Waveguide Evanescent-Field Microscopy for Label-Free Monitoring of Biological Nanoparticles: Fabrication, Characterization and Application
Doctoral thesis, 2018
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.
Waveguide scattering microscopy
Waveguide fluorescence microscopy
Supported lipid bilayer
Chalmers, Physics, Biological Physics
"Low-temperature fabrication and characterization of a symmetric hybrid organic-inorganic slab waveguide for evanescent light microscopy", Björn Agnarsson*, Mokhtar Mapar, Mattias Sjöberg, Mohammadreza Alizadehheidari and Fredrik Höök
Evanescent Light-Scattering Microscopy for Label-Free Interfacial Imaging: From Single Sub-100 nm Vesicles to Live Cells
ACS Nano,; Vol. 9(2015)p. 11849-11862
"Fabrication and Characterization of a Transparent Waveguide-Based Platform for Surface-Sensitive microscopy with microfluidic liquid handling", Mokhtar Mapar, Björn Agnarsson*, Fredrik Höök
"Effective refractive index and lipid content of extracellular vesicles revealed using optical waveguide scattering and fluorescence microscopy", Déborah L. M. Rupert#, Mokhtar Mapar#, Ganesh Vilas Shelke, Matthias Elmeskog, Karin Norling, Stephan Block, Björn Agnarsson, Jan O. Lötvall, Marta Bally, Fredrik Höök*
"Spatio-temporal kinetics of the formation of a lipid bilayer on silica via vesicle adsorption and rupture", Mokhtar Mapar, Silver Jõemetsa, Vladimir Zhdanov, Björn Agnarsson, Hudson Pace and Fredrik Höök*
Optics has a long history in biological science. Soon after the invention of the first compound optical microscope in 1595, it was used in studies of biological tissues and became a corner stone for many biological discoveries. Thanks to the early microscopes, scientists could lay their eyes on structures not larger than a few microns, such as bacteria and fungi. This ignited new speculations about the cause of various human and crops diseases, which in turn led to initiatives towards elimination of the wide spread plagues and famines at that time. Since then, the continuous improvement of optical microscopes and their broad use in various fields of science have had great impact on the quality of human life and our understanding of ourselves as biological beings.
Nowadays modern microscopy techniques can delve 1 000 times deeper than 400 years ago and offer information even at the single molecule level. This allows scientists to identify individual biomolecules and biological nanoparticles that are down to 10 000 times smaller than a thin hair, and study their origin, track their movements, learn about their role in the cellular machinery and cellular communication. This is the key knowledge needed to understand their complex functions, which in turn will aid the development of novel cures for many incurable diseases of today.
Most of these cutting-edge microscopy techniques owe their remarkable imaging quality to some “colorful” molecules often called labels, being attached to the object under investigation. However, the labeling process can be complicated, time consuming and expensive, and at times, these chemical labels may affect the function of the biomolecules that are being investigated. Although these labels offer possibilities beyond the capabilities of other existing technologies, the aforementioned complications have fueled a search for “label-free” techniques that, when possible, can deliver similar information without modifying the natural biomolecule with labels.
In the thesis in hand, in an interdisciplinary effort, we have brought together tools and knowledge from telecommunication, nanofabrication, optics, image processing, physics and biology, and introduce a new microscopy platform that, although fully compatible with conventional labeling schemes, also offers label-free imaging with nanometer resolution. In the thesis, I explain how the microscopy device, that is based on a concept commonly used in telecommunication, was materialized using various nanofabrication techniques. Further, image processing and theoretical representations were used to interpret results obtained when studying in particular cell-membrane mimics and their interactions with various proteins. The studies were also extended to include exosomes, a type of biological nanoparticle with promising potential in curing cancer and to provide new gene therapy solutions. By combining labeling and label-free imaging, we indeed obtained information that is invisible even to cutting edge microscopy techniques, and it is thus my hope that the work will serve as a seed for further development, which could possibly turn the concept it into a powerful multi-faceted characterization platform for single nanoparticle analytics and beyond.
Areas of Advance
Nanoscience and Nanotechnology (2010-2017)
Life Science Engineering (2010-2018)
Atom and Molecular Physics and Optics
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4397
Chalmers University of Technology
Kollektorn (A423), MC2 (Kemivägen 9), Chalmers
Opponent: Prof. Bo Liedberg, School of Materials Science and Engineering, Nanyang Technological University, Singapore