This PDF file contains the front matter associated with SPIE-OSA Proceedings Volume 6633, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

An optical switch exhibits two stable and selectively addressable states, a fluorescent state and a non-fluorescent state, which can be reversibly interconverted upon irradiation with different wavelengths of light. Efficient molecular optical switches are strongly desired for improved protein tracking in living cells and they are potentially very useful for far-field fluorescence imaging with improved resolution. Because suitable optical switches reversibly undergo light-induced transitions between two thermally stable states, and the transitions are saturable, a spatial intensity distribution of two laser wavelengths (one to switch off the fluorophore and another to reactivate fluorescence) featuring a local minimum might allow fluorescence imaging at the nanoscale. Conceptually similar to stimulated-emission depletion (STED) microscopy optical switches promise similar spatial resolution, but require much lower laser intensities. Here we review different concepts that use reversible saturable optical fluorescence transitions (RESOLFT) and discuss the requirements optical switches have to fulfill. In addition, we address the problem of selective in vivo labeling of target molecules with chemical switches, i.e. optical switches that are activated upon specific labeling to target molecules in living cells.

The approach of modeling intracellular networks of biochemical reactions in systems biology demands novel methods suited for acquiring quantitative data about transport and interaction of proteins and metabolites within the heterogeneous environment of living cells. Single-molecule fluorescence spectroscopy (SMFS) has proven a valuable tool for investigating complex structures and processes in biochemistry and molecular biology providing a rich set of methods for in vitro studies of protein/protein and protein/DNA interactions. Although especially designed to reveal spatial and temporal heterogeneities, few applications of SMFS to living cells were reported. Multi-parameter approaches like spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) have the unique advantage of acquiring individual photons from single molecules carrying along characteristic properties of the fluorescence emission, such as fluorescence lifetime and fluorescence emission spectrum. Herein we present different possibilities for data analysis that can be applied to single molecule data recorded on single photon bases. The photons be used to generate microscopic images with additional contrast based on different fluorescence lifetimes or emission spectra. Information about dynamic processes influencing the characteristic fluorescence signal, like local quenching or molecular diffusion, can be investigated by use of auto- or cross-correlation or on the basis of individual events. Additional information about the number of molecules contributing to the fluorescence emission can be obtained by use of photo-antibunching. Yet the versatile methods of SMFS need further development in view of acceleration and automation of data acquisition and analysis to meet the demands of their application in systems biology.

Cholesterol content is an important factor for membrane dynamics of living cells. With well defined protocols of depletion and enrichment the impact of cholesterol on membrane dynamics was examined by fluorescence microscopy. In addition, the intracellular cholesterol content was determined with biochemical methods. Changes of cholesterol amounts in cell membranes have previously been related to specific disease and may have some influence on the uptake of pharmaceutical agents. A combination of conventional and total internal reflection fluorescence microscopy was applied to the fluorescence marker laurdan, a polarity-sensitive probe, whose electronic excitation energy is different in polar and non-polar environment. Once incorporated into cell membranes, the fluorescence of laurdan shows a spectral shift towards longer wavelength when its molecules get into contact with adjacent water molecules, e.g. when a phase transition from the tightly packed gel phase to the liquid crystalline phase of membrane lipids occurs. The generalized polarization (GP, characterizing this spectral shift) as well as the fluorescence lifetime (τ) of laurdan revealed to be appropriate measures for membrane stiffness and fluidity. GP generally decreased with increasing temperature and was always higher for the plasma membrane than for intracellular membranes. Enrichment of cholesterol caused a pronounced increase, whereas depletion of cholesterol caused a decrease of GP. In addition, pronounced changes of the fluorescence lifetime pattern occurred in the subnanosecond range. GP, and τ were determined as integral values of single cells or small cell collectives and were also displayed as microscopic images.

We report a study for the development of tomographic imaging technique of fluorescence in biological tissue for assays of biological function. Ultrasonic modulation of light based on the acousto-optic effect (so called ultrasound 'tagging') is applied for imaging of fluorescence distribution in the light-scattering media. Sound-field characteristics that affect the light by modulating its amplitude through variation of the refractive index in the medium were determined. With using focused ultrasound, selectively modulated fluorescence on a depth-axis of the medium can be detected. Ultrasound tagging technique applied measuring the optical absorption in light scattering media is well known, and it is principally based on the modulation of speckle pattern. On the contrary, in the case of fluorescence, displacement of scattering particles and variation of the refractive index that is induced by density distribution in a sound field might produce the intensity modulation of scattered light. We have experimentally shown that ultrasound tagging technique is also available for fluorescence measurement. In this paper, we demonstrate the result of tomographic images of fluorescence in dense scattering media using porcine muscle as a biological tissue, and bovine adipose. Tissue samples had the dimension of 40 x 40 mm in section and fluorophore which had the 3mm size was embedded in the center of the tissue. The localized image of the fluorophore was determined with the spatial resolution of focus size of the ultrasound, suggesting the applicability of this technique for visualization of fluorescent probes in deep portion of living body.

Recent advances in tissue engineering (TE) aim to grow 3D volumes of tissue in bioreactor conditions. This has proved to be a difficult task thus far, notably due to the lack of non-invasive diagnostic tools to monitor the growth of a tissue and ensure its appropriate development. To fulfill part of this need, we currently develop a non-invasive imaging technique based on fluorescence diffuse optical tomography (FDOT) to image in 3D, via fluorescent tracers, processes relevant to tissue growth in a bioreactor. More particularly, here we are interested in imaging the formation of micro-blood vessels in tissue cultures grown on biodegradable scaffolds. Blood vessels are thought to play a fundamental role in tissue growth. Since a bioreactor possesses a known geometry (by design), we propose an FDOT configuration that uses fiber optics brought in contact with the boundary of the bioreactor to collect tomographic optical data. We describe an optical fibers-based set-up and experimental measurements that demonstrate the possibility of localizing a fluorophore-filled 500&mgr;m capillary immersed in a scattering medium contained in a cylindrically-shaped glass tube. These conditions are representative of experiments to be carried on real tissue cultures. In our particular implementation, time-resolved scattering- fluorescence measurements are made via time-correlated single photon counting. Numerical constant fraction discrimination applied to our time-resolved data allows to extract primary localization information.

