Novel strategies for super-resolution fluorescence microscopy

Abstract

<p>Minimally invasive technologies to characterize the structure and functionality of biological samples on the cellular and molecular scale are fundamental to life sciences. Optical fluorescence imaging at visible wavelengths is able to acquire to do so. Due to the wavelike nature of light the maximum achievable resolution in conventional microscopy is limited by diffraction, about half the wavelength of the considered light. Although this diffraction limit is fundamental, techniques have been developed to perform imaging at a resolution of two to a hundred times better than this. All these techniques come with their specific benefits and trade-offs and the presented thesis seeks to make a contribution in the filed of super-resolution microscopy. Structured illumination microscopy (SIM) is a technique that improves the image resolution by acquiring images under non-planar illumination and subsequent image reconstruction. This image reconstruction requires a prior parameter estimation of the illumination pattern from the acquired data. A contribution to improve this parameter estimation is presented and demonstrated using simulation frame work developed for this purpose. It can be shown, that the deviation of the pattern parameters from their actual value can be reduced by up to 80 percent as compared to a more conventional method. <p>A common way to generate sinusoidal illumination patterns in SIM is to interfere two coherent beams of light using the the imaging objective lens, the epifluorescence configuration. Two new methods to generate those interference patterns, namely transillumination structured illumination microscopy (tSIM), and chip structured illumination microscopy (cSIM) are presented. A pattern generation independent of the objective lens allows to improve the spacebandwidth product (SBP) of SIM by increasing the resolution without sacrificing the field of view (FoV). Imaging simulations are shown to demonstrate this effect when mirrors are used to generate the patterns in tSIM. A theoretical improvement of the SBP of almost five times the SBP of a conventional setup is discussed. Instead of using mirrors, optical waveguides may be used to generate those illumination patterns a presented in cSIM. Thesewaveguides are simulated using Comsol. Imaging is simulated according to expected pattern parameters and the improved imaging is illustrated. <p>The advantages of optical waveguides as discussion for cSIM are then examined regarding their implementation for light sheet fluorescence microscopy (LSFM). The image quality in wide-field imaging can be improved by limiting the sample illumination to the plane that is actually imaged. This is commonly done in LSFM and requires appropriate beam shaping. The implementation of different waveguide designs to perform beam shaping with waveguides are investigated performing a variety of simulations in Comsol. Chip based axicons with a total width of 20 micrometer and a wedge angle of six degree show to generate promising beam profiles with a propagation length of about 150 micrometer. A possible lattice light sheet generation using two counter propagation axicons is investigated as well as double axicon for the generation of bottle beams. The use of optical phased arrays to perform dynamic beam shaping using sets of 16 and 32 elements of 1 micrometer width is shown and the beam tilting and shifting capabilities a visible wavelength are presented.