnelle est prise en compte. Les résultats expérimentaux correspondent bien à la prédiction théorique : les bactéries dans les gouttelettes se séparent de manière asymétrique lorsqu'un cisaillement est généré dans le milieuIn his prescient article “More is different”, P. W. Anderson counters the reductionist argument by highlighting the crucial role of emergent properties in science. This is particularly true in biology, where complex macroscopic behaviours stem from communication and interaction loops between much simpler elements. As an illustration, I hereby present three different instances in which I developed and used quantitative methods in order to learn new biological processes.For instance, the regulation and eventual rejection of tumours by the immune system is the result of multiple positive and negative regulation networks, influencing both the behaviour of the cancerous and immune cells. To mimic these complex effects in-vitro, I designed a microfluidic assay to challenge melanoma tumour spheroids with multiple T cells and observe the resulting interactions with high spatiotemporal resolution over long (>24h) periods of time. Using advanced image analysis combined with mathematical modelling I demonstrate that a positive feedback loop drives T cell accumulation to the tumour site, leading to enhanced spheroid fragmentation. This study sheds light on the initiation if the immune response at the single cell scale: showing that even the very first contact between T cell and tumour spheroid increases the probability of the next T cell to come to the tumour. It also shows that it is possible to recapitulate complex antagonistic behaviours in-vitro, which paves the way for the elaboration of more sophisticated protocols, involving for example a more complex tumour micro-environment.Many biological processes are the result of complex interactions between cell types, particularly so during development. The foetal liver is the locus of the maturation and expansion of the hematopoietic system, yet little is known about its structure and organisation. New experimental protocols have been recently developed to image this organ and I developed tools to interpret and quantify these data, enabling the construction of a “network twin” of each foetal liver. This method makes it possible to combine the single-cell scale and the organ scale in the analysis, revealing the accumulation of myeloid cells around the blood vessels irrigating the foetal liver at the final stages of organ development. In the future, this technique will make it possible to analyse precisely the environmental niches of cell types of interest in a quantitative manner. This in turn could help us understand the developmental steps of crucial cell types such as hematopoietic stem cells.The interactions between bacteria and their environment is key to understanding the emergence of complex collective behaviours such a biofilm formation. One mechanism of interest is that of rheotaxis, whereby bacterial motion is driven by gradients in the shear stress of the fluid the cells are moving in. I developed a framework to calculate the semi-analytical equations guiding bacteria movement in shear stress. These equations predict behaviours that aren’t observed experimentally, but the discrepancy is solved once rotational diffusion is taken into account. Experimental results are well-fitted by the theoretical prediction: bacteria in droplets segregate asymmetrically when a shear is generated in the media.Although relating to very different topics, these three studies highlight the pertinence of quantitative approaches for understanding complex biological phenomena: biological systems are more than the sum of their constituents.a
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Sjögren, Florence. "Dermal cell trafficking : from microscopy to microdialysis /". Linköping : Univ, 2005. http://www.bibl.liu.se/liupubl/disp/disp2005/med883s.pdf.
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Samsuri, Fahmi B. "Single Cell analysis using AtomicForce Microscopy (AFM)". Thesis, University of Canterbury. Electrical and Computer Engineering, 2010. http://hdl.handle.net/10092/5516.
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Replication of biological cells for the purpose of imaging and analysis under
electron and scanning probe microscopy has facilitated the opportunity to
study and examine some molecular processes and structures of living cells in
a manner that were not possible before. The difficulties faced in direct
cellular analysis when using and operating Atomic Force Microscopy (AFM)
in situ for morphological studies of biological cells have led to the
development of a novel method for biological cell studies based on
nanoimprint lithography. The realization of the full potential of high
resolution AFM imaging has revealed some very important biological events
such as exocytosis and endocytosis. In this work, a soft lithography
Bioimprint replication technique, which involved simple fabrication steps,
was used to form a hard replica of the cell employing a newly developed
biocompatible polymer that has fast curing time at room temperature
essential for this process. The structure and topography of the rat muscle cell
and the endometrial (Ishikawa) cancer cell were investigated in this study.
Cells were cultured and incubated in accordance with standard biological
culturing procedures and protocols approved by the Human Ethics
Committee, University of Otago. An impression of the cell profile was
created by applying a layer of the polymer onto the cells attached to a
substrate and rapidly cured under UV-light. Fast UV radiation helps to lock
cellular processes within seconds after exposure and replicas of the cancer
cells exhibit ultra-cellular structures and features down to nanometer scale.
Elimination of the AFM tip damping effects due to probing of the soft
biological tissue allows imaging with unprecedented resolution. Highxx
resolution AFM imagery provides the opportunity to examine the structure
and topography of the cells closely so that any abnormalities can be
identified. Craters that resemble granules and features down to 100 nm were
observed. These represent steps on a transitional series of sequential
structures that indicate either an endocytotic or exocytotic processes, which
were evident on the replicas. These events, together with exocytosis, play a
very significant part in the tumorigenesis of these cancer cells. By forming
cell replica impressions, not only have they the potential to understand
biological cell conditions, but may also benefit in synthesizing three
dimensional (3-D) scaffolds for natural growth of biological cells and
providing an improvement over standard cell growth conditions. Further
examinations by observing the characteristic behaviour of the plasma
membrane when the cells were induced by certain compound such as cobalt
chloride (CoCl2) under control and stimulated conditions have brought in the
opportunity to examine the effect of this stimulant in inducing apoptosis in
many different kinds of cells. Numbers of pores formed on the cells
membrane were found to increase significantly after the cells where induced
with CoCl2 that correlated well with the level of vascular endothelial growth
factor (VEGF) receptors expression, which contributed to tumour growth.
This indicates CoCl2 has exaggerated the expression of the VEGF growth
factor. Investigations were also done to the cells using functionalized nanoparticles
as bio-markers to establish the connection between exocytosis with
nanopores found on the membrane surfaces of the cells. These microbeads
were found attached to sites surrounding the nucleus of the cell and higher
numbers of visible beads would confirm that there was an up-regulation of
the VEGF expression in cells induced by CoCl2. All these can contribute to
expanding the knowledge about exocytosis and fundamental physiology of
cells, and also assist in understanding diseases especially cancer.
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Sun, Mingzhai. "Cell mechanics studied using atomic force microscopy". Diss., Columbia, Mo. : University of Missouri-Columbia, 2008. http://hdl.handle.net/10355/5499.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2008.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on June 17, 2009) Vita. Includes bibliographical references.
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Nguyen, Tran Thien Dat. "Bayesian Multi-Object Tracking for Cell Microscopy". Thesis, Curtin University, 2021. http://hdl.handle.net/20.500.11937/86947.
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Cell tracking is an essential tool for studying how cells behave and divide under different conditions. This thesis proposes new approaches to track cells and their lineages using random finite set, which allows the tracking errors to be statistically quantified. Additionally, this thesis also explores criteria to rank performance of basic vision task algorithms (e.g., object detection, instance-level segmentation, and tracking), which have not been received proportionate attention from the scientific community.
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López, Ayón Gabriela. "Applying a commercial atomic force microscope for scanning near-field optical microscopy techniques and investigation of Cell-cell signalling". Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=92400.
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The field of research of this thesis is Condensed Matter Physics applied to Biology. Specifically it describes the development of different Atomic Force Microscopy techniques and tools towards the study of living cells in physiological solution. Particular interest is put into the understanding of the influence of noise in the determination of ordered liquid layers above a mica surface - as work towards the study of the role of water and ions in biological processes - and the influence of "diving bell" to boost the Q factor and allow stable imaging and force spectroscopy with tips based on Scanning Near-field Optical Microscopy [LeDue, 2010 and LeDue, 2008]. By combining SNOM techniques as a local illumination method (and thus avoiding photo bleaching of individual molecules) and high resolution AFM techniques we will be able to investigate mechano-transduction and associated signaling in living cells and individual proteins.Le domaine de recherche de cette thèse consiste en l'application de la physique de la matière condensée à la biologie. Plus précisément, ce travail décrit le développement de différentes techniques de Microscopie à Force Atomique (MFA) et d'outils permettant l'étude de cellules vivantes en solution physiologique. Un intérêt particulier est porté à la compréhension de l'influence du bruit dans la détermination de couches liquides ordonnées au-dessus d'une surface de mica - en tant que travail préalable à l'étude du rôle de l'eau et des ions dans les processus biologiques - et de l'influence d'une "cloche de plongée" pour renforcer le facteur Q ainsi que pour permettre l'imagerie stable et la spectrométrie de force avec des sondes basées sur la Microscopie Optique en Champ Proche (MOCP). En combinant des techniques MOCP, utilisées comme méthode d'éclairement local (évitant ainsi le photoblanchiment des molécules individuelles), et des techniques MFA haute résolution, nous serons capables d'investir la mécano-transduction et le signalement associé dans des cellules vivantes et dans des protéines individuelles.
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Makarchuk, Stanislaw. "Measurement of cell adhesion forces by holographic microscopy". Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAE034/document.
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Les forces mécaniques, générées par la cellule jouent un rôle crucial dans l'adhésion cellulaire, qui est un processus commun à un grand nombre de lignées cellulaires. Afin de mesurer la champ des forces pendant l'adhérence cellulaire, nous utilisons la microscopie de force de traction, où la cellule adhère à la surface plane d'un substrat souple dans le plan. Les forces sont calculées à partir du champ de déplacement mesuré à l'intérieur du substrat sous la cellule. Nous avons construit le microscope, dans lequel nous utilisons des billes sphériques en polystyrène pour mesurer le champ de déplacement. Les positions des marqueurs sont obtenues en analysant I' image interférentielle des particules. Avec cette technique, nous atteignons une précision nanométrique sur le champ de déplacement des particules, ce qui nous permet d'améliorer la résolution en force de ce type de microscope. Les premières mesures ont été effectuées avec la lignée de cellules cancéreuses SW 480Mechanical forces, generated by the cell plays crucial role in cell adhesion - common process for different cell lines. ln order to measure the force map during cellular adhesion, we use Traction Force Microscopy (TFM), where cell adheres to the soft substrate in 20 plane, and the forces are calculated from measured displacement field inside the substrate underneath the cell. We built the microscope, where instead of using fluorescent markers, we use spherical polystyrene beads in order to measure the displacement field. Positions of the markers are obtained by analyzing the interference pattern caused by the beads in bright-field light. With this technique, we reach nanometer accuracy of the microsphere position determination, that, respectively, influence accuracy of the calculated force field. With the microscope first measurements were performed with cancer cell line SW 480
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Magnusson, Klas. "Cell tracking for automated analysis of timelapse microscopy". Thesis, KTH, Signalbehandling, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-53772.