Membrane dynamics has a large impact on cellular uptake and release of various metabolites or pharmaceutical agents. For a deeper understanding of the cellular processes involved, we used U373-MG human glioblastoma cells as a model system. As conventional microscopy does not permit to investigate individual layers in living cells, we used structured illumination techniques and total internal reflection fluorescence microscopy (TIRFM) to analyse the plasma membrane and intracellular membranes of living cells selectively. Optical image sections provide a high resolution and the possibility of 3D reconstruction. Membranes of living cells were characterized by the membrane marker 6-dodecanoyl-2-dimethylamino naphthalene (laurdan). Due to its spectral and kinetic properties this fluorescence marker appears appropriate for measuring membrane stiffness and fluidity. After excitation with linearly polarized laser pulses, membrane fluidity of human glioblastoma cells was determined by measurements of steady-state and time-resolved fluorescence anisotropy r(t), since with increasing viscosity of the environment, the rotation of an excited molecule is impeded. The corresponding time constant &tgr;_r of molecular relaxation decreased with temperature and increased with the amount of cholesterol. In addition, fluorescence anisotropy r(t) values of the plasma membrane were larger than the values of intracellular membranes for all temperatures in the range of 16°C≤T≤41°C.

Fundamentals and advances of tissue optical properties control as a novel modality for the improvement of laser biomedical spectroscopy and imaging is discussed. As a major technology the optical immersion method at usage of exogenous optical clearing agents (OCAs) is described.

The goal was to develop a light-microscope platform concept, which allows characterization of live cells in microtiter plates or in live-cell slide chambers with a speed, sensitivity and versatility unattainable so far. The goal was achieved by combining several novel technological concepts: a model-based digital control for a voice coil focus drive; scanner technology to follow a continuously moving sample during image acquisition, thus avoiding the usual stop-and-go; fast sectioning capabilities by using slit-scan confocal concepts; motorized dual emission image registration; and integrated environmental control.

In connection with microscopy, digital holography provides contact-less, marker-free, quantitative phase-contrast imaging. Particularly, the feature of (subsequent) numerical auto focus adjustment enables applications in the field of life cell analysis. Here, prospects for long term time-lapse investigations in toxicology and cancer research as well as for monitoring of fast dynamic processes like shape variations are opened up. The evaluation of the obtained quantitative phase contrast provides data for thickness monitoring as well as for the observation of cell swelling kinetics due to osmotic stimulation. Results from investigations on living cells demonstrate digital holographic microscopy application fields for quantitative life cell analytics.

Digital-holographic metrology enables quantitative phase contrast microscopy of reflective and (partially) transparent samples. In this way, new application fields are opened up for nondestructive investigations of technical samples as well as for marker-free and time-resolved analysis of cell biological processes. Studies on long-term biological processes require permanent focus position readjustment to maintain an optimum image quality. Digital holographic microscopy permits subsequent numerical focusing by variation of the propagation distance. Here, the determination of the optimal propagation distance for a sharply focused image is of particular importance. At the Laboratory of Biophysics image definition quantification algorithms were adapted to the requirements of digital holographic microscopy. In order to obtain robust and reliable algorithms, the object-dependent optical absorption properties were taken into consideration. Automatic focus tracking is demonstrated on investigations with digital holographic microscopy on both technical amplitude objects and cytological pure phase objects.

Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram, quantitative phase image of living cell with interferometric accuracy. Specifically, the optical phase shift induced by the specimen on the transmitted wave front can be regarded as a powerful endogenous contrast agent, depending on both the thickness and the refractive index of the sample. We discuss some approaches allowing to directly obtain separate measurements of the thickness and the refractive index (RI) of a given living cell from the phase signal.

The actin cytoskeleton mediates a variety of crucial cellular functions as migration, intracellular transport, exocytosis, endocytosis and force generation. The highly dynamic actin fibers are therefore targets for several drugs and toxins. However the study of actin interfering processes by standard microscopy techniques fails in the detailed resolution of dynamic spatial alterations required for a deeper understanding of toxic effects. Here we applied digital holographic microscopy in the online functional analysis of the actin cytoskeleton disrupting marine toxin Latrunculin B. SEM and fluorescence microscopy showed rapid Latrunculin B induced alterations in cell morphology and actin fiber degradation in pancreas tumor cells. The dynamic digital holographic in vivo analysis of the drug dependent cellular processes demonstrated differences in the actin cytoskeleton stability of highly differentiated and dedifferentiated pancreas tumor cell lines. The spatial resolution of the morphological alterations revealed unequal changes in cell morphology. While cells with a low metastatic potential showed Latrunculin B induced cell collapse within 4 h the metastatic tumor cells were increased in cell volume indicating Latrunculin B effects also on cell water content. These data demonstrate that marker free, non-destructive online analysis of cellular morphology and dynamic spatial processes in living cells by digital holography offers new insights in actin dependent cellular mechanisms. Digital holographic microscopy was shown to be a versatile tool in the screening of toxic drug effects and cancer cell biology.

Most biological processes are governed by assemblies of several dynamically interacting molecules. We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. By comparing energy transfer of the different subpopulations, we can disentangle the reasons that lead to the occurrence of three-way junctions lacking one chromophore. A merit of the method is the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously.

An ultrafast video microscope (UVM), the frame rate of which reaches one million per second has been developed. Our UVM system provides pictures with high-contrast and high-resolution for differential interference contrast (DIC), phase contrast, or dark field imaging. It allows us to observe fast events that occur in live cells when irradiated by ultrashort laser pulses. Femtosecond laser pulses can be used to manipulate, stimulate, and destroy specific cells and organelles under the microscope. The irradiation of such an intense laser immediately results in some physical events, such as microbubble generation, plasma formation, and photoporation. We investigate biophysical mechanisms underlying the ultrafast processes. Our data will contribute to development of new bio-imaging modalities, which implement laser cell transfection. We also present a new method to observe side views of live cells on a substrate. We used a polymer material CYTOP as the substrate for HeLa cells. CYTOP has a refractive index of 1.34, which is close to 1.33 of water. We investigate generation of microbubbles beneath the plasma membranes with a time resolution of one microsecond for the purpose of improving the efficiency of photoporation.

