Spelling suggestions: "subject:"fluorescence lifetime imaging"" "subject:"afluorescence lifetime imaging""
11 |
High-Speed Time-Difference CircuitsLi, Shuo 01 January 2013 (has links) (PDF)
This thesis presents time difference (TD) circuits that are important for measuring fluorescence lifetime, building LIDAR systems, and optimizing digital systems. The contribution of this thesis is to present a systematic organization of TD circuits and to present novel designs for digital-to-time conversion (DTC) and time-to-digital conversion (TDC).
Four basic time difference circuits are presented: TD adder, arbiter, time-difference MUX, and time-difference memory. Specifications, symbols, and multiple circuit implementations are presented for each block. Then the basic blocks are combined to form two compound blocks: DTC and TDC. Novel designs are presented for both blocks along with detailed simulation results.
The DTC was fabricated in TSMC’s 0.35um high-voltage process. A printed circuit board was designed to interface the DTC chip to a computer and test instruments. The DTC demonstrated 80ps resolution.
|
12 |
CRACking the RiddleSchwarzer, Roland 31 July 2014 (has links)
In den vergangenen Jahren sind Lipide, Membranen und deren Organisationsformen mehr und mehr in den Fokus der biologischen Forschung gerückt. Es wurde vorgeschlagen, dass in zellulären Membranen selbstassemblierende, submikroskopische Aggregate aus Sphingolipiden, Cholesterol und bestimmten Proteinen existieren und man vermutet, dass insbesondere Viren diese “Lipid Rafts” für ihren Zusammenbau nutzen und auf diese Art ihre Proliferationseffizienz erhöhen. Gleichwohl sind die genaue biologische Funktion und auch die molekulare Basis der Assoziation bestimmter Protein mit Lipid Rafts auch weiterhin unbekannt. In der vorliegenden Arbeit wurde Fluoreszenz-Lebenszeit-Mikroskopie genutzt, um die Lipid-Raft-Anreicherung des HIV-1 Glycoproteins gp41 zu untersuchen. Förster-Resonanz-Energietransfer zwischen fluoreszenzmarkierten viralen und Raft-Marker-Proteinen wurde gemessen, um deren gemeinsame, lokale Aufkonzentrierung in Lipid Rafts nachzuweisen. Durch Verwendung verschiedener Deletions- und Mutationsvarianten des Proteins konnte nicht nur seine Lipid-Raft-Präferenz demonstriert, sondern auch das Cholesterol-Bindemotiv (CRAC) als entscheidender Faktor der lateralen Sortierung identifiziert werden. Wir haben in diesem Kontext auch eine systematische Zell-zu-Zell-Variabilität in unseren Daten bemerkt, die einen zugrundeliegenden zellbiologischen Mechanismus der Membranorganisation nahelegt. Mithilfe von Fluoreszenz-Polarisations-Mikroskopie konnte zudem eine klare CRAC-Abhängigkeit der gp41-Oligomerisierung aufgezeigt werden. Die von uns gewonnenen Daten erlauben einen tieferen Einblick in die molekulare Basis und die biologischen Folgen der cholesterol-abhängigen lateralen Proteinorganisation für Virusassemblierungsprozesse an biologischen Membranen. / In recent years, there has been a considerable interest in the molecular organization of biological membranes. It has been hypothesized that self-assembling, freely diffusing, submicroscopic domains consisting of sphingolipids, cholesterol and certain proteins exist and the prevailing view is that those lipid rafts serve as platforms for specific molecular interactions by the preferential exclusion and inclusion of proteins. It was presumed, that in particular viruses make use of plasma membrane lipid rafts to augment the infection process and spread efficiently. However, the exact biological function and physical basis of protein partitioning into microdomains remains an outstanding question in virus biology. In the present study, fluorescence lifetime imaging microscopy was used to study lipid raft partitioning of the HIV-1 glycoprotein gp41 by detecting Foerster Resonance Energy Transfer between fluorescently labeled viral and raft marker proteins in living cells. Plasma membrane microdomain association of gp41 was demonstrated and by introducing systematic mutations and truncations in different gp41 motifs, the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial determinant of the lateral sorting. Interestingly, we observed a systematic cell-to-cell variability in our raft related data that suggests underlying cell-biological mechanisms of membrane organization. Moreover, fluorescence polarization microscopy revealed a distinct CRAC requirement for gp41 oligomerization whereas other properties, such as intracellular distribution and expression efficiency were clearly demonstrated to be CRAC independent. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral protein sorting for virus assembly processes at cellular plasma membranes.