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This document presents an algorithm to automatically perform two dimensional tracking of cells in in-vitro cultures. The developed software handles all the necessary data processing, from preprocessing the images to automaticallytracking the cells and it also provides an interface to manually correct the obtained cell trajectories and functions to analyze the data. The system is developed for, and tested on, muscle stem cells (MuSCs) but it can also be applied to other cell types that look and behave similarly. The software was used in a bio-medical study to investigate the effects on mouse MuSC fate caused by culturing the cells on substrates of different rigidities. In this study the software enabled important findings about cell behavior. The software is capable of handling automatic track initialization, false detections, adhering cells, death and cell division. These are functionalities that can all be problematic to achieve. Cell tracking is normally done manually, which is very labor intensive and limits the parameters that can be analyzed. Having reliable systems to analyze a wide range of cell types automatically would therefore greatly benefit research in cell biology. The software package described here was named the Baxter Algorithm after the Donald E. & Delia B. Baxter Foundation that funded it’s development.
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Round, Andrew Neal. "Atomic force microscopy of plant cell wall polysaccharides". Thesis, University of East Anglia, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297475.
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Pulleine, Ellie Mui Mui. "Developing cell identification methods using atomic force microscopy". Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8074/.
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This body of work describes the development of a non-invasive and label-free method for characterization of cell surface markers. The motivation for such a method is the ability to measure cells whilst maintaining function, minimizing contamination and disturbance but enabling downstream applications. The technique would impact on life sciences applications including; phenotype identification of both individual and populations of cells, dynamic measurement of cellular response and monitoring cell-microenvironment interactions. The method described centers on molecular recognition interactions which are associated with specific binding forces. These specific forces can be measured in a highly sensitive manner using force instruments. In this study atomic force microscopy (AFM) was employed because of its powerful capability of highly sensitive force measurement at a nanoscale spatial resolution. The objective to develop a force based method for characterization of cell surface molecules may be considered in more specific aims; the development of a functional AFM probe for identification of specific molecules and establishment of quantitative measurement of surface markers. The probe developed has a colloidal geometry which encourages multivalent binding due to greater contact areas, which can reveal presence on cells in just few measurements. On non-deformable surfaces few interactions occur and regular force increments and probability of unbinding indicate presence of target molecules. With multivalent interactions on deformable samples other variables of adhesion indicate identification of interactions; namely distance of total separation, total peaks of unbinding and energy for total separation. With these variables, the identity of HeLa and HFF1 cells was indicated by cluster of differentiation markers 24, 44 and 98 in a semi-quantitative manner. Additionally individual mesenchymal stems cells are identified by the presence of cluster of differentiation marker 90 and dynamic measurement of Human Leukocyte Antigen. Single-cell force spectroscopy was employed to investigate cellular binding to cancerous matrices to gain greater understanding of tumour angiogenesis. Total internal reflection fluorescence microscopy was employed to inform the experimental setting of contact area and sampling density. The method developed illustrates the potential of force based measurement for label-free, non-invasive measurements on cells. Further development and automation may allow the dynamic measurement of multiple markers. This would allow for a number of applications; the identification of true stem cell clones which is of great importance for stem cells therapies, for monitoring of differentiation, where both short and long term activations could be investigated.
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Rong, Guoxin. "Probing cell membrane dynamics using plasmon coupling microscopy". Thesis, Boston University, 2013. https://hdl.handle.net/2144/12840.
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Thesis (Ph.D.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.The plasma membrane of mammalian cells is depicted as a two-dimensional hybrid material which is compartmentalized into submicron-sized domains. These membrane domains play a pivotal role in cellular signaling processes due to selective recruitment of specific cell surface receptors. The structural dynamics of the membrane domains and their exact biological functions are, however, still unclear, partially due to the wave nature of light, which limits the optical resolution in the visible light to approximately 400 nm in conventional optical microscopy. Here, we provide a non-fluorescence based approach for monitoring distance changes on subdiffraction limit length scales in a conventional far-field optical microscope. This approach, which is referred to as plasmon coupling microscopy (PCM), utilizes the distance dependent near-field coupling between noble metal nanoparticle (NP) labels to resolve close contacts on the length scale of approximately one NP diameter. We firstly utilize this PCM strategy to resolve interparticle separations during individual encounters of gold NP labeled fibronectin-integrin complexes in living Hela cells. We then further refine this ratiometric detection methodology by augmenting it with a polarization-sensitive detection, which enables simultaneous monitoring of the distance and conformation changes in NP dimers and clusters. We apply this polarization resolved PCM approach to characterize the structural lateral heterogeneity of cell membranes on submicron length scales. Finally, we demonstrate that PCM can provide quantitative information about the structural dynamics of individual epidermal growth factor receptor (ErbB1)-enriched membrane domains in living cells.
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Tomalik, Edyta. "Image-based Microscale Particle Velocimetry in Live Cell Microscopy". Thesis, Blekinge Tekniska Högskola, Institutionen för programvaruteknik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:bth-2564.
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Background: Nowadays, one of the medical problem is rolling cell adhesion. Rolling cell adhesion is a complex process that requires the analysis of the challenging environment such as body fluid and is the process responsible for recruiting the cell to specific organs. In order to explore the rolling cell adhesion, mathematical model is proposed. Different image processing methods are created, such as optical flow - Lucas Kanade algorithm, and other type of methods related to mechanical fluid, namely PIV (Particle Image Velocimetry). Aim: The aim of this master thesis is the identification of challenges while using PIV in live cell images and propose the algorithm, which may analyze the rolling cell adhesion problem. Methods: In order to understand properly the rolling cell adhesion problem from biological site, literature review combined with the expert consultation is performed. According to gather information, mathematical model is proposed. Particle Image Velocimetry is explained according to literature review, where at the beginning the expert recommends some books as a primary research. As a result of this research, PIV challenges are identified and generally PIV idea is explained. Then two experiments are performed. The first experiment evaluates detection algorithms and the second one, analyses track algorithm vs. PIV. In order to evaluate the mentioned algorithms, some evaluation method are selected and some criteria are defined. Unfortunately the found methods are not perfect, therefore a new method related to performance evaluation using time series is proposed. Thesis result: The result of this thesis is a proposition of the algorithm, which can be used in the rolling cell adhesion. The algorithm is formed according to the detailed exploration of the rolling cell adhesion and analysis of the selected algorithms related to the image analysis during the theoretical research and experiments.
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Morris, Sheila. "Atomic force microscopy studies of plant cell wall components". Thesis, University of Bristol, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420915.
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Bartolini, Luca <1989>. "Investigation of Cell-material Interactions by Scanning Probe Microscopy". Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2017. http://amsdottorato.unibo.it/8198/1/bartolini_luca_tesi.pdf.
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The morphology of cells changes consistently with the surface where they adhere. As reported in the literature, surfaces with micrometric and nanometric patterns affect the cell morphology, as well as surfaces with peculiar chemical functionalities. In order to control both morphology and chemistry of the surface, mono-molecular layers of small organic molecules (specifically Pentacene, α-sexithiophene and PDI8-CN2) were deposited on SiOx substrates by means of Organic Molecular Beam Epitaxy (OMBE). Through the partial annealing method, SiOx substrates were fully covered with a mono-molecular layer, as confirmed by Atomic Force Microscopy measurements (surface coverage of about 98%). Such molecules enable SiOx substrates to become biocompatible and to have flat morphologies with selective chemical functionalities. Epithelial cells were cultivated on such samples and their structure and shape has been investigated by optical and fluorescence microscope and Scanning Electrochemical Microscopy (SECM).
In the last years, Scanning Probe Microscopies (SPM) techniques have been increasingly used to investigate important biological issues. In order to best apply to the characteristics of these techniques in biological and medical fields, the probes used for imaging have to be: i) cheap and disposable as they can be easily contaminated and damaged during their use; ii) small enough to resolve the investigated biological phenomena or object; iii) reproducible in their aspect and imaging capability. In this work a new fabrication method was proposed and the probes obtained comply with all the aforementioned requirements.
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Arora, Bhupinder S. "Detection of polysaccharides on a bacterial cell surface using Atomic Force Microscopy". Link to electronic thesis, 2003. http://www.wpi.edu/Pubs/ETD/Available/etd-0826103-011111.
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Thesis (M.S.)--Worcester Polytechnic Institute.Keywords: Leuconostoc mesenteroides NIRC1542; Atomic Force Microscope; Pseudomonas putida KT2442; Adhesion. Includes bibliographical references (p. 75-83).
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Harvey, Taylor R. "Assessment of VE-Cadherin Stability at Endothelial Cell-Cell Junctions Using Photoconvertible Fluorescence Microscopy". Thesis, Albany Medical College, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=13422975.
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Regulation of barrier function is critical for patients who suffer from inflammatory diseases such as acute respiratory distress syndrome (ARDS) and sepsis. A major regulator of endothelial barrier function is vascular endothelial cadherin (VE-cad). Cellular levels of VE-cad are known to be regulated by p120 catenin. Loss of p120 leads to decreased barrier function as a result of the endocytosis of VE-cad. However, recent work from our lab shows that expression of an endocytic defective VE-cad mutant was not able to rescue barrier function, as measured using transendothelial electrical resistance (TEER). In contrast, expression of a non-phosphorylatable VE-cad mutant was able to restore barrier function independent of p120 binding. These results suggest that endocytosis is not the only mechanism regulating VE-cad localization to the cell-cell junctions, but rather the phosphorylation state of the protein may play a more critical role to stabilizing VE-cad at the junction. In order to investigate junctional stability of VE-cad, we created a recombinant form of VE-cad by cloning mEos2 into a plasmid containing the VE-cad gene. This fluorophore is photoconvertible, thus allowing for tracking protein movement at the cell-cell junction. The VE-cad proteins, labeled with mEos2 at the C-terminus, were introduced via adenoviral infection into human umbilical vein endothelial cells (HUVEC). Initially, mEos2 fluoresces green, in order to induce photoconversion, a 405nm laser is directed in a specific region of interest (ROI) at the junction. A conformational change in the mEos2 protein will cause irreversible red fluorescence. Tracking the change in fluorescence intensity in the ROI will provide insight into the localization of VE-cad at endothelial cell junctions. We now have a model that can be used to test junctional localization and stability of endocytic defective and non-phosphorylatable mutants of VE-cad.
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Kosmacek, Elizabeth Anne Ianzini Fiorenza Mackey Michael A. "Live cell imaging technology development for cancer research". [Iowa City, Iowa] : University of Iowa, 2009. http://ir.uiowa.edu/etd/388.
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Cheng, Eric. "Investigations into inkjet cell printing hydrodynamics through microscopy imaging techniques". Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/52776.