Cardiac failure is still one of the mayor reasons for death in the Western population but the pathophysiology of the molecular processes in the heart is far from being completely understood. Therefore further basic research is necessary. With recent developments of optical technologies novel tools to investigate cardiac physiology and pathophysiology became available. They comprise non-linear imaging techniques such as second harmonic generation imaging and fast two-photon excitation imaging of cardiac tissue. In addition, high-speed multi-beam two-photon imaging as well as ultra-high speed single beam single photon 2D-confocal imaging offer novel approaches to study cellular and subcellular signalling events in cardiac tissue and/or single cardiac myocytes. Here we introduce and discuss these new technologies and their practical application to study cardiac physiology and pathophysiology.

The application of gold nanoparticles as a contrast agent in optical bioimaging is well appreciated, but limited to a narrow excitation range due to its rather invariable optical resonance typically at 520 nm. Compared to gold nanoparticles, the optical response of gold nanoshells can be tuned to match the higher excitation wavelength of many promising clinical reflectance-based imaging modalities such as the optical coherence tomography (OCT). In this study, we demonstrate the tunability of gold nanoshells to improve the optical contrast of backscattering signal under confocal reflectance microscopy and OCT. The gold nanoshells were synthesized and conjugated to antibodies for in vitro demonstration of their selective optical contrast in cancer cells over normal cells under the confocal reflectance microscopy. The OCT signals from these gold nanoshells were compared to that from bare silica cores and intrinsic tissue scattering using 1% Intralipid. We have shown that gold nanoshells are able to elicit an optical contrast to discriminate between cancerous and normal cells under the confocal reflectance microscopy based on differences in molecular markers expression. Compared to bare silica core, the presence of the gold shell is able to effect a higher backscattered OCT signal with an apparent contrast over 1% Intralipid. This contrast can be made to be dependent on the molecular marker expression with antibody specificity.

Surface-enhanced Raman scattering (SERS) has received a great deal interest as an analytical tool due to its potential for obtaining Raman signals from single molecules. Many methods for preparing SERS-active substrate have been reported. These range from nano-particle based methods, which lack reproducibility, to highly reproducible nano-arrays requiring time consuming and costly preparation. We show that highly reproducible SERS can be achieved by applying a metallic coating to the brightly coloured regions of the graphium weiskei butterfly wing. Electron microscopy reveals the wing exhibit nanostructures with comparable dimensions to the roughness scale of SERS substrates. SERS measurements performed on wings coated with 60 nm of silver display enhancement factors of approximately 10⁷ with no apparent background contribution from the wing. To demonstrate effectiveness and reproducibility the substrate is coated with a monoclonal antibody.

In this paper, we briefly review earlier approaches for optical stretching of red blood cells (RBCs) and introduce a novel approach based on oscillatory optical tweezers. Preliminary experimental data for optical trap-and-stretch of RBCs by two approaches, namely the counter-propagating dual-beam trap-and-stretch and the oscillatory optical tweezers, are presented and discussed.

The red blood cell (RBC) viscoelastic membrane contains proteins and glycolproteins embedded in, or attached, to a fluid lipid bilayer and are negatively charged, which creates a repulsive electric (zeta) potential between the cells and prevents their aggregation in the blood stream. The basis of the immunohematologic tests is the interaction between antigens and antibodies that causes hemagglutination. The identification of antibodies and antigens is of fundamental importance for the transfusional routine. This agglutination is induced by decreasing the zeta-potential through the introduction of artificial potential substances. This report proposes the use of the optical tweezers to measure the membrane viscosity, the cell adhesion, the zeta-potential and the size of the double layer of charges (CLC) formed around the cell in an electrolytic solution. The adhesion was quantified by slowly displacing two RBCs apart until the disagglutination. The CLC was measured using the force on the bead attached to a single RBC in response to an applied voltage. The zeta-potential was obtained by measuring the terminal velocity after releasing the RBC from the optical trap at the last applied voltage. For the membrane viscosity experiment, we trapped a bead attached to RBCs and measured the force to slide one RBC over the other as a function of the relative velocity. After we tested the methodology, we performed measurements using antibody and potential substances. We observed that this experiment can provide information about cell agglutination that helps to improve the tests usually performed in blood banks. We also believe that this methodology can be applied for measurements of zeta-potentials in other kind of samples.

We have developed an automated microinjection system that can handle more than 500 cells an hour. Microinjection injects foreign agents directly into cells using a micro-capillary. It can randomly introduce agents such as DNA, proteins and drugs into various types of cells. However, conventional methods require a skilled operator and suffer from low throughput. The new automated microinjection techniques we have developed consist of a Petri dish height measuring method and a capillary apex position measuring method. The dish surface height is measured by analyzing the images of cells that adhere to the dish surface. The contrast between the cell images is minimized when the focus plane of an object lens coincides with the dish surface. We have developed an optimized focus searching method with a height accuracy of ±0.2 um. The capillary apex position detection method consists of three steps: rough, middle, and precise. These steps are employed sequentially to cover capillary displacements of up to ±2 mm, and to ultimately accomplish an alignment accuracy of less than one micron. Experimental results using this system we developed show that it can introduce fluorescent material (Alexa488) into adherent cells, HEK293, with a success rate of 88.5%.

