1,721,151 research outputs found
Excitation beyond the monochromatic laser limit: simultaneous 3-D confocal and multiphoton microscopy with a tapered fiber as white-light laser source
No full textConfocal and multiphoton microscopy are essential tools in modern life sciences. They allow fast and highly resolved imaging of a steadily growing number of fluorescent markers, ranging from fluorescent proteins to quantum dots and other fluorophores, used for the localization of molecules and the quantitative detection of molecular properties within living cells and organisms. Up to now, only one physical limitation seemed to be unavoidable. Both confocal and multiphoton microscopy rely on lasers as excitation sources, and their monochromatic radiation allows only a limited number of simultaneously usable dyes, which depends on the specific number of laser lines available in the used microscope. We have overcome this limitation by successfully replacing all excitation lasers in a standard confocal microscope with pulsed white light ranging from 430 to 1300 nm generated in a tapered silica fiber. With this easily reproducible method, simultaneous confocal and multiphoton microscopy was demonstrated. By developing a coherent and intense laser source with spectral width comparable to a mercury lamp, we provide the flexibility to excite any desired fluorophore combination
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Cell compliance: cytoskeletal origin and importance for cellular function.
Full text linkMechanical properties of cells, mainly defined by their cytoskeleton, are closely related to cell function and can be measured with a dual-beam laser trap (optical stretcher). Functional changes, which go hand in hand with changes of the cytoskeleton, also occur during differentiation of stem cells. This suggests monitoring differentiation by the changing compliance of the cells. During the course of my PhD I measured the compliance of three different types of stem cells before and after differentiation and was able to detect differences in some of the cell types. In order to relate rheological experiments to cell migration as a further example of functional change I investigated the migration behavior of cells that showed different compliance and found differences in migration. I was additionally able to show an altered migration behavior after I actively changed the mechanical behavior of one cell type using cytoskeletal drugs. These migration experiments have been carried out in 2D and 3D migration assays. Furthermore, the influence of the stiffness of the surrounding material on the migration behavior has been investigated. After relating functional changes to changes in compliance, I studied which mechanisms can be used to actually influence cell compliance and investigated the effect of cytoskeletal stabilizers or destabilizers as well as drugs acting on molecular motors. The effect of the surrounding temperature has been considered as well. Finally, I developed a new version of the optical stretcher measurement tool, which enables cell sorting and drug screening using a monolithic glass chip. With the results presented in this thesis I relate mechanical compliance to the cytoskeleton and specific cellular functions. I deliver insights how mechanical changes in cells can be used to identify and follow functional changes and how this knowledge can help to interfere with such functions, specifically in pathologies correlated to these functions. My modified optical stretcher would be developed to screen the effects of drugs on cell compliance and to sort cells with different mechanical properties. Such drug screening and cell sorting will offer diagnostic treatment options for various pathologies
Quantification of Molecular Interaction Strength in Protein Condensates by Brillouin Microscopy
No full textBiomolecular condensates have recently been identified as a crucial element of intracellular organization.
These membraneless organelles fulfill various roles, for example, in cellular stress response
or DNA damage repair. The basis for the formation of biomolecular condensates are molecular interactions,
driving a solution of biomolecules from a mixed into a phase-separated state and back.
These interactions also define the physical properties of such condensates, which are relevant for
their physiological function and pathology. However, the experimental toolbox for the assessment of
the physical properties of biological condensates and their underlying molecular interactions has not
evolved as rapidly as the field of biological phase separation. The lack of robust quantitative methods
poses a roadblock to the further development of the field. Only robust, dependable measurements
provide a solid ground for a fundamental explanation of so far mostly qualitative observations.
In order to address the shortage of experimental techniques, I used Brillouin microscopy to study the
physical properties of different protein condensates in vitro. Brillouin microscopy is an emerging tool
in the field of biomechanics that has been mostly used to investigate the elastic properties of cells
and tissues so far.