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Inkjet bioprinting technology aims to accurately and precisely dispense biological materials in a spatially predefined pattern within a three-dimensional space. The technology has a multitude of applications in the biomedical field such as in drug discovery and tissue or organ engineering. However, there are known limitations in an inkjet nozzle's capabilities in dispensing cells as the cell ejection rate does not follow any predictable distributions. In this work, the cell behaviors within a piezoelectric nozzle due to droplet ejection were classified through high speed brightfield imaging. With each ejected droplet, one of three cell behaviors was observed to occur: cell travel, cell ejection, or cell reflection. Cell reflection is an undesirable phenomenon which may adversely affect an inkjet's capability to reliably dispense cells. To further study how the hydrodynamics within a nozzle can influence the cell's behavior, µPIV was performed to identify the flow field evolution during droplet ejection. Through the study of cell motion, it was observed that the viscosity of the media in the cell suspension plays an important role in influencing the cell behavior. This was experimentally studied with the tracking of cells within the inkjet nozzle in a higher viscosity 10% w/v Ficoll PM400 cell suspension. As hypothesized, the addition of Ficoll PM400 was effective in preventing the occurrence of cell reflection which promises to increase the reliability in inkjet bioprinting systems.Applied Science, Faculty of
Graduate
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Panday, Namuna. "Scanning Ion Conductance Microscopy for Single Cell Imaging and Analysis". FIU Digital Commons, 2017. http://digitalcommons.fiu.edu/etd/3477.
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Most biological experiments are performed on an ensemble of cells under the assumption that all cells are identical. However, recent evidence from single cells studies reveals that this assumption is incorrect. Individual cells within the same generation may differ dramatically, and these differences have important consequences for the health and function of the entire living body. I have used Scanning Ion Conductance Microscopy (SICM) for imaging and analysis of topographical change of single cell membrane, which is difficult to be revealed by optical microscopes. Morphological change in the fixed and live HeLa cell membrane during endocytosis of conjugated polymer nanoparticles was studied. Results demonstrated SICM is a powerful tool to study the interaction between nanoparticle and cell membrane during internalization of nanoparticles through the membrane. This research can improve our fundamental understanding of cellular behavior and will be helpful for drug delivery applications.
Based on conventional SICM, we have developed a novel method to simultaneous map the topography and potential distributions of the single living cells membranes. At the first step, multifunctional nanopipettes (nanopore/nanoelectrode) have been fabricated and characterized. To demonstrate the potential sensing capability and understand the mechanism, I measured the ionic current and local electric potential change during translocation of 40 nm charged gold nanoparticles. Our results reveal the capability of the multifunctional probe for the highly sensitive detection of the ionic current and local electrical potential changes during the translocation of the charged entity through the nanopore. From the potential change, we revealed the dynamic assembly of GNPs before entering the nanopore. The experimental results are also nicely explained by the finite element method based numerical simulation results.
At the second step, I have measured the surface potential of living cell membrane at selected locations. Very recently, I have obtained results to show that we can map the extracellular membrane potential distribution of the complicated living cell membrane with sub-micron spatial resolution.This new imaging technique can help biologist to explore the extracellular potential distribution of varieties of cells quantitatively.These studies will have impacts on several biomedical applications such as regenerative repair and cancer treatment.
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CHIGHIZOLA, MATTEO. "INVESTIGATION OF CELL-MICROENVIRONMENT INTERACTIONS BY ATOMIC FORCE MICROSCOPY TECHNIQUES". Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/819943.
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The role of forces is fundamental in a wide variety of biological processes, such as cell adhesion, migration, proliferation and differentiation. The ability of cells to perceive the nanotopographical features of the surrounding microenvironment (i.e. the extracellular matrix, ECM), called mechanotransduction, is mediated by specific trans-membrane proteins, called integrins, clustered together in Integrin Adhesion Complexes (IAC). Unraveling which mechanical and nanoscale morphological properties of the ECM determine the IAC composition and tune specific cellular response, is particularly challenging.
Works have shown that biocompatible nanostructured thin films, grown by assembling zirconia nanoparticles (ns-ZrO2) on a substrate by Supersonic Cluster Beam Deposition technique, possess a morphological disorder on nanometer scale with structural features that mimic topographical properties of the ECM. Experiments performed with the neuron-like cell line PC12 demonstrated a link between the nanotopography of the ns-ZrO2 films and mechanotransductive events, which eventually foster neuronal differentiation.
During my three years of PhD, I have developed novel approach based on Atomic Force Microscopy (AFM) to quantify cell sensing of nanotopographical features of the microenvironment, represented by ns-ZrO2 films reproducing the nanostructured surface of the ECM. Using custom ns-ZrO2–coated colloidal probes, we have carried out a quantitative analysis of adhesion strength and distribution of IACs by AFM-based adhesive force spectroscopy.
We deposited ns-ZrO2 on custom AFM colloidal probes5. Bringing these nanostructured colloidal probes into contact with the body of living PC12 cells, it was possible to measure the strength, number and distribution of the IAC bonds by AFM force spectroscopy6, for different morphological properties of the interface. Furthermore, I used these functionalized probes to characterize the role of the surface pericellular layer, known as the Glycocalyx, in relation to the cell capability to react to external stimuli.
Eventually, along with my personal research project, I had the opportunity to participate to two external collaboration with the Istituto Nazionale dei Tumori and with Istitute of Nuclear Physiscs from the Polish academy of Science. These collaborations aimed to exploit the knowledge in the mechanobiolgy field to study the mechanical implications of cells and tissues in cancer development and survival.
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Altinoglu, Ipek. "Organization of Bacterial Cell Pole". Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS367/document.
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Chez les bactéries, les pôles cellulaires servent de domaines subcellulaires impliqués dans plusieurs processus cellulaires. Chez l’agent pathogène du choléra, Vibrio cholerae, en forme de bâtonnet incurvé, le pole contenant l’unique flagelle est impliqué dans la virulence. La protéine d’ancrage polaire HubP interagit avec plusieurs ATPases telles que ParA1 (ségrégation des chromosomes), ParC (localisation polaire du système de chimiotaxie) et FlhG (biosynthèse des flagelles), organisant ainsi l'identité polaire de V. cholerae. Cependant, les mécanismes moléculaires exacts de cet ancrage polaire doivent encore être élucidés. L’objectif de cette thèse est d’établir une vue d'ensemble de l'organisation de pôle cellulaire ce qui implique le mécanisme d’orchestration des différentes fonctions cellulaires par l’identification de l’ensemble des partenaires d'interaction de HubP ainsi que la cartographie fine du pôle cellulaire par microscopie à super résolution (PALM). Afin d’identifier de nouveaux partenaires d'interaction de HubP, j'ai étudié la différence de composition en protéines polaires entre les contextes HubP+ et HubP-. La composition en protéines polaires a été quantifiée de manière relative et absolue en ajoutant des Tag isobares aux protéines extraites de mini-cellules. Ces mini-cellules correspondent des petits compartiments cellulaires issus d’un évènement de division anormal proche du pole et sont enrichies en protéines polaires. Parmi ~800 protéines identifiées, ~ 80 protéines ont été considérées comme enrichies en contexte HubP+ incluant de nombreuses protéines attendues (FlhG, ParC et en aval des protéines de chimiotaxie). J'ai étudié la localisation de 14 protéines par microscopie à fluorescence et pu révéler 4 nouvelles protéines présentant une localisation polaire dépendant de HubP : VbrX, VbrY, et 2 protéines hypothétiques MotV et MotW. La délétion de motV et motW provoque un défaut significatif de propagation dans une gélose molle suggérant une implication dans la chimiotaxie et/ou la motilité. Alors que la microscopie électronique a montré que les deux mutants ont bien un flagelle polaire unique, le suivi-vidéo de leur déplacement a révélé que les deux mutants présentaient des défauts de nage assez distincts: ∆motV est plutôt affecté dans le changement de direction et ∆motW dans la vitesse de déplacement. Des expériences de microscopie fluorescente ont montré que MotV, MotW et HubP présentaient des dynamiques de localisation polaire distinctes au cours du cycle cellulaire. Pour une observation fine du pôle cellulaire par PALM, de nouveaux outils d’analyse d’image à haut débit étaient exigés. La précision des contours des petites cellules bactériennes faiblement contrastées n’est pas suffisante par l’observation en fond clair, j'ai développé une nouvelle technique de marquage avec des protéines fluorescentes photo-activables pour un tracé précis de la membrane interne ou du périplasme. En outre, nous avons créé un logiciel utilisant Matlab appelé Vibio qui intègre le contour de cellule et la liste des molécules obtenues par microscopie à super résolution. La capacité d’analyse à haut débit du logiciel permet d’étudier la distribution des molécules de l’échelle de la cellule unique à une population en orientant les cellules par leur courbure longitudinale. J’ai pu révéler que HubP est principalement localisé du côté convexe du pôle de la cellule, tandis que ses partenaires se situaient principalement au milieu du pôle. Mon travail de thèse a révélé avec succès de nouveaux partenaires d'interaction de HubP et la fonction de certaines protéines dans la motilité cellulaire. J'ai développé une nouvelle technique de microscopie pour une localisation subpolaire précise qui fonctionne bien pour l'analyse d'images PALM dans Vibio. J’ai ainsi pu faire progresser les connaissances de l’orchestration des fonctions polaires chez V. choleraeIn rod shaped bacteria, cell poles serve as important subcellular domains involved in several cellular processes including motility, chemotaxis, protein secretion, antibiotic resistance, and chromosome segregation. In the cholera pathogen Vibrio cholerae, vibrioid rod shape and single polarized flagellum involve in the virulence. Polar landmark protein HubP was shown to interact with multiple ATPases, such as ParA1 (chromosome segregation), ParC (polar localization of chemotaxis apparatus), and FlhG (flagella biosynthesis), thus organizing the polar identity of V. cholerae by tethering proteins to cell pole. However, the exact molecular mechanisms are yet to be elucidated. In this thesis, I tackled to unveil comprehensive view of the cell pole organization which implies the orchestration of different cellular functions, by identifying further interaction partners of HubP as well as drawing conceivable picture of the cell pole by super-resolution photoactivated localization microscopy. To identify new interaction partners of HubP, I used minicells in which cell poles were enriched as they derived from cell division near the cell pole. Difference in protein composition between HubP+ and HubP- minicells were examined by isobaric tags for relative and absolute quantitation. Among ~800 proteins identified, ~80 proteins were considered to be enriched in HubP+ minicells including many expected proteins (FlhG, ParC and downstream chemotaxis proteins). I chose 14 proteins to investigate their subcellular localization with fluorescent microscopy. In conclusion, I discovered 4 proteins that showed polar localization in a HubP-dependent manner. These proteins are VbrX, VbrY, and 2 hypothetical proteins MotV and MotW. ∆motV and ∆motW showed significant defect in a diameter of travel in soft agar plate that suggesting the possible involvement in chemotaxis and/or motility. Whereas electron microscopy showed that both mutants possess intact monotrichous flagellum, video-tracking revealed that the two mutants showed rather distinct defects during swimming: MotV is rather turning mutant while MotW is a speed mutant. Fluorescent microscopy experiments indicated that MotV, MotW and HubP showed distinct polar dynamics over cell cycle. For fine-scale observation of the cell pole by PALM, it was appreciated that novel tools for high-throughput analysis was demanded. Since brightfield images are not sufficient to have accurate contours of small and low contrast bacterial cells, I developed new labeling technique with photoactivatable fluorescent proteins for precise outlining at either inner membrane or periplasm. Furthermore, we created Matlab-based software called Vibio which integrates cell outline and the list of molecules obtained by super-resolution microscopy. High-throughput capability of the software enabled to analyze distribution of detected molecules from single cell to whole bunch of cells in a manner that cells are oriented by cell curvature. These allowed me to discover that HubP is mostly lopsided at the convex side of the cell pole, while its partners mostly located middle of the pole. Altogether, I successfully unveiled 4 novel interaction partners of HubP. I revealed of the function of hypothetical proteins that are involved in cell motility. I developed new labeling technique for precise polar localization that works well for PALM image analysis in Vibio. Therefore, I observed precise polar localization of HubP and other polar proteins
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Lähdesmäki, Ilkka Johannes. "Flow injection methods for drug-receptor interaction studies, based on probing cell metabolism /". Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/8590.