|
13 |
Giant vesiclesStöckl, Martin Thomas 14 January 2009 (has links)
In der vorliegenden Arbeit wird ein neuer Ansatz vorgestellt, um Lipiddomänen, die Bindungsorte peripherer und integraler Membranproteine darstellen können, zu charakterisieren. Insbesondere wurde die Analyse der Fluoreszenzlebenszeiten von NBD-markierten Lipidanaloga benutzt, um Lipiddomänen in Giant unilamellar vesicles (GUV) und darauf aufbauend, in der Plasmamembran von Säugerzellen zu untersuchen. Das typische Zeitfenster von Fluoreszenzlebenszeiten im Bereich von Nanosekunden ermöglicht es, auch sehr kurzlebige Lipiddomänen nachzuweisen. Mit Hilfe des Fluorescence lifetime imaging (FLIM) wurden für die liquid disordered (ld) und liquid ordered (lo) Domänen in GUV jeweils spezifische Werte für das Abklingen der Fluoreszenz gemessen. Sogar die Existenz von submikroskopischen Domänen in GUV konnte nachgewiesen werden. Die Fluoreszenzlebenszeit des Lipidanalogs C6-NBD-PC zeigte in der Plasmamembran von Säugerzellen eine breite Verteilung. Dies legt in Übereinstimmung mit FLIM-Experimenten an aus der Plasmamembran von HeLa-Zellen gewonnenen Giant vesicles nahe, dass in der Plasmamembran von Zellen eine Vielzahl verschiedener submikroskopischer Lipiddomänen existiert. Darauf aufbauend wurde die Fluoreszenzmikroskopie an GUV angewendet, um die Bindung von fluoreszenzmarkiertem alpha-Synuclein an mittels FLIM charakterisierte Lipiddomänen zu untersuchen. Die Experimente zeigten, dass das Protein mit hoher Affinität an negativ geladene Phospholipide unter der Vorraussetzung bindet, dass diese sich in ld Domänen befinden. Im Gegensatz dazu erfolgt keine Bindung wenn diese Lipide in lo Domänen lokalisiert sind. Im Vergleich zum wildtypischen alpha-Synuclein zeigte die Variante A30P eine geringere Affinität zur Membran, während die E46K-Variante eine stärkere Bindung zeigte. Dies deutet darauf hin, dass bei den erblichen Formen des Morbus Parkinson eine veränderte Assoziation des alpha-Synucleins mit der Membran eine Rolle spielen kann. / In the present study a novel approach to characterize lipid domains, which may provide binding sites for peripheral or integral membrane proteins, is demonstrated. In particular, analysis of fluorescence lifetimes of NBD-labeled lipid analogues was used to study lipid domains in Giant unilamellar vesicles (GUV) and – based on the GUV results – in the plasma membrane of mammalian cells. As fluorescence decays in a few nanoseconds it is possible to to detect also very short-lived lipid domains. Fluorescence Lifetime Imaging (FLIM) revealed that the fluorescence decay of NBD-lipid analogues showed domain dependent decay times in the liquid disordered (ld) and the liquid ordered (lo) phase of GUV. Even the existence of submicroscopic domains in lipid membranes could be detected by FLIM. A broad distribution of the fluorescence lifetime was found for C6-NBD-PC inserted in the plasma membrane of mammalian cells. In agreement with FLIM studies on lipid domain forming Giant vesicles derived from the plasma membrane of HeLa-cells this may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact mammalian cells. Based on that, fluorescence microscopy was used on GUV to study the binding of fluorescently labeled alpha-synuclein at lipid domains previously characterized by FLIM. The experiments suggested that alpha-synuclein binds with high affinity to negatively charged phospholipids, when they are embedded in a ld as opposed to a lo environment. When compared with wildtype alpha-synuclein, the disease-causing alpha-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that an altered association of alpha-synuclein with membranes may play a role in the inherited forms of Parkinson’s disease.