Here, I exploited the ability of Brillouin microscopy to investigate liquid materials. I established it as
a novel tool to quantify average molecular interaction strength as well as the dissipative properties
of protein condensates, represented by the Brillouin shift and the Brillouin linewidth, respectively.
Furthermore, I used quantitative phase imaging to measure the refractive index of the condensates,
giving access to their protein concentration and protein volume fraction. In contrast to many other
experimental methods, these optical techniques do not require extensive assumptions or modeling of
the system and work for liquid and solid materials.
I investigated two different phase-separating proteins from the family of heterogeneous nuclear ribonucleoproteins,
hnRNPA1 and the fused in sarcoma protein (FUS). I monitored the physical changes in
the protein condensates in response to altered temperature and ion concentration, which have been
shown to affect phase separation. Conditions favoring phase separation (e.g., low temperatures)
increased Brillouin shift, Brilouin linewidth, and protein concentration. In contrast to solidification
by chemical crosslinking, the physical aging process of FUS condensates had only a small impact
on the Brillouin shift, which was consistent with the establishment of an elastic network within the
condensates. This process was suppressed at a high ion concentration. Finally, I compared different
variants of the hnRNPA1 protein featuring a broad range of different saturation concentrations. My
experiments showed, that parameters with an impact on the phase separation behavior also alter
the physical properties of the respective condensates. These results provide a new experimental
perspective on the time-dependent physical properties of protein condensates and their sensitivity to
temperature, solution conditions, and amino acid sequence of the respective protein
High-Throughput Viscoelastic Characterization of Cells in Microchannels
Full text linkExtensive research has showcased the potential of cell viscoelastic properties as intrinsic markers of cell state, functionality, and disease. While various techniques have been developed to measure cell viscoelasticity, most lack the throughput required for clinical diagnostics. Microfluidic techniques were designed to enhance throughput, but available designs lead to intricate stress distributions on the cell surface, complicating the analysis of a stress-strain relation.
This thesis aims to bridge this gap by presenting a high-throughput framework offering a straightforward stress-strain relation for a direct derivation of viscoelastic properties.
Microfluidic cell deformability techniques frequently employ a carrier solution of methyl cellulose (MC). These solutions exhibit complex stresses in flow and a complete description of shear and normal stresses at high shear rates was not reported before. In a comprehensive study, the shear rheology of MC solutions was measured at shear rates up to 150 000 1/s, providing the first results at these shear rates and revealing the viscoelastic nature of these solutions. I analyzed these measurements to derive constitutive equations for calculating the stresses of MC solutions in microchannel flow.
These results were employed to model the flow in real-time deformability cytometry (RT-DC), a prominent microfluidic technique for measuring cell elasticity. I developed a protocol and analysis pipeline to extend RT-DC for viscoelasticity measurements. Simultaneously, simulations were created to match experimental conditions, revealing discrepancies between simulation and experiment. The main reason for the mismatch was identified as an incomplete modeling of the viscoelastic contributions of the MC solutions.
Furthermore, the simulations unveiled a complex stress-strain relation for RT-DC experiments. To address this, I proposed a hyperbolic channel design to create a well-defined and constant stress along the flow axis, simplifying the viscoelastic analysis. I validated this approach by determining deformation timescales through measurements on oil droplets with varying viscosities and the stiffness of mechanical calibration particles from polyacrylamide (PAAm) microgels. Subsequently, the hyperbolic channels were then employed on human leukemia cell line HL60 cells, demonstrating that changes in cell viscoelasticity due to interference with the cytoskeleton could be measured. This showcases the approach as a streamlined and time-efficient solution for assessing the viscoelastic properties of large cell populations and other microscale soft particles.
