The diffraction patterns of fixed fluorophores are characteristic of the orientation of the molecules' underlying
dipole. Fluorescence localization microscopy techniques such as PALM and STORM achieve super-resolution by
sequentially imaging sparse subsets of fluorophores, which are localized by means of Gaussian-based localization.
This approach is based on the assumption of isotropic emitters, where the diffraction pattern corresponds to a
section of the point spread function. Applied to fixed fluorophores, it can lead to an estimation bias in the range
of 5-20nm.
We introduce a method for the joint estimation of position and orientation of single fluorophores, based on
an accurate image formation model expressed as a 3-D steerable filter. We demonstrate experimental estimation
accuracies of 5 nm for position and 2 degrees for orientation.
We introduce an efficient, image formation model-based algorithm that extends super-resolution fluorescence localization
to include orientation estimation, and report experimental accuracies of 5 nanometers for position estimation and 2 degrees
for dipole orientation estimation.
We present the results on two-photon total-internal-reflection fluorescence correlation spectroscopy. The combination of
liquid crystal spatial light modulator, providing radial polarization, with ultrafast laser (picosecond Nd:GdVO4 laser)
allowed us to take the advantage of nonlinear optical contrast mechanisms to suppress the side-lobe energy specific for
radial polarization and reduce the effective excited volume twice compared to one-photon evanescent wave excitation in
fluorescence correlation spectroscopy.
Odorant receptors are an excellent example of natural superiority in specifically binding specific, small and hydrophobic
molecules. They are of particular interest in the development of a sensor platform for G protein-coupled receptors
(GPCRs). Odorant receptors (OR5) of Rattus norvegicus were incorporated into model membranes by in vitro synthesis
and vectorial incorporation for achieving natural receptor function. The vectorial insertion of OR5 into the planar membrane
and their lateral distribution, their interactions and their mobility within the membrane are of great importance for
ligand-receptor interaction. We applied total internal reflection fluorescence (TIRF) microscopy and image analysis to
assess the insertion and the OR5 distribution as well as the lateral mobility of these receptors at the single molecule level.
The vectorial incorporation of OR5 into planar lipid membranes was investigated with TIRF microscopy and image segmentation.
With increasing expression time, the OR5 incorporation density and aggregation increased linearly by about
0.02μm-2min-1. The expression and incorporations of single OR5s were completed within about 8 minutes. The mobility
of the incorporated receptors was measured with fluorescence correlation spectroscopy (FCS) and fluorescence recovery
after photo-bleaching (FRAP). These measurements revealed that the incorporated receptors were immobilized with this
class of lipid membranes.
We present a method for fast calculation of the electromagnetic field near the focus of an objective with a high numerical
aperture (NA). Instead of direct integration, the vectorial Debye diffraction integral is evaluated with the fast Fourier
transform for calculating the electromagnetic field in the entire focal region. We generalize this concept with the chirp z
transform for obtaining a flexible sampling grid and an additional gain in computation speed. Under the conditions for the
validity of the Debye integral representation, our method yields the amplitude, phase and polarization of the focus field
for an arbitrary paraxial input field in the aperture of the objective. Our fast calculation method is particularly useful for
engineering the point-spread function or for fast image deconvolution.
We present several case studies by calculating the focus fields of high NA oil immersion objectives for various amplitude,
polarization and phase distributions of the input field. In addition, the calculation of an extended polychromatic
focus field generated by a Bessel beam is presented. This extended focus field is of particular interest for Fourier domain
optical coherence tomography because it preserves a lateral resolution of a few micrometers over an axial distance in the
millimeter range.
Micro- and nano-optical structures offer the possibility to control light on a wavelength scale. This allows further miniaturization of integrated optical circuits. Planar photonic crystal waveguides and microcavities are considered basic building blocks for applications such as microlasers, filters, multiplexers and optical switches. The possibility to tune or switch photonic crystal devices by various ways such as temperature, refractive index change using liquid crystals, free charge carrier density or non linear material effects increases their functionality to form multifunctional, intelligent devices. High-Q cavities in planar photonic crystals exhibit highly localised fields and narrow transmission bands. Due to their strong light confinement even a small perturbation of the localized field can change their transmission properties of the cavity. We present different ways of perturbing the optical environment near a photonic crystal cavity enabling tuning and modulation of the in-plane transmission. Optical switching and wavelength tuning is obtained by means of induced thermo and plasma dispersion effects when focusing a laser onto the cavity structure. The feasibility of high-speed optical integrated circuits based on silicon photonic crystal structures is shown. On the other hand, an AFM tip is used for mechanically tuning and damping the inplane transmission. A future challenge is the integration of more than one silicon tip to combine filter and tuning functionalities and to create a chip-based device.
Recent results of our studies into optical effects where sub-micron length scales play a pivotal role are presented. We start with a discussion of fine optical features produced by relatively large objects, and then move on to consider the big effects that can be produced by sub-micron structures. Topics covered include fine structure in the optical field of microlenses and gratings, and then further down in length scale from microstructured surfaces to resonant filters, photonic crystal waveguides and metallic nanoparticles. For each step we demonstrate potential applications in which such a length scale can present important advantages, as well as discussing some of the disadvantages and challenges in the design and fabrication of such elements. We particularly highlight the sensitivity of many of the structures to small variations in optical situation (e.g. geometry, orientation, material, polarization) leading significant optical effects for small-scale changes. Methods for the characterization of optical fields produced by objects at these smaller dimensions are also presented.
We present recent applications of one-dimensional (1D) and two-dimensional (2D) periodic structures. The structures were designed using rigorous diffraction theory and produced by modern micromachining techniques (electron beam writing, optical lithography). In addition, interferometric recording of periodic structures was investigated in order to fabricate periodic structures with arbitrary profile shapes.
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