|
14 |
Influence of ABCA1 and ABCA7 on the lipid microenvironment of the plasma membranePlazzo, Anna Pia 02 July 2009 (has links)
Der ABC-Transporter ABCA1 ist unmittelbar in die zelluläre Lipidhomeostasie einbezogen, in dem er die Freisetzung von Cholesterol an plasmatische Rezeptoren, wie ApoA-I, vermittelt. Trotz intensiver Untersuchungen ist dieser molekulare Mechanismus nicht verstanden. Verschiedene Studien deuten daraufhin, dass durch die Aktivität von ABCA1 bedingte Veränderungen in der Lipidphase der äußeren Hälfte der Plasmamembran (PM) wichtig für die Freisetzung des Cholesterols sind. In der vorliegenden Arbeit wird die Lipidumgebung von ABCA1 in der PM lebender Säugetierzellen unter Anwendung der Fluoreszenzlebenszeitmikroskopie von fluoreszierenden Lipidsonden untersucht. Es wurde eine breite Verteilung der Fluoreszenzlebenszeiten der Sonden gefunden, die sensitiv gegenüber Veränderungen der lateralen und transversalen Organisation der Lipide ist. Im Einklang mit Studien an riesengroßen unilamellaren Vesikeln und Plasmamembranvesikeln weisen unsere Ergebnisse die Existenz einer größeren Vielfalt submikroskopischer Lipiddomänen auf. Die FLIM-Untersuchungen an ABCA1 exprimierenden HeLa-Zellen weisen eine die Lipidphase destabilisierende Funktion des Transportes aus. Dieses wurde unterstützt durch die Lipidanalyse von Fraktionen der PM. Auf der Basis unserer Untersuchungen und früheren Daten stellen wir die Hypothese auf, dass die Exponierung von Phosphatidylserin (PS) auf der Zelloberfläche ein zentrales Ereignis der ABCA1 bedingten Veränderungen ist. Allerdings zeigen vergleichende Studien an ABCA7 exprimierenden Zellen, dass dies nicht ausreicht, um die ABCA1 verursachten Veränderungen in der Lipidpackung der PM zu erklären. Unsere Ergebnisse beweisen, dass die Fähigkeit von ABCA1, den Cholesterolefflux zu vermitteln, auf durch den Transporter bedingte Veränderungen in der LP der PM zurückzuführen sind, die unabhängig von der Bindung von ApoA-1 sind und dieser vorausgehen. Diese Veränderungen sind notwendig für die Lipidierung von ApoA-1 und der Generierung von HDL-Partikeln. / The ABCA1 transporter organizes cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors such as ApoA-I. Despite intensive investigation, the molecular mechanism of such a process has not yet been clarified. In the present study we report on the analysis of the ABCA1 lipid microenvironment at the plasma membrane of living cells, by a novel approach based on fluorescence lifetime imaging microscopy (FLIM). In the plasma membrane of mammalian cells, a broad fluorescence lifetime distribution sensitive to treatments interfering with the membrane lateral and transbilayer organization was found. In agreement with investigations in giant unilamellar vesicles and giant plasma membrane vesicles, our results are consistent with the existence of a large variety of submicroscopic lipid domains. Based on that, FLIM in HeLa cells expressing ABCA1 revealed the destabilizing function of the transporter on the lipid arrangement at the membrane, indicating that lipid packing was a primary target of ABCA1 activity. This was corroborated by the analysis of plasma membrane fractions isolated by density fractionation. On the basis of our analysis and previous data, we speculate that the exposure of phosphatidylserine on the cell surface is a central event for ABCA1-dependent modifications. However, a comparative study of cells expressing ABCA7, the member of the ABCA subfamily with the highest homology to ABCA1, revealed that exposure of PS alone cannot account for the detected effects. Collectively, our data suggest that the ability of ABCA1 to promote cholesterol efflux is independent and precedes its actual binding to ApoA-I. Rather, ABCA1-induced plasma membrane modifications are necessary for the lipidation of ApoA-I and the generation of high density lipoprotein particles.