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Zhang, Weimin. "Topics in living cell miultiphoton laser scanning microscopy (MPLSM) image analysis". Texas A&M University, 2006. http://hdl.handle.net/1969.1/4412.
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Multiphoton laser scanning microscopy (MPLSM) is an advanced fluorescence
imaging technology which can produce a less noisy microscope image and minimize the
damage in living tissue. The MPLSM image in this research is the dehydroergosterol
(DHE, a fluorescent sterol which closely mimics those of cholesterol in lipoproteins and
membranes) on living cell's plasma membrane area. The objective is to use a statistical
image analysis method to describe how cholesterol is distributed on a living cell's
membrane. Statistical image analysis methods applied in this research include image
segmentation/classification and spatial analysis. In image segmentation analysis, we
design a supervised learning method by using smoothing technique with rank statistics.
This approach is especially useful in a situation where we have only very limited
information of classes we want to segment. We also apply unsupervised leaning methods
on the image data. In image data spatial analysis, we explore the spatial correlation of
segmented data by a Monte Carlo test. Our research shows that the distributions of DHE
exhibit a spatially aggregated pattern. We fit two aggregated point pattern models, an
area-interaction process model and a Poisson cluster process model, to the data. For the area interaction process model, we design algorithms for maximum pseudo-likelihood
estimator and Monte Carlo maximum likelihood estimator under lattice data setting. For
the Poisson Cluster process parameter estimation, the method for implicit statistical
model parameter estimate is used. A group of simulation studies shows that the Monte
Carlo maximum estimation method produces consistent parameter estimates. The
goodness-of-fit tests show that we cannot reject both models. We propose to use the area
interaction process model in further research.
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Lindmark, Sofia. "Cell Tracking in Microscopy Images Using a Rao-Blackwellized Particle Filter". Thesis, Uppsala universitet, Avdelningen för visuell information och interaktion, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-236769.
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Analysing migrating cells in microscopy time-lapse images has already helped the understanding of many biological processes and may be of importance in the development of new medical treatments. Today’s biological experiments tend to produce a huge amount of dynamic image data and tracking the individual cells by hand has become a bottleneck for the further analysis work. A number of cell tracking methods have therefore been developed over the past decades, but still many of the techniques have a limited performance. The aim of this Master Project is to develop a particle filter algorithm that automatically detects and tracks a large number of individual cells in an image sequence. The solution is based on a Rao-Blackwellized particle filter for multiple object tracking. The report also covers a review of existing automatic cell tracking techniques, a review of well-known filter techniques for single target tracking and how these techniques have been developed to handle multiple target tracking. The designed algorithm has been tested on real microscopy image data of neutrophils with 400 to 500 cells in each frame. The designed algorithm works well in areas of the images where no cells touch and can in these situations also correct for some segmentation mistakes. In areas where cells touch, the algorithm works well if the segmentation is correct, but often makes mistakes when it is not. A target effectiveness of 77 percent and a track purity of 80 percent are then achieved.
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Siamantouras, Eleftherios. "Nanomechanical investigation of soft biological cell adhesion using atomic force microscopy". Thesis, University of Warwick, 2014. http://wrap.warwick.ac.uk/62745/.
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Cell-to-cell adhesion is critically important for the improved secretory function of endocrine pancreatic beta (β)-cells and for the progression of fibrosis in the renal proximal tubule in Diabetic Nephropathy. In this research project the effects of specific biochemical treatment on functional cell-to-cell adhesion and single cell mechanics were systematically investigated. Atomic Force Microscopy (AFM) Single Cell Force Spectroscopy was applied to quantitatively characterise E-cadherin mediated surface ligation and cytoskeletal reorganisation in the pancreatic mouse insulinoma MIN6 and human kidney proximal tubule HK2 cell model. AFM tipless cantilevers were functionalised with a single cell or a spherical microbead for performing cell-to-cell adhesion and single cell indentation experiments respectively. The impact of elastic deformation of single cells into cell-to-cell adhesion was examined by per-forming adhesion experiments at various retraction speeds. The results illustrate that both adhesive and mechanical properties of single cells constitute important underlying factors of the physiological and pathological conditions under investigation since they were significantly affected by biochemical changes. More specifically, it is suggested that the enhanced secretory function of MIN6 cells upon calcium-sensing re-ceptor activation is owned to a combination of increased E-cadherin mediated cell-to-cell adhesion and decreased elastic (E)-modulus of single cells. In addition, it was shown that treatment of HK2 with the cytokine TGF-β1 decreased E-cadherin mediated cell-to-cell adhesion and increased E modulus of single cells, suggesting a mechanism that initiates early fibrotic changes in the tubular epithelia. Overall, both studies demonstrate that alterations of biological states evoke complex interactions between E-cadherin and actin cytoskeleton as manifested by the interplay between the mechanistic behaviour and surface binding of the cells. Therefore single cell mechanics have profound effects on cell-to-cell adhesion characterisation, particularly when physiological versus pathological states are to be investigated.
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Harriman, Oliver Leon Jacobs. "A system-level approach to single-molecule live-cell fluorescence microscopy". Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:81425bd2-6bc3-489e-b159-a2590ffffbb1.
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In this work a system-level approach was taken to the single-molecule fluorescence microscopy of living cells. This primarily involved the unification of relevant information within appropriately structured artefacts that were used to inform and enhance experimentation. Initially the diversity of emerging single-molecule techniques was reviewed and presented with a novel article structure to suit the purpose of designing an experiment (Harriman and Leake 2011). Techniques were grouped by the type of information they could access, rather than the standard organisation centred on the techniques themselves. A bespoke microscope was conceived and built with reference to knowledge and tools from the fields of Architecture and Systems-Engineering. The microscope layout would enable multiple experiment types through independent control of multiple illumination beams. A technique was developed enabling the prescription of evanescent field penetration depth for each incident beam. The various empirical and theoretical results that are used to understand and modify a microscopy experiment were integrated into an internally consistent simulation model (Harriman and Leake. 2013). This was used to inform the selection of experimental components and parameters and ultimately acquire higher data quality as measured by functions such as signal-to-noise ratio (SNR). The combined experimental system of microscope and simulation model was applied in two live-cell investigations. In Escherichia coli, the spatial distribution of membrane bound proteins was investigated and a novel technique was applied to the analysis of colocalisation. Results indicate that NADH dehydrogenase and ATP synthase follow uncorrelated trajectories. This supports the hypothesis of spatial decoupling of molecules that energise the membrane and molecules that use membrane energy. In human carcinoma cells, the mechanism of ligand-receptor binding was investigated. Data was collected prior to and periodically after the addition of ligands, and fluorescence images were acquired of both ligands and receptors. Analyses based on single particle tracking are currently being carried out by a collaborator to extract information on stoichiometry and dynamics at the single-molecule level.
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MacKay, Gillian E. "Analysis of cell allocation in GFP chimeric blastocysts by confocal microscopy". Thesis, University of Edinburgh, 2003. http://hdl.handle.net/1842/29238.
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This study combined confocal microscopy with the use of a tau-GFP (green fluorescent protein) transgenic mouse strain to study cell fate in two main types of chimeric blastocysts. In each case one component of the chimera was known to contribute poorly to the fetal lineage at later stages. The experiments were designed to test whether this was due to non-random allocation to different tissues at the blastocyst stage. The initial part of this thesis involved the establishment of confocal microscopy techniques and the characterisation of two novel tau-GFP transgenic mouse strains. A method of culturing embryos on the confocal microscope was established for use in further studies. Two tau-GFP transgenic mouse lines, TgTP6.3 and TgTP6.4, were evaluated for their use in following chimera studies by assessing the timing of the onset of GFP expression during preimplantation development and the viability of heterozygote and homozygote mice. The remaining studies involved the use of tau-GFP chimeric embryos. Mouse tetraploid↔diploid chimeras have previously been used as a model of confined placental mosaicism (CPM). Approximately 2% of human conceptuses investigated by chorionic villus sampling contain chromosomally abnormal cells that are confined to the placenta. This condition, known as human CPM, can lead to incorrect prenatal diagnosis. Animal models would be useful for investigating the mechanisms responsible for the exclusion of abnormal cells from the fetus. As spontaneous chromosomal mosaicism is rare in mouse embryos, mouse aggregation chimeras have been used as a model. Previous results have shown that tetraploid cells are excluded from the epiblast derivatives, including the fetus, of mid gestation tetraploid↔diploid chimeras. Tetraploid cells have been shown to be preferentially allocated to the trophectoderm, in particular the mural trophectoderm, of mouse tetraploid↔diploid blastocysts. However, tetraploid cells are present within the inner cell mass region of the blastocyst. Therefore, the current study used tau-GFP tetraploid↔diploid aggregation chimeras to determine if tetraploid cells are present within the epiblast and lost later or are excluded from the epiblast region by preferential allocation to the hypoblast. Tetraploid↔diploid chimeras were produced using TgTP6.3 embryos. Analysis of these chimeras at E3.5 and E4.5 has confirmed that tetraploid cells are preferentially allocated to the mural trophectoderm. However, tetraploid cells were present within the region of the blastocyst that forms the epiblast. Analysis of expanded chimeric blastocysts at E5.5 and E7.5, produced by transferring them to delayed implantation females, also showed that tetraploid cells were present within the epiblast region. This suggests that tetraploid cells are initially present within the epiblast region but lost from the epiblast later by some mechanism of cell selection against tetraploid cells. Embryos from some inbred strains, such as BALB/c, also tend to contribute poorly to chimeras, so producing 'unbalanced chimeras'. Therefore unbalanced BALB/c chimeras could be a possible model of CPM. BALB/c↔GFP aggregation chimeras were analysed using the established time-lapse technique. This was to determine if BALB/c cells are underrepresented in mid-gestation BALB/c chimeras by preferential allocation of BALB/c cells to the mural trophectoderm. These results showed that BALB/c cells were not preferentially allocated to the mural trophectoderm and indicate that a general cell selection mechanism takes place.