Objective: To automatically segment cell nuclei in histology images of bladder and skin tissue for karyometric analysis. Materials/Methods: The four main steps in the program were as follows: 1) median filtering and thresholding, 2) segmentation, 3) categorizing, and 4) cusp correction. This robust segmentation technique used properties of the image histogram to optimally select a threshold and create closed four-way chain code nuclei segmentations. Each cell nucleus segmentation was treated as an individual object with properties of segmentation quality. A segmentation was placed in one of the following three categories based on its properties: throw away, salvageable, or good. Erosion/dilation and rethresholding were performed on salvageable nuclei to correct cusps. Results: Ten bladder histology images were segmented both by hand and using this automatic segmention algorithm. The automatic segmentation resulted in a sensitivity of 76.4%. The average difference between hand and automatic segmentations over 42 nuclei, calculated for each of the 95 features used in karyometric analysis, ranged between 0 and 48.3%, with an average of 2.8%. The same procedure was performed on 10 skin histology images with a sensitivity of 83.0%. Average differences over 44 nuclei ranged between 0 and 200%, with an average of 10.0%. Conclusion: The close agreement in karyometric features with hand segmentation shows that automated segmentation can be used for analysis of bladder and skin histology images. Average differences between hand and automatic segmentations were smaller in bladder histology images because these images contained less contrast, and therefore the range of the karyometric feature values was smaller.

We investigated different kinds of human cutaneous ex-vivo skin samples by combined two photon intrinsic fluorescence (TPE), second harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), spectral lifetime imaging (SLIM), and multispectral two photon emission detection (MTPE). Morphological and spectroscopic differences were found between healthy and pathological skin samples, including tumors. In particular, we examined tissue samples from normal and pathological scar tissue (keloid), and skin tumors, including basal cell carcinoma (BCC) and malignant melanoma (MM). By using combined TPE-SHG microscopy we investigated morphological features of different skin regions, as BCC, tumor stroma, healthy dermis, fibroblastic proliferation, and keloids. A score, based on the SHG to autofluorescence aging index of dermis (SAAID), was assigned to characterize each region. We found that both BCC and surrounding dermis have a negative SAAID value, tumor stroma has a positive SAAID value, whereas fibroblastic proliferation and keloids have a SAAID value close to the unit. Further comparative analysis of healthy skin and neoplastic samples was performed using FLIM, SLIM, and MTPE. In particular, BCC showed a blue-shifted fluorescence emission, a higher fluorescence response at 800 nm excitation wavelength and a slightly longer mean fluorescence lifetime. MM showed an emission spectrum similar to the corresponding healthy skin emission spectrum, and a mean fluorescence lifetime distribution shifted towards shorter values. Finally, the use of aminolevulinic acid as a contrast agent has been demonstrated to increase the constrast in BCC border detection. The results obtained represent further support for in-vivo non-invasive imaging of diseased skin.

Recently, two-photon microscopy has been used for high spatial resolution imaging of the intact neocortex in living rodents. In this work we used near-IR femtosecond laser pulses for a combination of two-photon microscopy and microdissection on fluorescently-labeled neuronal structures in living mice. Three-dimensional reconstructions of dendrites expressing the green fluorescence protein were made in the cortex of GFP-M and YFP-H transgenic mice. Afterwards, single dendrites were laser-dissected irradiating the structure with a high femtosecond laser energy dose. We report that laser dissection can be performed with micrometric precision and without any visible collateral damage of the surrounding neuronal structures. After laser irradiation, one part of the severed dendrite underwent degeneration and disappeared within 5 hours. Using a chronically implanted glass window, we performed long-term imaging in the area of the dissected dendrite. Images of the long-term morphological changes in the neuronal network after dendritic lesioning will be provided. Laser microdissection of selected structures of the neuronal branching in vivo represents a promising tool for neurobiological research.

A miniaturized photoplethysmographic sensor system which utilizes the principle of pulse oximetry is presented. The sensor is designed to be implantable and will permit continuous monitoring of important human vital parameters such as arterial blood oxygen saturation as well as pulse rate and shape over a long-term period in vivo. The system employs light emitting diodes and a photo transistor embedded in a transparent elastic cu. which is directly wrapped around an arterial vessel. This paper highlights the specific challenges in design, instrumentation, and electronics associated with that sensor location. In vitro measurements were performed using an artificial circulation system which allows for regulation of the oxygen saturation and pulsatile pumping of whole blood through a section of a domestic pig's arterial vessel. We discuss our experimental results compared to reference CO-oximeter measurements and determine the empirical calibration curve. These results demonstrate the capabilities of the pulse oximeter implant for measurement of a wide range of oxygen saturation levels and pave the way for a continuous and mobile monitoring of high-risk cardiovascular patients.

The purpose of this study is to investigate the kinetics of precorneal tear film stabilization process after eye blink and the process of creating the break-up of the tear film layer. The tear film of patients were examined in vivo by used the lateral shearing interferometer. The information about the distribution and stability of the tear film over the cornea is carried by the wave front reflected from the surface of tears and coded in interference fringes. Smooth and regular fringes indicate the smooth surface of tears over the cornea. Immediately after eye blink the interference fringes are observed on background of bright and dark areas. The contrast of this structure fades with time slowly and after 1-3 sec the background of interference fringes becomes uniform. The vertical orientation and instability of this structure suggests connection with eyelid movement and the spread of tears. If the eye is kept open for a long time, bright lines appear in the background of fringes after a dozen seconds. The slowly appearing structure might signify the tear film break-up. In case of eyes after a LASIK surgery the shape of the background structure has different nature and might be stable in time suggesting the stability of the corneal surface irregularities.

We have investigated the effect of application of gold nanoparticles with a diameter of 50 nm and nanoshells with a 150 nm silica core size and 25 nm thick gold shell on optical properties of skin. We have analyzed the possibility of using these particles as a contrasting agent for optical coherence tomography (OCT). As the first step in the study, effects of gold nanoparticles after one application to skin were studied using OCT. Then we evaluated the effects of multiple applications of 50 nm gold nanoparticles to skin in 30-minute intervals. Biopsy of relevant skin areas was performed under local anaesthesia and samples for light and electron microscopy were prepared. Identification of skin layers on OCT images was made by comparing with histology. Application of gold-silica nanoshells caused increase in intensity of useful signal, brightness of the superficial part of the dermis and contrast between the superficial and deep parts of the dermis 30 minutes after application on skin. After 24 hours the changes in OCT images became more pronounced as the brightness of the superficial part of the dermis and the contrast between the superficial and deep parts of the dermis further increased. In addition, the border between the superficial and deep parts of the dermis became more distinct, continuous and well discernible, permitting to accurately differentiate these layers. Besides that, the application of nanoshells caused contrasting of hair follicles and glands. In order to give interpretation to the obtained experimental OCT-images of skin and understand the mechanisms of contrasting a set of Monte Carlo calculations was performed in order to simulate the images of skin before and after application of the nanoparticles for skin model close to that in the experiment. The results of the simulation exhibit good qualitative agreement with the experimental images and prove that the contrasting originates from the nanoparticles added while contrasting of hair bulb originates from the absence of nanoparticles in it with their presence in surrounding area.

