1,720,970 research outputs found
Waveguide Evanescent-Field Microscopy for Label-Free Monitoring of Biological Nanoparticles: Fabrication, Characterization and Application [Elektronisk resurs]
No full textThe recent development of microscopy methods, biological assays and bioanalytical sensors has significantly advanced the understanding of biological systems. Surface-based bioanalytical sensors have in recent years gained increased interest thanks to improvements in sensitivity and simplicity to use. However, most of them, such as quartz crystal microbalance (QCM) and surface plasmon resonance (SPR), provide information based on ensemble averaging of biomolecular interactions. In contrast, with surface-sensitive microscopy methods, biological processes can be resolved down to the level of individual molecular interactions. Total internal reflection fluorescent microscopy is one commonly used surface-sensitive method, reaching sensitivities down to the level of single molecules, but it requires fluorescent labeling of at least one of the interaction partners and is often also hampered by photo bleaching processes. In this thesis, we introduce a new wide-field surface-sensitive microscopy platform, based on a nanofabricated planar optical waveguide design that is capable of label-free evanescent-field microscopy of biological nanoparticles well below 100 nm in diameter. The waveguide generates an evanescent-field at the interface between the core of the waveguide and an aqueous solution, providing a thin sheet of illumination that offers imaging with low background disturbance. The device is presented in two designs, being compatible with either upright or inverted microscopes. The work presented demonstrates how simultaneous monitoring of fluorescence and scattering signals can offer new information about the relation between scattering intensity, refractive index and lipid content of biological nanoparticles, such as exosomes. Further, the microfluidic design allowed not only for convenient liquid handling with dead volumes of a few microliter, it is also showed to aid label-free investigations of the interaction between proteins and individual lipid vesicles, with the latter serving as cell-membrane mimic. With the device also being compatible with formation of fluid supported lipid bilayers, preliminary results suggest that the design will open up a possibility to simultaneously determine the size, scattering intensity and fluorescence emission at the level of individual biological nanoparticles. With this realized, we foresee a broad applicability of the microscopy platform as multidimensional characterization tool for biological nanoparticles and beyond
Fabrication, development and characterization of a waveguide microscopy device for biological applications
No full textOur understanding of biological systems has advanced significantly thanks to the development of microscopy methods and suitable biological assays. Surface based methods have increasingly gained interest thanks to their superior sensitivity and ease of use. Still, most of them, such as quartz crystal microbalance (QCM) and surface plasmon resonance (SPR), provide information based on ensemble averaging of biomolecular interactions. In contrast, surface-sensitive microscopy methods provide the possibility to study biological processes on an individual biomolecular basis. Total internal reflection fluorescent (TIRF) microscopy is a surface-sensitive microscopy method that reaches sensitivities down to the level of single molecules, but it is limited by the need to fluorescently label the interaction partners. On the contrary, waveguide-based evanescent-light scattering microscopy provides a label-free surface-sensitive imaging technique that can also benefit from fluorescent labeling if desired. Here we present ongoing efforts to further develop a waveguide-based platform for evanescent light-scattering microscopy. By adopting the fabrication and processing steps to a transparent substrate and adding compatible microfluidics, we can now use high NA oil-immersion objectives thereby collecting more light and resolve details that could not be resolved when fabricated on an opaque substrate. Further, well-controlled microfluidic handling makes it possible to perform more complicated experiments and extract data on interaction dynamics. Moreover an image processing code has been developed that can be applied to the different experimental sequences, including both waveguide fluorescent and scattering modes, and thereby extract otherwise hidden information in the experimental data
Waveguide Evanescent-Field Microscopy for Label-Free Monitoring of Biological Nanoparticles: Fabrication, Characterization and Application
Get PDFThe recent development of microscopy methods, biological assays and bioanalytical sensors has significantly advanced the understanding of biological systems. Surface-based bioanalytical sensors have in recent years gained increased interest thanks to improvements in sensitivity and simplicity to use. However, most of them, such as quartz crystal microbalance (QCM) and surface plasmon resonance (SPR), provide information based on ensemble averaging of biomolecular interactions. In contrast, with surface-sensitive microscopy methods, biological processes can be resolved down to the level of individual molecular interactions. Total internal reflection fluorescent microscopy is one commonly used surface-sensitive method, reaching sensitivities down to the level of single molecules, but it requires fluorescent labeling of at least one of the interaction partners and is often also hampered by photo bleaching processes.In this thesis, we introduce a new wide-field surface-sensitive microscopy platform, based on a nanofabricated planar optical waveguide design that is capable of label-free evanescent-field microscopy of biological nanoparticles well below 100\ua0nm in diameter. The waveguide generates an evanescent-field at the interface between the core of the waveguide and an aqueous solution, providing a thin sheet of illumination that offers imaging with low background disturbance. The device is presented in two designs, being compatible with either upright or inverted