The concepts outlined here are already routinely used for the analysis of RT-DC experiments. The results presented in this thesis offer valuable insights for the fields of cell mechanics, deformability cytometry, and fluid mechanics of complex liquids, with wide-ranging applications in cell research or diagnostics.Umfangreiche Forschung hat das Potenzial viskoelastischer Zelleigenschaften zur Charakterisierung von Zellzustand, -funktionalität oder Krankheiten aufgezeigt. Obwohl verschiedene Messmethoden zur Bestimmung der Zellviskoelastizität entwickelt wurden, fehlt es den meisten an der erforderlichen Durchsatzleistung für die klinische Diagnostik. Mikrofluidische Methoden wurden entwickelt, um den Durchsatz zu erhöhen, aber vorhandene Designs führen zu komplexen Spannungsverteilungen auf der Zelloberfläche, was die Analyse einer Spannungs-Dehnungs-Beziehung erschwert.
Das Ziel dieser Arbeit ist es, diese Lücke zu schließen, indem ein Hochdurchsatz-Ansatz vorgestellt wird, der eine einfache Spannungs-Dehnungs-Beziehung für eine direkte Ableitung viskoelastischer Eigenschaften bietet.
Mikrofluidische Zelldeformationsmethoden verwenden häufig eine Trägerlösung aus Methylzellulose (MC). Diese Lösungen zeigen komplexe Spannungen im Kanalfluss, und eine umfassende Beschreibung von Scher- und Normalspannungen bei hohen Scherraten war bisher nicht bekannt. In einer umfassenden Studie wurde die Scherrheologie von MC-Lösungen bei Scherraten von bis zu 150 000 1/s gemessen, wodurch die ersten Ergebnisse bei diesen Scherraten gezeigt und die viskoelastischen Eigenschaften dieser Lösungen aufgedeckt wurden. Diese Messungen wurden von mir analysiert und ich habe Gleichungen zur Berechnung der Kräfte von MC-Lösungen im Mikrokanalfluss hergeleitet. Diese Ergebnisse wurden verwendet, um den Fluss in Real-Time Deformability Cytometry (RT-DC) zu modellieren, einer verbreiteten, mikrofluidischen Methode zur Messung der Zellelastizität. Ich habe ein Protokoll und eine Analyse-Pipeline entwickelt, um RT-DC für viskoelastische Messungen zu erweitern. Gleichzeitig wurden Simulationen entwickelt, die die experimentellen Bedingungen nachbilden, wobei Diskrepanzen zwischen Simulation und Experiment auftraten. Die unvollständige Modellierung der viskoelastischen Eigenschaften der MC-Lösungen wurde als der Hauptgrund für diese Unterschiede ausgemacht.
Darüber hinaus zeigten die Simulationen ein komplexes Spannungs-Dehnungs-Verhältnis für RT-DC-Experimente. Um die viskoelastische Analyse zu vereinfachen, entwickelte ich ein hyperbolisches Kanaldesign, um eine gut definierte und konstante Spannung entlang der Flussachse zu erzeugen. Ich validierte diesen Ansatz durch Messungen der Verformungszeitskalen von Öltropfen mit unterschiedlichen Viskositäten und der Steifigkeit von mechanischen Kalibrierungspartikeln aus Polyacrylamid (PAAm)-Mikrogelen. Anschließend wurden Zellen der humanen Leukämiezelllinie HL60 in den hyperbolischen Kanälen gemessen und gezeigt, dass Veränderungen der Zellviskoelastizität aufgrund von Veränderungen des Zytoskeletts auftraten. Dies unterstreicht die Effektivität und Zeitersparnis dieses Ansatzes zur Messung der viskoelastischen Eigenschaften großer Zellpopulationen und anderer verformbarer Partikel im Mikrometerbereich.