The aim of the study is to evaluate the ability of a visible light based spectroscopic sensor system to monitor caries activity in saliva. In this study an optical sensor is utilized to monitor the bacterial-mediated acidogenic profile of stimulated saliva using a photosensitive pH indicator. Microbiological assessment of the saliva samples were carried out using the conventional culture methods. In addition, the shifts in the pH of saliva-sucrose samples were recorded using a pH meter. The absorption spectra obtained from the optical sensor showed peak maxima at 595nm, which decreased as a function of time. The microbiological assessment showed increase in the bacterial count as a function of time. A strong positive correlation was also observed between the rates of decrease in the absorption intensity measured using the optical sensor and the decrease in pH measured using the pH meter. This study highlights the potential advantages of using the optical sensor as a sensitive and rapid chairside system for monitoring caries activity by quantification of the acidogenic profile of saliva.

A photosensitizer formulation and strategy was developed based on the photophysical, photochemical and photobiological characteristics of methylene blue (MB) for the disinfection of root canal using light activated therapy. Disinfection of matured E. faecalis biofilms on root canal dentine was tried with the newly developed 'Advanced Non- Invasive Light Activated Disinfection' (ANILAD), conventional photodynamic therapy, and conventional root canal therapy alone or in combination with ANILAD. The results showed that, although complete disinfection of nonmatured biofilm is possible by ANILAD alone, a combination of conventional root canal treatment (RCT) with ANILAD could achieve significantly higher bacterial killing (6log₁₀-7log₁₀ bacterial reduction) compared to any other tested treatment in matured biofilm (p<0.001).

An upcoming problem in Europe is the protection of water resources and control of water quality. Coastal areas, rivers, ground water, wetlands, and especially drinking water require permanent monitoring to avoid pollution by small organic molecules or especially endocrine disrupting compounds. Biosensors have demonstrated the proof-of-principle of immunochemistry for these applications. It turns out that especially optical methods based on fluorescence detection can be successfully used for the development of fast, sensitive, cost-effective, and easy-to-use analytical systems meeting the requirements given by European Community Directives and national legislation. Results obtained with the RIANA and AWACSS systems are discussed here.

The identification of bacteria is necessary as fast as possible e.g. to provide an appropriate therapy for patients. Here the cultivation time should be kept to a minimum. Beside microbiological identification methods Raman spectroscopy is a valuable tool for bacteria identification. UV-resonance Raman spectroscopy enables selective monitoring of the cellular DNA/RNA content and allows for a genotaxonomic classification of the bacteria. Since UV excitation may lead to sample destruction the measurements are performed on rotated bacterial films. For a faster identification avoiding the cultivation step single bacteria analysis is necessary. Using micro-Raman spectroscopy a spatial resolution in the size range of the bacteria can be achieved. With this Raman excitation the chemical components of the whole cell are measured which leads to a phenotypic classification. For localization of bacteria inside complex matrices fluorescence labeling is achieved.

In the recent years, it has been shown that terahertz (or T-ray) spectroscopy is a versatile tool for biosensing and safety applications. This is due to the fact that the THz-spectra of many biomolecules show very characteristic, distinct spectroscopic features. Furthermore, most non-metallic packaging materials are nearly transparent in this frequency range (0.1 - 6 THz, 3 cm^-1 - 200 cm^-1), so that it is possible to non-invasively identify even sealed substances like pharmaceuticals, illicit drugs or explosives by their spectroscopic signatures. This opens a significant potential for a wide range of applications from quality control of pharmaceutical substances via safety applications through to biomedical applications. The individual spectroscopic features below approximately 5 THz that spurred the increased world wide interest in T-ray spectroscopy are mainly due to intermolecular rather than intramolecular vibrations in the polycrystalline samples. The spectra of more complex biomolecules, like proteins and nucleotides, typically show less or even no sharp features, due to the lack of long- range intermolecular order. Furthermore, due to the typically significantly smaller sample amount, the signal to noise ratio is strongly increased. Water shows a strong absorption in this frequency range, which all together makes real biomedical applications of T-ray spectroscopy rather difficult. Yet, by combining a careful sample preparation, novel experimental techniques and an advanced signal processing of the experimental data we can still clearly distinguish between even complex biomolecules and therefore demonstrate the potential the technique holds for biomedical applications.

In this research poly (d,l-lactide-coglycolide acid) (PLGA) as polymeric nanospheres, poly(vinyl alcohol) (PVA) with 87-89% hydrolysis degree as surfactant and distilled water as suspending medium were used. The encapsulated drug was Bethametasone. The nanospheres were prepared by an emulsion-solvent evaporation method. The nanospheres characterized by photon correlation spectroscopy (PCS) and scanning electron microscopy (SEM). The amount of drug release was determined by HPLC. In emulsion-solvent evaporation technique, time of ultrasound exposure, surfactant content in the formulation and evaporation rate of organic solvents were considered as formulation variables.

We have developed a non-invasive thermal image analyzer for deceptive detection (TAD²) where the far-infrared data around the periorbital and nostril areas are simultaneously analyzed. Measured change in maximum skin temperature around two periorbital regions is converted to a relative blood flow velocity. A respiration pattern is also simultaneously determined via the ratio of the measured maximum and minimum temperatures in the nostril area. In addition, our TAD² employs a simple normalized cross correlation scheme to independently track locations of the two periorbital and nostril areas. Our field case study from 7 subjects in two real crime scenes and with the use of our baseline classification criteria shows two-fold improvement in classification rate compared to our analysis using either the periorbital or nostril area alone.