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Wong, Tsz-wai Terence, e 黃子維. "Optical time-stretch microscopy: a new tool for ultrafast and high-throughput cell imaging". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hub.hku.hk/bib/B5066234X.
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The exponential expansion in the field of biophotonics over the past half-century has been leading to ubiquitous basic science investigations, ranging from single cell to brain networking analysis. There is also one biophotonics technology used in clinic, which is optical coherence tomography, mostly for high-speed and high-resolution endoscopy. To keep up such momentum, new biophotonics technologies should be aiming at improving either the spatial resolution or temporal resolution of optical imaging. To this end, this thesis will address a new imaging technique which has an ultra-high temporal resolution. The applications and its cost-effective implementations will also be encompassed.
In the first part, I will introduce an entirely new optical imaging modality coined as optical time-stretch microscopy. This technology allows ultra-fast real-time imaging capability with an unprecedented line-scan rate (~10 million frames per second). This ultrafast microscope is renowned as the world’s fastest camera. However, this imaging system is previously not specially designed for biophotonics applications. Through the endeavors of our group, we are able to demonstrate this optical time-stretch microscopy for biomedical applications with less biomolecules absorption and higher diffraction limited resolution (<2 μm). This ultrafast imaging technique is particularly useful for high-throughput and high-accuracy cells/drugs screening applications, such as imaging flow cytometry and emulsion encapsulated drugs imaging.
In the second part, two cost-effective approaches for implementing optical time-stretch confocal microscopy are discussed in details. We experimentally demonstrate that even if we employ the two cost-effective approaches simultaneously, the images share comparable image quality to that of captured by costly specialty 1μm fiber and high-speed ( >16 GHz bandwidth) digitizer. In other words, the cost is drastically reduced while we can preserve similar image quality.
At the end, I will be wrapping up my thesis by concluding all my work done and forecasting the future challenges concerning the development of optical time-stretch microscopy. In particular, three different research directions are discussed.published_or_final_version
Electrical and Electronic Engineering
Master
Master of Philosophy
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Dahan, Sophie. "Intracellular proteinmembrane trafficking : evaluation of the Golgi and endosomal apparatus by cryoimmune electron microscopy". Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=29387.
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Protein trafficking events in the secretory and endosomal apparatus were evaluated in rat liver hepatocytes by EM immunogold cytochemistry. The cellular distribution of apolipoprotein E and other abundant hepatic secretory and endosomal proteins was quantitatively examined in ultrathin cryosections of liver hepatocytes. Under steady-state conditions, apoE was concentrated within the Golgi apparatus, along sinusoidal plasma membrane microvilli and within all components of the endosomal apparatus, as evaluated immunocytochemically and confirmed by quantitative immunoblotting of organelles isolated from liver homogenates.Within the secretory pathway, the hepatic Golgi apparatus was a site of protein concentration as evaluated by the gold labeling density of another major secretory protein of liver hepatocytes, albumin, which was concentrated $ sim$10-fold in the Golgi apparatus relative to the ER. Sorting of this secretory protein within pre-Golgi compartments was not observed. Within the Golgi apparatus, apoE was concentrated within Golgi saccular distensions while being predominantly absent from flattened saccular components; apoB was similarly segregated within peripheral distensions. In contrast albumin, as well as two other monomeric proteins, transferrin (Tf) and the polymeric immunoglobulin receptor (pIg-R) were distributed homogeneously throughout Golgi stacks. In an attempt to assess a key prediction of the vesicular transport hypothesis, small 60-90 nm vesicles in the immediate vicinity of Golgi apparatus, postulated to mediate intersaccular transport were examined for their content of cargo secretory or plasma membrane proteins. Lack of immunoreactive apoE, apoB, albumin, Tf, or pIgR, within small vasicular profiles suggests limits to current models of vesicle-mediated intra-Golgi transport.
Along the endocytic pathway, at the cell surface, apoE and pIgR were dispersely distributed along the sinusoidal microvilli. Quantitative analysis of the immunolabeling distribution of these proteins did not reveal concentration within plasma membrane pits. These findings which were confirmed by observations of cell surface labeling of two other ligands, Tf and apoB, are consistent with receptors and ligands gaining access to the endocytic machinery likely without receptor/ligand preclustering or prolonged clustering events within plasma membrane pits. Intracellularly, apoE was concentrated within endocytic structures which were double-labeled for apoE and internalized HRP. Large endocytic vesicles closely juxtaposed to Golgi stacks also revealed a high content of apoE. Together the endosomal labeling distribution of apoE as well as morphological features of endosomal components are consistent with the maturation model for endosomes.
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Khanduja, Nimisha. "Processive Acceleration of Actin Barbed End Assembly by N-WASP". Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/54933.
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Actin-based cell motility plays crucial roles throughout the lifetime of an organism. The dynamic rearrangement of the actin cytoskeleton triggers a plethora of cellular processes including cellular migration. Neural Wiskott Aldrich syndrome protein (N-WASP) is involved in transduction of signals from receptors on the cell surface to the actin cytoskeleton. N-WASP activated actin polymerization drives extension of invadopodia and podosomes into the basement layer. In addition to activating Arp2/3 complex, N-WASP binds actin filament barbed ends, and both N-WASP and barbed ends are tightly clustered in these invasive structures.
We used nanofibers coated with N-WASP WWCA domains as model cell surfaces and single actin filament imaging to determine how clustered N-WASP affects Arp2/3-independent barbed end assembly. Individual barbed ends captured by WWCA domains of N-WASP grew at or below their diffusion limited assembly rate. At high filament densities, overlapping filaments formed buckles between their nanofiber tethers and myosin attachment points. These buckles grew 3.4-fold faster than the diffusion-limited rate of unattached barbed ends. N-WASP constructs with and without the native poly-proline (PP) region showed similar rate enhancements. Increasing polycationic Mg2+ or Spermine to enhance filament bundling increased the frequency of filament buckle formation, consistent with a requirement of accelerated assembly on barbed end bundling.
Our preliminary data shows that tethered N-WASP construct containing one WH2 domain does not generate processive bundles or filament loops leading us to believe that tandem WH2 is required for processivity. We propose that this novel N-WASP assembly activity provides an Arp2/3-independent force that drives nascent filament bundles into the basement layer during cell invasion. Discovery of this bundle mediated unique pathway involved in invasion and metastasis will provide new targets for therapeutic development.Ph. D.
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Balanant, Marie-Anne. "Experimental studies of red blood cells during storage". Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/119221/1/Marie-Anne_Balanant_Thesis.pdf.
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This project used mechanical testing and imaging techniques to understand how red blood cells age in an in vitro environment, and identified markers of red blood cell product quality. The damage caused to the cells by cold storage could lead to a reduced transfusion efficiency and potential improvements to current storage protocols were proposed as a result of this research.
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Tobin, Mark James. "Hormone-receptor interaction by time resolved fluorescence and image analysis". Thesis, University of Salford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.238743.
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Olofsson, Per Erik. "Microscopy-based single-cell in vitro assays for NK cell function in 2-D and 3-D". Doctoral thesis, KTH, Cellulär biofysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-199571.
