During open brain surgery we acquire perfusion images non-invasively using laser Doppler imaging. The regions of
brain activity show a distinct signal in response to stimulation providing intraoperative functional brain maps of
remarkably strong contrast.
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:GdVO<sub>4</sub> 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 <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 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
We present the development and first application of a novel dual-color total internal reflection (TIR) fluorescence system for single-molecule coincidence analysis and fluorescence cross-correlation spectroscopy (FCCS). As a performance analysis, we measured a synthetic DNA-binding assay, demonstrating this dual-color TIR-FCCS approach to be a suitable method for measuring coincidence assays such as biochemical binding, fusion, or signal transduction at solid/liquid interfaces. Due to the very high numerical aperture of the epi-illumination configuration, our setup provides a very high fluorescence collection efficiency resulting in a two- to three-fold increase in molecular brightness compared to conventional confocal FCCS. Further improvements have been achieved through global analysis of the spectroscopic data.
We present a semi-analytical model of optical coherence tomography (OCT) taking into account multiple scattering. The model rests on the assumptions that the measured portion of the backscattered sample field is spatially coherent and that the sample is motionless relative to measurement time. This allows modeling an OCT signal as a sum of spatially coherent fields with random phase arguments-constant during measurement time-caused by multiple scattering. We calculate the mean OCT signal from classical results of statistical optics and a Monte Carlo simulation. Our model is shown to be in very good agreement with a whole range of experimental data gathered in a comprehensive study of cross-talk in wide-field OCT realized with spatially coherent illumination. The study consists of depth scan measurements of a mirror covered with an aqueous suspension of microspheres. We investigate the dependence of cross-talk on important optical system parameters, as well as on some relevant sample properties. We discuss the more complex OCT models based on the extended Huygens-Fresnel principle, which rest on different assumptions since they assume partially coherent interfering fields.
Monitoring biological relevant reactions on the single molecule level by the use of fluorescent probes has become one of the most promising approaches for understanding a variety of phenomena in living organisms. By applying techniques of fluorescence spectroscopy to labelled molecules a manifold of different parameters becomes accessible i.e. molecular dynamics, energy transfer, DNA fingerprinting, etc... can be monitored at the molecular level.
However, many of these optical methods rely on oversimplified assumptions, for example a three-dimensional Gaussian observation volume, perfect overlap volume for different wavelength, etc. which are not valid approximations under many common measurement conditions. As a result, these measurements will contain significant, systematic artifacts, which limit their performance and information content.
Based on Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Spectroscopy we will present representative examples including a thorough signal analysis with a strong emphasis on the underlying optical principles and limitations. An outlook to biochip applications, parallel FCS and parallel Lifetime measurements will be given with cross links to optical concepts and technologies used in industrial inspection.