Laser ablation for the formation of apodized patterns on intraocular lenses, as an alternative of the conventional injection molding, has been proved to be a very promising new technique. For the precise lenses ablation, the use of suitable laser wavelength and pulse duration, resulting in a small optical penetration depth in the lens and in confinement of the energy deposition in a small volume, as well as the reduced thermal damage to the surrounding tissue, is essential. Mid-infrared laser wavelengths, at which the organic biological simulators absorption coefficient is large, meet well the above conditions. Towards the complete understanding of the intraocular lens ablation procedure and therefore the choice of the optimum laser beam characteristics for the most accurate, efficient and safe surgical application, the comparative study of various mid-infrared laser sources is of great interest. In this work we investigate the potential of the development of three different mid-infrared laser sources, namely the Yb:YAG, the Cr:Tm:Ho:YAG and the Er:Tm:Ho:YLF laser, operating at 1029 nm, 2060 nm and 2080 nm respectively and their ability in forming patterns on biomaterials. Pumping was achieved with conventional Xe flash lamps in a double elliptical pump chamber. A properly designed Pulse-Forming- Network capable of delivering energy up to 800 J, in variable lamp illumination durations is used. Several hundreds of mJoules were achieved from the Yb:YAG laser oscillator and several Joules from the Ho:YAG and Ho:YLF laser oscillators. Free running and Q-switched laser operation studies and preliminary experiments on laser and biomaterials (biopolymers and animal tissues) interactions will be reported.

The present paper recorded spontaneous ultra weak photon emission of five subjects at palm and dorsal sides of both left and right hands in a 24 h period. Data demonstrate that intensity as well as left-right symmetry varies diurnally. Emission intensity is low during the day, rises during the evening and is high during the night. Time patterns for left and right hand are different. The left-right symmetry shifts in the evening. Data are explained within the concept of a regulatory role of the photon field in human physiology. However, other explanations cannot be excluded.

The UVA induced Delayed Luminescence (DL), has been measured in vivo in the forearm skin of some healthy volunteers of different sex and age during several periods of the year. An innovative instrument able to detect, in single photon counting mode, the spectrum and the time trend of the DL emission has been used. The measured differences in the time trends of the spectral components may be related to the sex and the age. The potential development of a new analysis technique based on this phenomenon is discussed.

In recent years Fluorescence Resonance Energy Transfer (FRET) has been widely used to determine distances, observe distance dynamics, and monitor molecular binding at the single-molecule level. A basic constraint of single-molecule FRET studies is the limited distance resolution owing to low photon statistics. We demonstrate that by confining molecules in nanopipettes (50-100 nm diameter) smFRET can be measured with improved photon statistics reducing the width of FRET proximity ratio distributions (PRD). This increase in distance resolution makes it possible to reveal subpopulations and dynamics in biomolecular complexes. Our data indicate that the width of PRD is not only determined by photon statistics (shot noise) and distance distributions between the chromophores but that photoinduced dark states of the acceptor also contribute to the PRD width. Furthermore, acceptor dark states such as triplet states influence the accuracy of determined mean FRET values. In this context, we present a strategy for the correction of the shift of the mean PR that is related to triplet induced blinking of the acceptor using reference FCS measurements.

Experiments based on Förster resonance energy transfer (FRET) are widely used to obtain information on conformational dynamics of biomolecular systems. To reliably measure FRET, accurate knowledge of photophysical properties of the used fluorophores is indispensable. In high FRET constructs donor (D) and acceptor (A) fluorophores can approach each other close enough that electronic interactions might occur. When separated by distances on the order of van der Waals radii, photophysical properties can be changed reversibly, opening new non-radiative relaxation pathways, or irreversibly, chemically altering the fluorophores. Even transient contacts can thus compromise accurate FRET measurements. To study FRET and competing D-A contact-induced processes we labeled the amino acid cystein (Cys) with two fluorophores. A donor (D; TMR or Cy3B) was attached to the thiol group and an acceptor (A; Atto647N) to the amino group of Cys. Absorption spectroscopy, steady-state fluorescence spectroscopy, and time-correlated single-photon counting (TCSPC) were used to characterize the different A-Cys-D complexes at the ensemble level. In addition, we performed single-molecule FRET experiments using alternating-laser excitation to study the heterogeneity of the FRET-systems. We identified competing quenching processes severely changing D and A quantum yields upon fluorophore contact. The results are applicable for quantitative analysis of FRET in dynamic molecular systems that allow transient contact between D and A fluorophores.

In this work we propose and demonstrate that time-resolved optical spectroscopy in the spectral region 700-1040 nm, on a picosecond time scale, is a valuable technique for non-invasive wood characterization. Two different wood types have been considered, fir and oak chestnut as an example of softwood and hardwood, respectively. Wood samples have been measured in three different conditions: dry, wet and degraded by an ozone treatment. The two types of wood show different absorption and scattering spectra according to the treatment, revealing both chemical and structural changes.

Laser-induced fluorescence spectroscopy (LIF) and Raman spectrum of serum for diagnosis of colon cancer and rectum cancer were investigated in this paper. The aim of this study was that using Raman spectrum and LIF analysis the serum of colon cancer and rectum cancer for found the difference compared to normal, the difference was found. For example: the intensity and red shift both different In this paper we investigated 82 colon cancers, 69 rectum cancers and obtained 80.7%, 82.5% accuracy to rectum cancer and colon cancer separately compared to clinical diagnostic. It is exploring that use Raman spectrum and LIF to detection of cancer.

In this contribution we describe the realization of MUSES, a novel research equipment able to detect and identify photons emitted, after laser irradiation, from biological samples (like micro-organisms and human cells) for fast ultraweak luminescence analysis. MUSES has been entirely designed and realised at LNS-Southern National Laboratory of the Italian INFN-Nuclear Physics National Institute. The excellent performances in terms of timing, wavelength and angular identification make this multi-detector a unique device in biophotonics research field.