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Natural killer (NK) cells are effector cells of the innate immune system that are responsible for mediating cellular cytotoxicity against virally infected or neoplastically transformed cells. NK cell subsets are defined by their expression of certain cell-surface markers, and are usually related to activation and developmental status. However, how distinct NK cell phenotypes correlate with behavior in NK-target interactions is less widely characterized. There is therefore a need to study NK cell behavior down at the single-cell level. One aim of this thesis is to approach methods that quantitatively describe these single-cell-level behavioral differences of NK cells. Additionally, the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment is crucial for NK cell trafficking and immune surveillance. Traditional imaging studies of NK cell migration and cytotoxicity do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. Therefore, it is desirable to implement 3-D in vitro migration and killing assays that better mimic in vivo conditions. Another aim of this thesis is to develop a microwell-based assay for 3-D time-lapse imaging of NK cell migration and cytotoxicity. Using a newly developed single-cell imaging and screening assay, we trap small populations of NK and target cells inside microwells, where they are imaged over extended periods of time. We have performed experiments on resting, IL-2-activated, educated, and non-educated NK cells and quantified their migration behavior and cytotoxicity. One major discovery was that a small population of NK cells mediate a majority of the cytotoxicity directed against target cells. A particularly cytotoxic group of cells, termed serial killers, displayed faster and more effective cytotoxicity. Serial killers were more prevalent in IL-2-activated and educated NK cells, but were also present in a small fraction of resting and non-educated NK cells. IL-2-activated and educated NK cells displayed more dynamic migration behavior than resting and non-educated NK cells. Additionally, IL-2-activated and educated NK cells spent more time in NK–target cell conjugates and post-conjugation attachment than resting and non-educated NK cells. To more closely approximate in vivo conditions, we have combined our microwell assay with an interstitial ECM-like matrix. The microwells allow for long-term imaging of NK–target cell interactions within a confined 3-D volume. NK cells were tracked and interactions with target cells were scored for duration and outcome. The developed microwell-based assay is suitable for 3-D time-lapse imaging of NK cell migration and cytotoxicity. As it allows for experiments with human cells, it could be used as a complement to in vivo imaging. We have quantified NK cell behavioral heterogeneity and developed tools that can be used to further study and elucidate differences in the behavior of single immune cells. These tools advance current methods for single-cell analysis, which will likely play an even more important role in the study of immune responses in the future.NK-celler är effektorceller tillhörande det ospecifika immunförsvaret och har till uppgift att avdöda virusinfekterade och neoplastiska celler. Subpopulationer av NK-celler klassificeras på basis av uttryck av ytmolekyler och är vanligtvis relaterade till cellernas aktiverings- och utvecklingsstatus. Hur dessa fenotypiskt distinkta subpopulationer korrelerar med beteende i NK–målcellinteraktioner är inte lika välstuderat. Det finns därför ett behov att studera NK-cellbeteende ner på encellsnivå. Ett mål med denna avhandling är att närma sig metoder som kvantitativt beskriver dessa skillnader i NK-cellbeteende på encellsnivå. NK-cellers förmåga att migrera genom extracellulär matris är avgörande för deras celltrafik och immunövervakning. I traditionella avbildningsstudier av NK-cellers migration och cytotoxicitet återskapas inte de strukturella och mekaniska faktorer som formar NK-cellmigration in vivo. Det är därför önskvärt att implementera migrationsassays i 3-D som bättre efterliknar in vivo-situationer. Ett annat mål med denna avhandling är att utveckla en mikrobrunnsbaserad assay för 3-D-avbildning av NK-cellmigration och -cytotoxicitet. Genom att använda en nyligen utvecklad plattform för encellsavbildning och -screening fångar vi små populationer av NK- och målceller inuti mikrobrunnar, där de kan avbildas under längre tider. Vi har genomfört experiment på vilande och IL-2-aktiverade NK-celler, samt undersökt NK-cellutbildning, och kvantifierat dessa cellers migration och cytotoxiska beteende. En huvudsaklig upptäckt var att en liten population av de studerade NK-cellerna avdödade en majoritet av målcellerna. En särskilt cytotoxisk grupp celler, som benämnes seriemördare, uppvisade en snabbare och mer effektiv cytotoxicitet. Seriemördare var mer vanligt förekommande hos IL-2-aktiverade och utbildade NK-celler än hos vilande och icke-utbildade NK-celler. IL-2-aktiverade och utbildade NK-celler uppvisade mer dynamiskt migrationsbeteende än vilande och icke-utbildade NK-celler. Dessutom tillbringade IL-2-aktiverade och utbildade NK-celler en länge tid i målcellskonjugat och var i kontakt med målceller längre efter konjugering än vilande och icke-utbildade NK-celler. För att närmare återskapa in vivo-tillstånd har vi kombinerat vår mikrobrunnsassay med en matris som liknar interstitiell extracellulär matris. Mikrobrunnarna möjliggör långtidsavbildning av NK–målcellinteraktioner inom en avgränsad volym. NK-cellerna spårades och längden och utfallet av målcellinteraktioner utvärderades. Den utvecklade mikrobrunnsassayen är lämplig för 3-D-avbildning av NK-cellmigration och -cytotoxicitet. Eftersom den tillåter experiment med humana celler kan den komplettera avbildning in vivo. Vi har kvantifierat funktionell NK-cellheterogenitet och utvecklat verktyg som kan användas för att ytterligare studera och bringa klarhet i hur enskilda immuncellers beteende skiljer sig åt. Dessa verktyg är en vidareutveckling av nuvarande metoder för encellsanalys, som sannolikt kommer att spela en större roll i studiet av immunsvar i framtiden.
自然杀伤细胞是先天免疫系统自带的效应细胞，主要通过调解其细胞毒性对抗病毒感染和细胞瘤变。自然杀伤细胞的亚型主要通过其表面抗原性质来定义并通常与一些激活和进展状态相联系。然而，关于自然杀伤细胞表型与其目标反应之间的相互联系的研究依然比较匮乏。因此，在单细胞层面对自然杀伤细胞表现的研究是十分必要的。 本论文的研究目的之一就是寻找方法来定量分析单细胞层水平NK细胞的行为差异。 此外，自然杀伤细胞在细胞外基质微环境中的迁移对自然杀伤细胞的移动和免疫监督非常重要。 关于自然杀伤细胞迁移和细胞毒性的传统成像研究并不能合理地呈现触发此细胞在体内迁移的形变和应变响应过程。因此，关于细胞迁移和细胞杀伤的体外三维研究对探索NK细胞的体内反应机制尤为重要。 本论文的另一个目的就是构建基于微孔试验来研究NK细胞迁移和细胞毒性随时间在三维空间中随时间的变化。 通过新型的单细胞成像和筛选方法，我们将少量NK细胞和靶细胞放入微孔内，同时进行长期的图像观察。 我们实验观察并测定了不同NK细胞的迁移和细胞毒性，包括静止型，IL-2 激活型，诱导型和非诱导型NK细胞。 一个重要发现是少量NK细胞实际上介导了其对靶细胞的主要细胞毒性。 一个具有特别细胞毒性的群体，称为连续杀伤细胞/持续杀伤细胞，表现出了更快更有效的细胞毒性。连续杀伤细胞在IL-2 激活型，诱导型细胞中出现得更多，但是在静止型和非诱导型细胞中也少量释放。前两者比后两者表现出了更活跃的迁移性能，但需要较长的结合时间。 为了更接近在体状态，我们把基于微孔的实验与细胞外基质类似结构结合来研究NK细胞的活动。微孔有效地把NK细胞控制在一个三维小空间内，以便长时间观察NK细胞与目标细胞的反应。NK细胞可以被一直追踪并进一步测定了其与目标细胞的反应时间和反应结果。这种基于微孔的测试对研究NK细胞在三维空间内随时间的迁移和细胞毒性的图像研究非常有效。 它也适应于人类细胞的研究，可以为体内细胞成像研究提供良好辅助平台。 综上，本论文研究中，我们量化分析了NK细胞行为的异质性，并开发了实验方法可用于进一步研究和阐明不同单一免疫细胞的行为的方法。 这些实验手段进一步提升了单细胞研究分析能力，并且未来将在免疫响应研究进一步起到更加重要的作用。
QC 20170110
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Stayton, Isaac Alexander. "Investigation of the interactions between selected nanoparticles and human lung carcinoma cells at the single cell and single particle level". Diss., Rolla, Mo. : Missouri University of Science and Technology, 2009. http://scholarsmine.mst.edu/thesis/pdf/Stayton_09007dcc8065344d.pdf.
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Thesis (Ph. D.)--Missouri University of Science and Technology, 2009.Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed April 29, 2009) Includes bibliographical references.
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Joensuu, Jenny. "Online Image Analysis of Jurkat T Cells using in situ Microscopy". Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-153313.
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Cell cultivation in bioreactors would benefit from developed monitoring systems with online real-time imaging to evaluate cell culture conditions and processes. This opportunity can be provided with the newly developed in situ Microscope also called ISM. The ISM probe is mounted into the wall of a bioreactor and consists of a measurement zone with an illuminating light source to obtain real-time images of moving cells in suspension. The instrument is linked to advanced imaging analysis software which can be specifically adapted for the objects in study. The aim of this project is to analyze the T lymphocyte cell line Jurkat T cells using the ISM equipment and identify specific features of the cells that can be obtained. The results show that the equipment and linked software are suitable for monitoring cell density, cell size distribution and cell surface analysis of the Jurkat cells during cultivation. The ISM could also detect induced changes in cell size caused by osmotic shifts and the course of an infection occurring in the cell suspension using a developed software for online real-time monitoring.
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Spanoudis, Catherine M. "Cell Division Regulation in Staphylococcus aureus". Scholar Commons, 2017. http://scholarcommons.usf.edu/etd/7090.
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Cell division is a fundamental biological process that occurs in all kingdoms of life. Our understanding of cell division in bacteria stems from studies in the rod-shaped model organisms: Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. The molecular underpinnings of cell division regulation in non-rod-shaped bacteria remain to be studied in detail. Rod-shaped bacteria possess many positive and negative regulatory proteins that are essential to the proper placement of the division septa and ultimately the production of two identical daughter cells, many of which are absent in cocci. Given that essential cell division proteins are attractive antibacterial drug targets, it is imperative for us to identify key cell division factors especially in pathogens, to help counter the emergence of multi-drug resistance. In Staphylococcus aureus, a spherical Gram-positive opportunistic pathogen that causes a range of diseases from minor skin infections to life-threatening sepsis, we have identified the role of an essential protein, GpsB, in the regulation of cell division. We discovered that GpsB preferentially localizes to cell division sites and that overproduction of GpsB results in cell enlargement typical of FtsZ inhibition, while depletion of GpsB results in cell lysis and nucleoid-less minicell formation. The identification of GpsB’s interaction partners will allow us to understand the molecular mechanism by which GpsB regulates cell division.
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Thomas, Gawain M. "The Role of Integrins in Cellular Response to Mechanical Stimuli". Digital WPI, 2017. https://digitalcommons.wpi.edu/etd-theses/114.
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Tissue cells exhibit varying responses according to the stiffness of their extracellular matrix (ECM). The mechanism of this stiffness sensing is not fully understood; however, it is known that cells probe stiffness by applying intracellular force to the ECM via integrin-mediated focal adhesions. The bonds between integrins and ECM have been described as “catch bonds�, and it is unclear how ECM viscoelasticity affects these bonds. We have observed the effects of ECM stiffness on the binding strength of integrins to ECM ligands by measuring the dissociation force of individual integrin-ligand bonds of 3T3 fibroblasts on collagen-coated polyacrylamide gels using atomic force microscopy. Results show that integrins exhibit higher rates of activation on stiff substrates. Furthermore, increased matrix stiffness results in the occurrence of larger, multi-bond dissociation events, which suggests that substrate stiffness may affect the cellular response by promoting integrin clustering as well as by modulating the maximum possible force between individual integrins and the ECM.
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Shojaeizadeh, Mina. "An improved approach for cell traction force microscopy using a continuous hydrogel". Digital WPI, 2013. https://digitalcommons.wpi.edu/etd-theses/1199.
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"In this thesis, a cell traction force microscopy method is developed for measuring traction forces of connective tissue cells. This method includes an improved methodology in traction force microscopy of live cells cultured on an elastic substrate. Tissue cells, such as skin and muscle cells respond to the mechanical stimuli of their microenvironment by adhering to their substrate and exerting forces on the proteins of the extracellular matrix (ECM). These forces are called cell traction forces. Fibroblasts are grown on polyacrylamide (PA) gels embedded with fluorescent beads and coated with different types of ECM ligands. Traction forces of NIH 3T3 fibroblasts are calculated from the measured deformations of PA gels by using a 3-D finite element method. The advantages of this method compared to the traditional methods of cell traction force microscopy (CTFM) are that this method takes into account the finite thickness of the substrate by applying a 3-D FEM analysis to reduce the errors of using an infinite half space approximation for a substrate with a finite thickness and that it uses a novel method for embedding the substrate with fluorescent markers that decreases the measurement uncertainties. In our approach fluorescent beads were embedded on the top of substrate instead of getting mixed with the gel. This decreases the effect of out-of-focus fluorescent beads on the measured deformation fields which enhances the accuracy of cell traction force measurements."