|
15 |
High-sensitivity spectral fluorescence lifetime imaging for resolving spectroscopically overlapping speciesCrawford, Justin Lee 01 August 2009 (has links)
The capability to resolve the contributions from spectroscopically overlapping fluorophores has enabled significant breakthroughs in cellular imaging. However, commercial microscopes for this purpose use analog light detection with least squares curve-fitting analysis and improvements in sensitivity are needed. To this end, a microscope has been constructed with high throughput and single-photon detection capability. The fluorescence is separated through use of a prism spectrometer or a series of dichroic mirrors into four spectral bands and detected using four single-photon avalanche diode (SPAD) detectors, which provide high-quantum efficiency in the red spectral region. The detectors are connected to a time-correlated single photon counting module to provide sub-nanosecond temporal resolution for distinguishing fluorophores with different fluorescence lifetimes. Maximum-likelihood (ML) methods have been developed for analyzing the temporally and spectrally resolved photon count data from the SPADs to find the contributions from different fluorescent species and from background. Commercially available SPADs exhibit a count-rate dependent time shift in the impulse response function, and hence the instrument incorporates custom modified SPADs with improved timing stability. Nevertheless, there is still some time shift, and hence the ML-analysis has been extended to include this as an adjustable parameter for each individual SPAD. Monte Carlo simulations have also been developed to enable studies of the number of photons needed to resolve specific fluorophores.
|
16 |
High-sensitivity spectral fluorescence lifetime imaging for resolving spectroscopically overlapping speciesCrawford, Justin Lee 01 August 2009 (has links)
The capability to resolve the contributions from spectroscopically overlapping fluorophores has enabled significant breakthroughs in cellular imaging. However, commercial microscopes for this purpose use analog light detection with least squares curve-fitting analysis and improvements in sensitivity are needed. To this end, a microscope has been constructed with high throughput and single-photon detection capability. The fluorescence is separated through use of a prism spectrometer or a series of dichroic mirrors into four spectral bands and detected using four single-photon avalanche diode (SPAD) detectors, which provide high-quantum efficiency in the red spectral region. The detectors are connected to a time-correlated single photon counting module to provide sub-nanosecond temporal resolution for distinguishing fluorophores with different fluorescence lifetimes. Maximum-likelihood (ML) methods have been developed for analyzing the temporally and spectrally resolved photon count data from the SPADs to find the contributions from different fluorescent species and from background. Commercially available SPADs exhibit a count-rate dependent time shift in the impulse response function, and hence the instrument incorporates custom modified SPADs with improved timing stability. Nevertheless, there is still some time shift, and hence the ML-analysis has been extended to include this as an adjustable parameter for each individual SPAD. Monte Carlo simulations have also been developed to enable studies of the number of photons needed to resolve specific fluorophores.
|
17 |
Etude de la spore de Bacillus subtilis : caractérisation des structures impliquées dans sa résistance / Study of Bacillus subtilis spore's : characterication of stuctures implied in its resistanceLoison, Pauline 10 October 2013 (has links)
La spore bactérienne est une forme microbienne multicouche extrêmement résistante aux perturbations environnementales. Cette résistance est notamment liée à sa structure unique qui est particulièrement peu perméable et compacte. Ce travail de thèse a pour but d’identifier et de caractériser les structures sporales impliquées dans ces propriétés. Des méthodes d’investigations globales comme la RMN ou l’anisotropie de fluorescence ont permis de montrer que le cortex des spores de Bacillus subtilis est modifié par la température, pour des valeurs proches de celle de l’activation de la germination. Ceci aura pour conséquence de modifier l’accès à la membrane interne. Un outil d’étude à l’échelle de la spore, l’imagerie en temps de vie de fluorescence (FLIM) couplé à l’utilisation d’un rotor moléculaire, a également été mis au point. Cet outil a permis de mettre en évidence que la membrane interne de B. subtilis possède une très forte viscosité, environ deux fois plus importante que celle de la membrane d’une cellule végétative. Cette viscosité n’est modifiée par la température qu’au-delà de 65 °C, correspondant également à l’activation de la germination. Une perturbation connue pour modifier l’intégrité de la structure de la spore a également été étudiée : l’éthanol couplé à une