The detection of single bacteria should be improved by lowering the acquisition time via the application of SERS (surface enhanced Raman spectroscopy). Nano structured colloids or surfaces consisting of gold or silver can be used as SERS active substrates. However, for biological applications mostly gold is used as SERS active substrate since silver is toxic for bacterial cells. Furthermore, the application of gold as a SERS-active substrate allows the usage of Raman excitation wavelengths in the red part of the electromagnetic spectrum. For the SERS investigations on bacteria different colloids (purchased and self prepared, preaggregated and non-aggregated) are chosen as SERS active substrates. The application of different gold colloids under gently mixing conditions to prevent the bacterial damage allowed the recording of reproducible SERS spectra of bacteria. The SERS spectra of B. pumilus are dominated by contributions of ingredients of the outer cell wall, e.g. the peptidoglycan layer. SEM images of the coated bacteria demonstrate the incomplete adsorption most probably due to variations within the binding affinities between different outer cell components and the gold colloids.

Based on their various interesting properties metal nanoparticles show the potential as analytical tool in electronic, optical, and catalytical applications. The different properties depending on composition, shape, and size of the single particles were utilized in many different approaches such as optics, magnetics and laser technology¹. We present a way for enzymatic deposition of silver nanoparticles and a bioanalytical application in DNA microarray technology for this method. The technology consists of a microstructured chip with 10&mgr;m broad electrode gaps on the surface and specially designed readout device². In principle we immobilize gold nanoparticle-labelled DNA in a gap between two electrodes. Afterwards a silver deposition on the bound gold nanoparticles generates a conductive layer between the electrodes. The measured drop in the resistance serves as signal for the chip-based electrical detection of DNA³. To further optimize this system the gold nanoparticles as seed are replaced by the enzyme horseradish peroxidase. For a better understanding of the enzymatically silver deposition process the formed silver particles were analyzed by spectroscopic characterization on a single particle level. Further investigations of these particles by AFM and SEM should give a hint to the connection between size/shape and the plasmonic properties at individual particles.

Fluoroquinolones are important antibacterial drugs. They were found to interfere with the gyrase-DNA complex which causes cell death. However, the detailed mode of action on a molecular level is so far not understood. In this contribution Raman spectroscopy is chosen as a non-invasive technique to first characterize the individual involved components: fluoroquinolone drugs, and the biological targets DNA and gyrase; and second to study the influence of the fluoroquinolones on bacteria in in-vivo experiments. The use of UV resonance Raman spectroscopy with excitation at 244 nm allows the investigation of the drugs and the biological targets in aqueous solution at biological low concentrations (a few μM). Raman bands associated with the action of the enzyme gyrase could be identified in in-vitro mixing experiments. In-vivo experiments with bacteria experiencing varying drug concentrations revealed changes in the vibrational bands of the protein and DNA components within the bacterial cell caused by the action of the drug. Due to the complexity of the bacterial spectra advanced multivariate statistics in combination with variable selection methods proved to be useful in the data analysis.

Understanding the cellular mechanisms of energy supply to neurons following physiological activation is still challenging and has strong implications to the interpretation of clinical functional images based on metabolic signals such as Blood Oxygen Level Dependent Magnetic Resonance Imaging or ¹⁸F-Fluorodexoy-Glucose Positron Emission Tomography. Intrinsic Optical Signal Imaging provides with high spatio temporal resolution in vivo imaging in the anaesthetized rat. In that context, intrinsic signals are mainly related to changes in the optical absorption of haemoglobin depending on its oxygenation state. This technique has been validated for imaging of the rat olfactory bulb, providing with maps of the actived olfactory glomeruli, the functional modules involved in the first step of olfactory coding. A complementary approach would be autofluorescence imaging relying on the fluorescence properties of endogenous Flavin Adenine Dinucleotide (FAD) or Nicotinamide Adenine Dinucleotide (NADH) both involved in intracellular metabolic pathways. The purpose of the present study was to investigate the feasibility of in vivo autofluorescence imaging in the rat olfactory bulb. We performed standard Monte Carlo simulations of photons scattering and absorption at the excitation and emission wavelengths of FAD and NADH fluorescence. Characterization of the fluorescence distribution in the glomerulus, effect of hemoglobin absorption at the excitation and absorption wavelengths as well as the effect of the blurring due to photon scattering and the depth of focus of the optical apparatus have been studied. Finally, optimal experimental parameters are proposed to achieve in vivo validation of the technique in the rat olfactory bulb.

The wide-spread use of fluorescent dyes in molecular diagnostics and fluorescence microscopy together with new developments such as single-molecule fluorescence spectroscopy provide researchers from various disciplines with an ever expanding toolbox. Single-molecule fluorescence spectroscopy relies to a large extent on extraordinary bright and photostable organic fluorescent dyes such as rhodamine- or cyanine- derivatives. While in the last decade singlemolecule equipment and methodology have significantly advanced and in some cases reached theoretical limits (e.g. detectors approaching unity quantum yields), instable emission ("blinking") and photobleaching become more and more the bottleneck of further development and spreading of single-molecule fluorescence studies. In recent years, agents and recipes have been developed to increase the photostability of conventional fluorescent dyes. Here, we investigate some of these strategies at the single-molecule level. In particular, we focus on the dye selection criteria for multi-color applications. We investigate fluorescent dyes from the rhodamine, carborhodamine, cyanine, and oxazine family and show that within one dye class the photophysical properties are very similar but that dyes from different classes show strikingly different properties. These findings facilitate dye selection and provide improved chemical environment for demanding fluorescence microscopic applications.