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Ray, Lucille Alexandria. "Live single cell fluorescence microscopy; from antibiotic resistance detection to mitochondrial dysfunction". University of Akron / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=akron1597342775751888.
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Karuna, Arnica. "Applications of coherent anti-Stokes Raman scattering (CARS) microscopy to cell biology". Thesis, Cardiff University, 2016. http://orca.cf.ac.uk/94088/.
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Traditionally, many advances in the field of biology have been driven by optical microscopy based techniques which reveal morphological information about the samples under study [1, 2, 3, 4]. The scope of the applications of these methods is limited due to the lack of contrast from most biological materials (cells and tissues) which are transparent to visible light. The introduction of extraneous materials (such as fluorescent quantum dots or other fluorescent proteins/labels) with affinity towards certain sub-cellular components which are then imaged, has emerged as a popular and powerful method to image biological materials. Fluorescence microscopy using visible wavelengths in its simplest application includes the identification and imaging of interesting features of a sample which are fluorescently labelled in a structurally/ chemically specific way. In more recent developments, fluorescence lifetimes have been imaged. Fluorescence based techniques can also be applied to track protein dynamics or drug delivery in live samples. Despite the benefits of imaging samples with labels, and a host of associated applications, the issues of photobleaching and induced phototoxicity remain. Another very important aspect which gains relevance in live sample imaging is that the system dynamics may be influenced by the introduction of fluorescent labels. Spectroscopy techniques, which rely on the material resonances are chemically specific, sensitive, and if combined with microscopy, bridge the gap between non-invasive imaging and fluorescence microscopy. Instead of an extraneous label, the contrast generated originates from molecular transitions of the chemical species in the sample. Coherent Raman scattering (CRS) based techniques rely on the chemical contrast generated due to molecular vibrations and have been applied to biology [5]. One CRS technique, stimulated Raman scattering (SRS) has been used to distinguish between the macromolecular constituents of cells [6, 7] and tissues [8]. Additionally, quantitative hyperspectral SRS has been demonstrated in polymer and lipid mixtures [6]. Another type of CRS, coherent anti-Stokes Raman scattering (CARS) [9] was reported in 1965, nearly half a decade before SRS. However, due to difficulties in implementation, CARS was not readily put to application. Since its revival in 1999 [10], CARS has emerged as a label-free, chemically specific microscopy technique and has been applied to image various biological materials [11, 12]. In addition to the studies of lipid rich samples [13, 14], spectral differences between the cytosol and the nucleus have been reported using CARS microscopy [15, 16]. However, none of the previous works in this field present full 3D hyperspectral data, or make quantitative volumetric estimates of the various chemical components present in the sample. With respect to biomedical/biochemical application based studies, literature suffers from a paucity of examples investigating the effects of drugs on biological materials using CARS microscopy. This project aims to overcome these shortcomings. In this work, CARS microscopy is applied to single cells (osteosarcoma, U-2OS which are lipid poor due to their functional profile) with volumetric quantitative analysis to determine the absolute masses of the component species. For the first time in our knowledge, full 3D hyperspectral data has been acquired and analyzed. Correlative fluorescence imaging to ascertain the origin of various components of the cells as imaged with CARS was also performed. Furthermore, reports of no observed (with CARS) correlation in protein content in the intranuclear region with the mitotic stage in cells one publication [16] have been disproved, shown in this thesis. Osteosarcoma is a rare type of cancer, most commonly diagnosed in children and adolescents. However, due to its rarity, it is not well researched. The usual line of treatment includes surgery followed by chemotherapy, of which Taxol (microtubule stabilizer) and ICRF-193 (topoisomerase II poison) form an important part. The effects of these drugs on cells are often investigated in literature using a range of techniques, of which, the most non-invasive one is chemically non-specific optical microscopy [17, 18]. Among the chemically specific methods used to perform such studies, flow cytometry [19, 20, 21] is one of the most commonly employed; and the most invasive methods, also in widespread use, are Western blotting and gel electrophoresis [22]. This means that in the best case scenario, we can perform optical microscopy on the cells with no chemical specificity or sensitivity, or sacrifice non-invasiveness for chemical information. Identifying a need for label-free methods to study the effects of these drugs, we applied CARS microscopy to study the effects of Taxol and ICRF-193 on U-2OS cells. This was done to determine whether CARS microscopy is suitable for population phenotyping and profiling the effects of anticancer drugs over a period of time, following treatment. Also for the first time, in this project, CARS microscopy has been demonstrated with chemical specificity and sensitivity on structurally and functionally multicellular 3D assemblies, organoids. In the past, other groups have reported studies on organoids, their metabolism and drug interactions using fluorescence microscopy [23, 24, 25], with the already mentioned shortcomings and pitfalls of photodamage and photobleaching. This thesis is structured into five chapters. The required background is given in the first two chapters. The first chapter introduces optical spectroscopy with emphasis on CARS, including a discussion of the theory and the various implementations of CARS microscopy. The second chapter contains the biology background in cells and cell division requisite for this project. An overview of the current state of the art in imaging techniques is also given. The set-up and analysis techniques used to acquire and analyze the data presented in this work are described in the third chapter, along with a characterization of the effects of the imaging optics and the sample’s refractive index on the analysis method and the quantitative calculations, using polystyrene and polymethymethacrylate beads of different sizes. The next two chapters describe the applications of CARS microscopy to fixed U- 2OS cells and organoids. In chapter four, the results of CARS imaging and spectral analysis of U-2OS cells correlated with two-photon fluorescence are shown. Furthermore, applications of CARS to study the effects of two kinds of anti-cancer drugs i.e, Taxol and ICRF-193 on U-2OS cells are demonstrated. Additionally, a side project not related to CARS microscopy, but presenting a simple method to quantitatively investigate the number of eGFP molecules attached to cyc-B, across the cell cycle is also described in this chapter. Chapter five demonstrates the suitability of CARS microscopy to image higher levels of biological organization, specifically organoids which are miniature lab grown 3D models of organs. The summary and outlook of this project are given in the last chapter, which is followed by the Appendices including additional detailed information referenced in the thesis. All 3D data are available as videos in the data DOI related to this thesis.
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Huh, Seungil. "Toward an Automated System for the Analysis of Cell Behavior| Cellular Event Detection and Cell Tracking in Time-lapse Live Cell Microscopy". Thesis, Carnegie Mellon University, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3538985.
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Time-lapse live cell imaging has been increasingly employed by biological and biomedical researchers to understand the underlying mechanisms in cell physiology and development by investigating behavior of cells. This trend has led to a huge amount of image data, the analysis of which becomes a bottleneck in related research. Consequently, how to efficiently analyze the data is emerging as one of the major challenges in the fields.
Computer vision analysis of non-fluorescent microscopy images, representatively phase-contrast microscopy images, promises to realize a long-term monitoring of live cell behavior with minimal perturbation and human intervention. To take a step forward to such a system, this thesis proposes computer vision algorithms that monitor cell growth, migration, and differentiation by detecting three cellular events—mitosis (cell division), apoptosis (programmed cell death), and differentiation—and tracking individual cells. Among the cellular events, to the best our knowledge, apoptosis and a certain type of differentiation, namely muscle myotubes, have never been detected without fluorescent labeling. We address these challenging problems by developing computer vision algorithms adopting phase contrast microscopy. We also significantly improve the accuracy of mitosis detection and cell tracking in phase contrast microscopy over previous methods, particularly under non-trivial conditions, such as high cell density or confluence. We demonstrate the usefulness of our methods in biological research by analyzing cell behavior in scratch wound healing assays. The automated system that we are pursuing would lead to a new paradigm of biological research by enabling quantitative and individualized assessment in behavior of a large population of intact cells.
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Wang, Ruixing. "STED-fluorescence correlation spectroscopy for dynamic observations in cell biology : from theoretical to practical approaches". Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0163/document.
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Les techniques de super-résolution offrent un nouvel aperçu de la description de l'organisation moléculaire dynamique de la membrane plasmique. Parmi ces techniques, la microscopie par déplétion d'émission stimulée (stimulated emission depletion, STED) dépasse la limite de diffraction optique et atteint une résolution de quelques dizaines de nanomètres. Il est une technique polyvalente qui peut être combinée avec d'autres techniques telles que la spectroscopie par corrélation de fluorescence (fluorescence correlation spectroscopy, FCS), fournissant des résolutions spatiales et temporelles élevées pour explorer les processus dynamiques qui se produisent dans les cellules vivantes. Ce projet de doctorat vise à mettre en œuvre un microscope STED, puis à combiner ce module STED avec la technique FCS pour les applications biologiques. Des études théoriques du STED et de la technique combinant STED et FCS ont permis dans les aspects spatio-temporels. Une solution analytique pour la fonction d'autocorrélation FCS a été dérivée dans l'état de déplétion STED incomplet. et un nouveau modèle d'ajustement FCS a été proposé. La méthode de variation du volume d’observation FCS (spot variation FCS, svFCS) a démontré sa capacité à identifier la présence de nanodomaines limitant la diffusion latérale des molécules dans la membrane plasmique. L’approche STED-FCS permet d’étendre l’application de la svFCS à l'échelle nanométrique afin d’évaluer la persistance plus ou moins importante de tels nanodomaines. Dans ce contexte, des simulations préliminaires de Monte Carlo ont été réalisées figurant des molécules diffusant en présence d'auto-assemblage/désassemblage dynamique des nanodomainesSuper-resolution techniques offer new insight into the description of the dynamic molecular organization at the plasma membrane. Among these techniques, the stimulated emission depletion (STED) microscopy breaks the optical diffraction limit and reaches the resolution of tens of nanometer. It is a versatile setup that can be combined with other techniques such as fluorescence correlation spectroscopy (FCS), providing both high spatial and temporal resolutions to explore dynamic processes occurring in live cells. This PhD project aims at implementing a STED microscope, and then at combining this STED module with FCS technique for biological applications. Detailed theoretical studies on STED and the combined STED-FCS technique in spatio-temporal aspects were performed. An analytical solution for FCS autocorrelation function was derived in the condition of incomplete STED depletion and a new FCS fitting model was proposed to overcome this problem. The spot variation FCS (svFCS) method has demonstrated its capability to identify the presence of nanodomains constraining the lateral diffusion of molecules at the plasma membrane. The STED-FCS can extend the svFCS approach to the nanoscale evaluating the long-lasting existence of such nanodomains. Within this frame, preliminary Monte Carlo simulations were conducted mimicking molecules diffusing in the presence of dynamic self-assembling/disassembling nanodomains
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Everett, William Neil. "Evanescent wave and video microscopy methods for directly measuring interactions between surface-immobilized biomolecules". Thesis, [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1585.