Die hier dargelegten Konzepte werden bereits routinemäßig zur Analyse von RT-DC-Experimenten verwendet. Die in dieser Arbeit präsentierten Ergebnisse bieten wertvolle Einblicke in die Bereiche Zellmechanik, Verformbarkeitszytometrie und Strömungsmechanik komplexer Flüssigkeiten, mit vielfältigen Anwendungen in der Zellforschung oder Diagnostik
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
A quantitative analysis of the optical and material properties of metaphase spindles
Full text linkDie Metaphasenspindel ist eine selbstorganisierende molekulare Maschine, die die entscheidende Funktion erfüllt, das Genom während der Zellteilung gleichmäßig zu trennen. Spindellänge und -form sind emergente Eigenschaften, die durch komplexe Wechselwirkungsnetzwerke zwischen Molekülen hervorgerufen werden. Obwohl erhebliche Fortschritte beim Verständnis der einzelnen molekularen Akteure erzielt wurden, die ihre Länge und Form beeinflussen, haben wir erst kürzlich damit begonnen, die Zusammenhänge zwischen Spindelmorphologie, Dynamik und Materialeigenschaften zu untersuchen.
In dieser Arbeit untersuchte ich zunächst quantitativ die Rolle zweier molekularer Kraftgeneratoren - Kinesin-5 und Dynein - bei der Regulierung der Spindelform von Xenopus-Eiextrakt. Eine Störung ihrer Aktivität veränderte die Spindelmorphologie, ohne die Gesamtmasse der Mikrotubuli zu beeinflussen. Um die Spindelform physikalisch zu stören, wurde ein Optical Stretcher (OS) -Aufbau entwickelt. Obwohl das OS Vesikel in Extrakten verformen könnte, konnte keine Kraft auf Spindeln ausgeübt werden. Die Untersuchung des Brechungsindex der Struktur mittels optischer Beugungstomographie (ODT) ergab, dass es keinen Unterschied zwischen Spindel und Zytoplasma gab. Korrelative Fluoreszenz- und ODT-Bildgebung zeigten, wie sich die Materialeigenschaften innerhalb verschiedener Biomoleküle räumlich unterschieden. Die Gesamttrockenmasse der Spindel skalierte mit der Länge, während die Gesamtdichte konstant blieb. Interessanterweise waren die Spindeln in HeLa-Zellen dichter als das Zytoplasma. Schließlich deckte eine störende Mikrotubulusdichte auf, wie die Gesamttubulinkonzentration die Spindelgröße, die Gesamtmasse und die Materialeigenschaften regulierte.
Insgesamt bietet diese Studie eine grundlegende Charakterisierung der physikalischen Eigenschaften der Spindel und hilft dabei, Zusammenhänge zwischen der Biochemie und der Biophysik einer aktiven Form weicher Materie zu beleuchten.The metaphase spindle is a self-organising molecular machine that performs the critical function of segregating the genome equally during cell division. Spindle length and shape are emergent properties brought about by complex networks of interactions between molecules. Although significant progress has been made in understanding the individual molecular players influencing its length and shape, we have only recently started exploring the links between spindle morphology, dynamics, and material properties. A thorough analysis of spindle material properties is essential if we are to comprehend how such a dynamic structure responds to forces, and maintains its steady-state length and shape.
In this work, I first quantitatively investigated the role of two molecular force generators– Kinesin-5 and Dynein in regulating Xenopus egg extract spindle shape. Perturbing their activity altered spindle morphology without impacting total microtubule mass. To physically perturb spindle shape, an Optical Stretcher (OS) setup was developed. Although the OS could deform vesicles in extracts, force could not be exerted on spindles. Investigating the structure’s refractive index using Optical Diffraction Tomography (ODT) revealed that there was no difference between the spindle and cytoplasm. Correlative fluorescence and ODT imaging revealed how material properties varied spatially within different biomolecules. Additionally, spindle mass density and the microtubule density were correlated. The total dry mass of the spindle scaled with length while overall density remained constant. Interestingly, spindles in HeLa cells were denser than the cytoplasm. Finally, perturbing microtubule density uncovered how total tubulin concentration regulated spindle size, overall mass and material properties.
Overall, this study provides a fundamental characterisation of the spindle’s physical properties and helps illuminate links between the biochemistry and biophysics of an active form of soft matter
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