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 <i>Rattus norvegicus</i> 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<sup>-2</sup>min<sup>-1</sup>. 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 novel Fourier domain method for microscopic imaging - so-called k-microscopy
- with lateral resolution independent of the detection numerical aperture. The
concept is based on sample illumination by a lateral fringe-pattern of varying spatial
frequency, which probes the lateral spatial frequency or k- spectrum of the sample
structure. The illumination pattern is realized by interference of two collimated coherent
beams. Wavelength tuning is employed for modulation of the fringe spacing. The
uniqueness of the proposed system is that a single point detector is sufficient to collect
the total light corresponding to a particular position in the sample k-space. By shifting
the phase of the interference pattern, we get full access to the complex frequencies. An
inverse Fourier transformation of the acquired band in the frequency- or k-space will
reconstruct the sample. The resulting lateral resolution will be defined by the temporal
coherence length associated with the detected light source spectrum as well as by the
illumination angle. The feasibility of the concept has been demonstrated in 1D.
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