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Ball, Francis John. "Development of a microfluidic device for single cell analysis using FT-IR microscopy". Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/development-of-a-microfluidic-device-for-single-cell-analysis-using-ftir-microscopy(595222c5-f908-49a2-8fa9-6cfddc96e462).html.
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Prostate cancer is the second most common cause of cancer fatalities in males in the UK (2006) [1]. Therefore any advances in the diagnosis or screening for this form of cancer will yield significant benefits in the treatment of this disease. FT-IR has already been successfully used to assess and grade prostate biopsies by Gazi et al 2006 [2]. The collection of prostate biopsy is however a highly invasive procedure and as current screening methods are highly sensitive, but not very specific, large numbers of patients are referred for biopsy procedures that later come back as negative for prostate cancer [3]. Harvey et al used Raman spectroscopy to classify live cells of a number of prostate cell lines as a first step towards a possible urine screening protocol for prostate cancer [3]. Due to the complementary nature of Raman and FT-IR spectroscopy a similar live cell study should be possible using FT-IR and the combination of this technique with a high-throughput microfluidic device could lead to a useful screening tool for prostate cancer.The aim of the project was therefore to develop a microfluidic system which would enable higher through-put FT-IR analysis of live single cells in an aqueous carrier solution such as PBS or urine than has been previously possible. The design of the microfluidic device must also account for the fact that the materials used to produce the analysis chamber must be highly transparent to mid-IR radiation. The microfluidic device and peripheral systems must be easily transportable as it will be necessary to perform experiments in multiple locations. A design and manufacturing protocol for such a device has been developed.The development of a spectral contribution removal algorithm for the aqueous carrier fluid will also be necessary in order to allow the accurate interpretation of the IR data obtained. A least squares fitting based spectral subtraction algorithm was developed and validated for this purpose.Although it did not prove possible during the project to investigate the possible application of this device to a prostate cancer screening protocol other applications in cell line classification and drug cell interaction studies were performed and yielded encouraging results.
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Myers, Janette Bernadette. "Application of Single Particle Electron Microscopy to Native Lens Gap Junctions and Intrinsically Disordered Signaling Complexes". PDXScholar, 2019. https://pdxscholar.library.pdx.edu/open_access_etds/5008.
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Gap junctions are a class of membrane proteins that facilitate cell-to-cell communication by forming channels that directly couple the cytoplasm of neighboring cells. The channels are composed of monomers called connexins. Humans express 21 connexin isoforms in a cell-type specific fashion, and each isoform has distinct mechanisms of permeation and regulation. Co-assembly of multiple isoforms into a single intercellular channel can change channel properties, such as conductance and selectivity to substrates (e.g., ions, metabolites and signaling molecules). However, the mechanistic basis for this functional diversity has remained poorly understood. This lack of mechanistic insight has been due in large part to the lack of high-resolution (atomic-level) structural knowledge on this class of proteins. Prior to this work, the only high-resolution information available on gap junction structure came from a single connexin isoform, connexin-26 (cx26).
CryoEM has recently transformed from a low-resolution technique into one capable of rivaling the atomic-level resolutions achieved by x-ray crystallography -- but without the necessity for crystal formation, which has hindered progress towards understanding many classes of proteins (ie, membrane proteins, intrinsically disordered cell signaling complexes and other structurally dynamic systems). For my thesis research, I applied novel methods in single particle electron cryo-microscopy (CryoEM) to study a class of membrane proteins called gap junctions isolated from native lens tissue, as well as two signaling complexes not amenable to other structural techniques. I determined the structure of the lens gap junction, which contains connexin-46 (cx46) and connexin-50 (cx50), to a resolution of 3.4 Å and generated atomic models for both connexin isoforms. Structural analysis paired with molecular dynamics gave insight into energetic features of these channels that determine their isoform-specific conductance and selectivity to electrically charged ions. The cx46/50 gating domain was found to be stabilized by hydrophobic anchors, and also seems to adopt a more stable open state than found in cx26. Genetic mutations associated with congenital cataract formation were found to map to hot-spots of conserved structural and functional importance, rationalizing their disease-causing effects.
As part of collaborative efforts, I used methods in single particle EM to characterize two separate signaling complexes that had proven difficult to study with x-ray crystallography and NMR spectroscopy. One system, Ca2+/Calmodulin Kinase II (CaMKII), is a signaling complex in the brain involved in memory formation. Characterization of the CaMKII complex by single particle EM revealed an extended state, which was also shown to be prevalent in cells -- giving more depth to our understanding of how this signaling molecule functions. The second collaboration characterized the multimeric binding sites of the hub protein LC8, which interacts with the disordered region of a transcription factor (ASCIZ). This provided support for a novel model of transcription regulation, wherein LC8 fine-tunes its own transcription levels through multi-valent binding to the disordered region of its own regulatory transcription factor.
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Distasi, Matthew R. "The 3D characterization of the annulate lamellae : the development of a new methodology incorporating 3D-anaglyph techniques and serial transmission electron microscopy". Virtual Press, 2003. http://liblink.bsu.edu/uhtbin/catkey/1266020.
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Wang, Congchao. "Automated Tracking of Mouse Embryogenesis from Large-scale Fluorescence Microscopy Data". Diss., Virginia Tech, 2021. http://hdl.handle.net/10919/103595.
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Recent breakthroughs in microscopy techniques and fluorescence probes enable the recording of mouse embryogenesis at the cellular level for days, easily generating terabyte-level 3D time-lapse data. Since millions of cells are involved, this information-rich data brings a natural demand for an automated tool for its comprehensive analysis. This tool should automatically (1) detect and segment cells at each time point and (2) track cell migration across time. Most existing cell tracking methods cannot scale to the data with such large size and high complexity. For those purposely designed for embryo data analysis, the accuracy is heavily sacrificed. Here, we present a new computational framework for the mouse embryo data analysis with high accuracy and efficiency. Our framework detects and segments cells with a fully probability-principled method, which not only has high statistical power but also helps determine the desired cell territories and increase the segmentation accuracy. With the cells detected at each time point, our framework reconstructs cell traces with a new minimum-cost circulation-based paradigm, CINDA (CIrculation Network-based DataAssociation). Compared with the widely used minimum-cost flow-based methods, CINDA guarantees the global optimal solution with the best-of-known theoretical worst-case complexity and hundreds to thousands of times practical efficiency improvement. Since the information extracted from a single time point is limited, our framework iteratively refines cell detection and segmentation results based on the cell traces which contain more information from other time points. Results show that this dramatically improves the accuracy of cell detection, segmentation, and tracking. To make our work easy to use, we designed a standalone software, MIVAQ (Microscopic Image Visualization, Annotation, and Quantification), with our framework as the backbone and a user-friendly interface. With MIVAQ, users can easily analyze their data and visually check the results.Doctor of Philosophy
Mouse embryogenesis studies mouse embryos from fertilization to tissue and organ formation. The current microscope and fluorescent labeling technique enable the recording of the whole mouse embryo for a long time with high resolution. The generated data can be terabyte-level and contains more than one million cells. This information-rich data brings a natural demand for an automated tool for its comprehensive analysis. This tool should automatically (1) detect and segment cells at each time point to get the information of cell morphology and (2) track cell migration across time. However, the development of analytical tools lags far behind the capability of data generation. Existing tools either cannot scale to the data with such large size and high complexity or sacrifice accuracy heavily for efficiency. In this dissertation, we present a new computational framework for the mouse embryo data analysis with high accuracy and efficiency. To make our framework easy to use, we also designed a standalone software, MIVAQ, with a user-friendly interface. With MIVAQ, users can easily analyze their data and visually check the results.
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CRESTANI, MICHELE. "MECHANOPROPERTIES, HETEROGENEITY AND CELL MIGRATION IN GLIOBLASTOMA". Doctoral thesis, Università degli Studi di Milano, 2022. https://hdl.handle.net/2434/946015.
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Glioblastomas (GBMs) are primary brain tumors endowed with inter- and intra-patient
heterogeneity and extreme di↵usivity. As heterogeneity is studied with genomic and
transcriptomic analysis, little is known on how it is reflected on cell migration, mechanoproperties
and motility modes. Generally, the tumor cells invade the brain moving on
brain vasculature or white matter tracks: Patient-Derived Xenograft (PDX) has been a
standard to reproduce them in order to study GBM invasion. However, PDX presents
many disadvantages, including time consumption, hard standardization, high cost and
ethical concerns.
The present PhD thesis report aims at summarizing the existing literature through
an historical journey that gradually walks the reader towards the state of the art in
the biological knowledge, therapeutic treatments, and bioengineering of GBM. It also
reports the results of this PhD work. They include novel bioengineering tools for
studying the mechanoproperties in GBM and the development of methods to dissect
their migration and motility modes. Finally, a stand-alone assay aims at fostering a
discussion on how the scientific mindset and science have evolved and are evolving to
drive technological innovation in nowadays’ world.
The main goal of this PhD work was to develop bioengineering tools to crack
mechanoproperties and GBM motility. Initially, by utilizing clones of patient-derived
GBM cells that were either highly proliferative or highly invasive, I co-studied their cellular
architecture, migratory, and biophysical properties. One of the milestones of this
PhD work consists in the link between that invasiveness and cellular fitness. The most
invasive cells were sti↵er, developed higher mechanical forces on the substrate, and
moved stochastically. The mechano-chemical-induced expression of the formin FMN1
supports the mechanical cohesion of the cytoskeleton and enhances cell’s mechanoproperties,
leading to a higher motility and invasive phenotype.
In order to scale up the motility screen to several GBM clones, I co-developed SP2G
(SPheroid SPreading on Grids), the live imaging of GBM spheroids spreading on grid
micropatterns mimicking the brain vasculature. To counteract the issues in PDX and
rapidly identify the most invasive sub-populations hidden in heterogeneous GBMs, we
developed an in vivo mimicry platform named SP2G (SPheroid SPreading on Grids).
Live imaging of tumor-derived spheroids spreading on gridded micro patterns imitating
the brain vasculature mimicked 3D motility features observed in brain or 3D matrices.
Using patient-derived samples coupled with a semi-automated ImageJ/Fiji macro suite,
SP2G easily characterized and sorted di↵erences in cell migration and motility modes
through a set of 6 parameters (area expansion, di↵usivity, boundary speed, collective
migration, directional persistence, hurdling). Moreover, SP2G exposed the hidden
intra-patient heterogeneity in cell motility that correlated molecularly to specific integrins.
Thus, SP2G is intended as a versatile and potentially pan-cancer workflow to
identify the invasive tumor sub-populations in patient-derived specimens. SP2G represents
an integrative tool, available as open-source Fiji macro suite, for therapeutic
evaluations at single patient level.