Fluorescence microscopy for interference lithography: Set-up design and pattern characterization by fluorescence modulation

Abstract

In the course of this thesis, a novel approach for creating a regular and well-defined interference pattern in the focal plane of a microscope’s objective is presented. The structured illumination pattern was created by two-beam interference within a self-built epi-fluorescence microscope. Beam separation was accomplished by two Wollaston prisms installed in separate rotation mounts. It was shown that the orientation of the interference lines in the focal plane of the objective was directly related to the beams’ position at the back focal plane and that the distance between two interference fringes was linearly proportional to the inverse of the beams’ distance at the back aperture. The structured illumination pattern was used as an interference lithography set-up by bleaching it into a self-built fluorescent photoresist. Fluorescent dyes which were located within or close to the nodes of the interference pattern remained intact thus creating a negative imprint of the pattern within the photoresist, consisting of defined lines containingfluorescent dyes. The line widths of the nodes were assessed by single molecule localization of individual fluorescent dyes using an alternating variable search method (AVM) based algorithm. The fluorescence data for the AVM algorithm was recorded by using fluorescence modulation. AVM results on single molecule localizations provided evidence that the dyes’ positions were distributed on sub-diffractional dimensions for non-modulated as well as for modulated fluorescence data. The full width at half maximum (FWHM) values of each distribution (approx. 70 nm) were much smaller than the diffraction limit of light with respect to the dye’s emission wavelength. Furthermore, it was shown that the temporal domain of fluorescence modulation allowed the spatial distinction of selected molecule pairs which were located in close proximity to one another. The photo-selection of regular modulation was substantially narrowed by applying a second de-excitation beam whose polarization plane was oriented perpendicular with respect to the excitation light's polarization vector. The effect named excitation polarization angle narrowing (ExPAN) increased the photo-selectivity of excitation and improved the temporal separability of dye pairs or trios compared to regular modulation.