microscopes.The work presented demonstrates how simultaneous monitoring of fluorescence and scattering signals can offer new information about the relation between scattering intensity, refractive index and lipid content of biological nanoparticles, such as exosomes. Further, the microfluidic design allowed not only for convenient liquid handling with dead volumes of a few microliter, it is also showed to aid label-free investigations of the interaction between proteins and individual lipid vesicles, with the latter serving as cell-membrane mimic. With the device also being compatible with formation of fluid supported lipid bilayers, preliminary results suggest that the design will open up a possibility to simultaneously determine the size, scattering intensity and fluorescence emission at the level of individual biological nanoparticles. With this realized, we foresee a broad applicability of the microscopy platform as multidimensional characterization tool for biological nanoparticles and beyond
Fabrication, development and characterization of a waveguide microscopy device for biological applications [Elektronisk resurs]
No full textOur understanding of biological systems has advanced significantly thanks to the development of microscopy methods and suitable biological assays. Surface based methods have increasingly gained interest thanks to their superior sensitivity and ease of use. Still, most of them, such as quartz crystal microbalance (QCM) and surface plasmon resonance (SPR), provide information based on ensemble averaging of biomolecular interactions. In contrast, surface-sensitive microscopy methods provide the possibility to study biological processes on an individual biomolecular basis. Total internal reflection fluorescent (TIRF) microscopy is a surface-sensitive microscopy method that reaches sensitivities down to the level of single molecules, but it is limited by the need to fluorescently label the interaction partners. On the contrary, waveguide-based evanescent-light scattering microscopy provides a label-free surface-sensitive imaging technique that can also benefit from fluorescent labeling if desired. Here we present ongoing efforts to further develop a waveguide-based platform for evanescent light-scattering microscopy. By adopting the fabrication and processing steps to a transparent substrate and adding compatible microfluidics, we can now use high NA oil-immersion objectives thereby collecting more light and resolve details that could not be resolved when fabricated on an opaque substrate. Further, well-controlled microfluidic handling makes it possible to perform more complicated experiments and extract data on interaction dynamics. Moreover an image processing code has been developed that can be applied to the different experimental sequences, including both waveguide fluorescent and scattering modes, and thereby extract otherwise hidden information in the experimental data
Optical Manipulation of Nanoparticles’ Flow through Porous Membranes
No full textBy invention of laser as a high-intensity narrow-band light source and pioneering works of Ashkin, the optical tweezers emerged as a versatile tool for manipulating biological samples and mirco/nano-scale objects. Since then the optical forces have been a mean to trap, levitate and push particles or measure the forces at piconewton scale. To get to this point, many researches have been conducted to study the optical forces and optimize the trapping conditions for different particle sizes and materials. Nevertheless the effect of optical forces on the flow of particles has not been addressed before. The purpose of this thesis is to investigate the effect of light on the flow of nanoparticles. The work includes some nanofabri-cation and silicon work to create a porous membrane using colloidal lithography. PDMS soft lithography has been assisted to form a microfluidic backbone for the silicon device, responsible for delivering colloidal solution to the fabricated membrane and controlling the flow. Then the effect of light has been investigated by studying the flow of nanoparticles through the porous membrane in presence of a converging laser beam in darkfield microscopy setup. The general trend in the experimental results has been postulated by analytical solutions and simulations
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Evanescent Light-Scattering Microscopy for Label-Free Interfacial Imaging: From Single Sub-100 nm Vesicles to Live Cells
No full textAdvancement in the understanding of biomolecular interactions has benefited greatly from the development of surface-sensitive bioanalytical sensors. To further increase their broad impact, significant efforts are presently being made to enable label-free and specific biomolecule detection with high sensitivity, allowing for quantitative interpretation and general applicability at low cost. In this work, we have addressed this challenge by developing a waveguide chip consisting of a flat silica core embedded in a symmetric organic cladding with a refractive index matching that of water. This is shown to reduce stray light (background) scattering and thereby allow for label-free detection of faint objects, such as individual sub-20 rim gold nanoparticles as well as sub-100 nm lipid vesicles. Measurements and theoretical analysis revealed that light-scattering signals originating from single surface-bound lipid vesicles enable characterization of their sizes without employing fluorescent lipids as labels. The concept is also demonstrated for label-free measurements of protein binding to and enzymatic (phospholipase A2) digestion of individual lipid vesicles, enabling an analysis of the influence on the measured kinetics of the dye-labeling of lipids required in previous assays. Further, diffraction-limited imaging of cells (platelets) binding to a silica surface showed that distinct subcellular features could be visualized and temporally resolved during attachment, activation, and spreading. Taken together, these results underscore the versatility and general applicability of the method, which due to its simplicity and compatibility with conventional microscopy setups may reach a widespread in life science and beyond
- …