température importante (65 ou 70°C). Ce traitement est responsable d’une perméabilisation et d’une inactivation des spores. L’éthanol conduit notamment à l’altération de la membrane interne, dont la viscosité et la perméabilité sont modifiées. Ces résultats apportent de nouvelles données pour la compréhension des mécanismes responsables de l’inactivation des spores. Ils permettent d’envisager des applications, pour lesquelles une maitrise des modifications structurales est nécessaire, comme la microencapsulation. / The bacterial spore is a multilayer microbial form which is extremely resistant to environmental perturbations. This resistance is especially due to its unique structure which is particularly compact and weakly permeable. This work aims to identify and characterize the spore structures involved in these properties. Overall investigation methods, such as NMR and fluorescence anisotropy, have shown that the cortex of Bacillus subtilis spores is modified by temperature for level similar to that of the activation of germination. This will result in changes to the access to the inner membrane. A tool at the spore’s scale, the fluorescence lifetime imaging microscopy (FLIM) in conjunction with the use of a molecular rotor, has been set up. This tool allowed demonstrating that inner membrane of B. subtilis has a very high viscosity, about two times greater than that of the membrane of a vegetative cell. This viscosity is changed by temperature near 65 °C, which corresponds to activation of germination. A stress known to modify the structural integrity of the spore has also been studied: ethanol combined with significant temperature (65 ou 70 °C). This treatment is responsible for inactivation of spores in parallel with their permeabilization. Ethanol especially leads to alteration of the inner membrane for which the viscosity and permeability are changed. These results provide new understanding of mechanisms implicated in spores’ destruction. They allow considering some new applications, for which it is necessary to control structural changing, for example microencapsulation.
|
18 |
Kolagenní struktury od buněčných kultur k šlaše / Collagen structures from cell culture to intact tendonHadraba, Daniel January 2017 (has links)
CHARLES UNIVERSITY and HASSELT UNIVERSITY / tUL Doctoral dissertation Collagen structures from cell culture to intact tendon ABSTRACT Author: Daniel Hadraba Promoters: Assoc. Prof. Karel Jelen | Charles University Prof. Marcel Ameloot | Hasselt University Co-promoters: Dr. Frantisek Lopot | Charles University Prof. Virginie Bito | Hasselt University Annotation Author: Ing. Mgr. Daniel Hadraba Doctoral thesis title: Collagen structures from cell culture to intact tendon Year: 2010 - 2017 Doctoral program: Doctor of Biomechanics at Charles University Doctor of Biomedical Science at Hasselt University / transnational University Limburg Departments: Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Dept. Biophysics | Hasselt University Promoters: Assoc. Prof. Karel Jelen | Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Prof. Marcel Ameloot | Hasselt University / transnational University Limburg Co-promoters: Dr. Frantisek Lopot | Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Prof. Virginie Bito | Hasselt University / transnational University Limburg Bibliography details: Pages 102 Figures 30 Tables 2 Equations 17 Keywords: tendon, collagen, crimps, orientation, aging,...
|
19 |
Kolagenní struktury od buněčných kultur k šlaše / Collagen structures from cell culture to intact tendonHadraba, Daniel January 2017 (has links)
CHARLES UNIVERSITY and HASSELT UNIVERSITY / tUL Doctoral dissertation Collagen structures from cell culture to intact tendon ABSTRACT Author: Daniel Hadraba Promoters: Assoc. Prof. Karel Jelen | Charles University Prof. Marcel Ameloot | Hasselt University Co-promoters: Dr. Frantisek Lopot | Charles University Prof. Virginie Bito | Hasselt University Annotation Author: Ing. Mgr. Daniel Hadraba Doctoral thesis title: Collagen structures from cell culture to intact tendon Year: 2010 - 2017 Doctoral program: Doctor of Biomechanics at Charles University Doctor of Biomedical Science at Hasselt University / transnational University Limburg Departments: Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Dept. Biophysics | Hasselt University Promoters: Assoc. Prof. Karel Jelen | Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Prof. Marcel Ameloot | Hasselt University / transnational University Limburg Co-promoters: Dr. Frantisek Lopot | Dept. Anatomy and Biomechanics | Faculty of Physical Education and Sport | Charles University Prof. Virginie Bito | Hasselt University / transnational University Limburg Bibliography details: Pages 102 Figures 30 Tables 2 Equations 17 Keywords: tendon, collagen, crimps, orientation, aging,...
|
20 |
Single-Molecule Metal-Induced Energy Transfer: From Basics to ApplicationsKaredla, Narain 02 June 2016 (has links)
No description available.
|
Page generated in 0.0986 seconds