For successful uptake and distribution of drugs from transdermal formulations, it is important to understand the skin barrier function. Innovative advances in modern microscopy have provided valuable tools to study the interaction between the skin and xenobiotics. Two-photon microscopy (TPM) allows non-invasive visualization of fluorescent compounds in the skin. The advantages of TPM over conventional confocal microscopy are better light penetration into highly scattering and absorbing tissue such as human skin, improved detection efficiency, limited out of focus photobleaching and reduced phototoxic effects. We present TPM as an alternative non-invasive in vitro method to study chemical penetration enhancement of fluorescent model drugs. The permeability of sulforhodamine B (SRB) through human epidermis was measured with vertical diffusion cells. The absorption was visualized using TPM after 24 h passive diffusion. We have evaluated variations in physicochemical parameters controlling dermal drug uptake induced by the penetration enhancer oleic acid according to methods previously described by Yu et al. Optical sectioning by TPM was compared with cryosectioning. Oleic acid significantly increased penetration of sulforhodamine. TPM images demonstrate a four-fold increase in the partition coefficient. In addition, a six-fold increase in the concentration gradient was found over stratum corneum. Better light penetration and detection efficiency increase maximum imaging depth in TPM compared to conventional confocal microscopy, however loss of signal due to scattering and absorption is still significant and will affect distribution profiles generated by optical sectioning. A true concentration profile cannot be established without better knowledge about signal losses in the skin.

Due to its extremely low fluorescence quantum yield, in the conventionally (one-photon) excited autofluorescence of skin tissue, melanin fluorescence is masked by several other endogenous and possibly also exogenous fluorophores (e.g. NADH, FAD, Porphyrins). A first step to enhance the melanin contribution had been realized by two-photon fs-pulse excitation in the red/near IR, based on the fact that melanin can be excited by stepwise two-photon absorption, whereas all other fluorophores in this spectral region allow only simultaneous two-photon excitation. Now, the next and decisive step has been realized: Using an extremely sensitive detection system, for the first time twophoton fluorescence of skin tissue excited with pulses in the ns-range could be measured. The motivation for this step was based on the fact that the population density of the fluorescent level resulting from a stepwise excitation has a different dependence of the pulse duration than that from a simultaneous excitation (&Dgr;t² vs. &Dgr;t). Due to this strong discrimination between the fluorophores, practically pure melanin fluorescence can be obtained. Examples for in-vivo, ex-vivo as well as paraffin embedded skin tissue will be shown. The content of information with respect to early diagnosis of skin deseases will be discussed.

Nowadays the artificial neural network (ANN), an effective powerful technique that is able denoting complex input and output relationships, is widely used in different biomedical applications. In present study the applying of ANN for the determination of characteristics of random highly scattering medium (like bio-tissue) is considered. Spatial distribution of the backscattered light calculated by Monte Carlo method is used to train ANN for multiply scattering regimes. The potential opportunities of use of ANN for image reconstruction of an absorbing macro inhomogeneity located in topical layers of random scattering medium are presented. This is especially of high priority because of new diagnostics/treatment developing that is based on the applying gold nano-particles for labeling cancer cells.

Fluorescence based enzyme analysis is commonly done by FRET-probes, natural enzyme substrates flanked by two corresponding fluorophores, showing spectral changes upon distance variations between the fluorophores. However, the use of double labeled substrates displays several limitations such as reduction of sensitivity and high background signal accompanied by high costs for synthesis. Therefore, the development of new probes avoiding these factors is of general interest in enzyme research. A promising approach represents smart probes, i.e. singly labeled quenched enzyme substrates that increase fluorescence intensity upon enzymatic cleavage. Smart probes use the fact that certain rhodamine and oxazine dyes are selectively quenched upon contact formation with guanine or tryptophan residues via photoinduced electron transfer (PET). The rapid response time of the probes enables real-time monitoring of enzyme activity in ensemble as well as in single molecule measurements, which is an important prerequisite for the improved understanding of enzyme mechanisms. We present the design of smart probes for the detection of the two hydrolases, DNaseI and Carboxypeptidase A (CPA) with respect to stability and substrate specificity in ensemble measurements. Furthermore, we investigate the influence of the attached fluorophore on hydrolysis efficiency in case of CPA and demonstrate first applications of smart probes in single enzyme experiments.

Cataract is one of the most frequent reasons of blindness all around the world. Its treatment relies on removing the pathologically altered crystalline lens and replacing it with an artificial intraocular lens (IOL). There exists plenty of types of such implants, which differ in the optical materials and designs (shapes). However one of the important features, which is rather overlooked in the development of the intraocular implants is the chromatic aberration and its influence on the retinal image quality. In this study authors try to estimate the influence of the design and optical material of the implant on the retinal image quality in the polychromatic light, taking into consideration several exemplary types of IOLs which are commercially available. Authors also propose the partially achromatized hybrid IOLs, the longitudinal chromatic aberration (LCA)of which reduces the total LCA of the phakic eye to the level of a healthy eye's LCA. Several image characteristics, as the polychromatic Point Spread Function (PSF) and the Modulation Transfer Function (MTF) and the polychromatic encircled energy are estimated. The results of the simulations show the significance of the partial chromatic aberration correction.

Intestinal tumors exhibit cell surface properties that differ from neighboring healthy epithelia and thus allow tumor cell-specific molecular targeting. Ganglioside G_M1 is such a discriminatory target. Although expressed in the apical membrane of all intestinal epithelial cells it is accessible for particle conjugated ligands on tumor cells only. In order to exploit this phenomenon we want to develop a nanoparticulate optical contrast agent equipped with a peptidic G_M1 binding ligand. For identification of ligand peptides a novel screening platform was devised where potential ganglioside GM1-binding peptides are generated on glass capillary plates using microfluidic non-contact arraying techniques and screened in situ for binding of fluorophor-labeled G_M1. These three-dimensional supports are easy to handle and show better sensitivity than either flat glass or membrane supports because of their large inner surface and low interference with readout systems. A custom fluorescence reader was designed to comply with the specific optical behaviour of peptide arrays synthesized on microcapillary plates. This reader uses a small numerical aperture for excitation and a large numerical aperture for detection in epifluorescence-mode. Background noise from fluorescence and Raman scattering is reduced by time gated photon counting. Peptides showing affinity to ganglioside G_M1 will be conjugated to a nano-particulate carrier bearing a fluorescent dye. The resulting optical contrast agent shall be used for fluorescence endoscopic intestinal tumor screening.

A rotationally invariant algorithm was developed to evaluate the orientation direction and orientation coherence of features in a two-dimensional image. The algorithm was validated on test images and was applied on in vivo confocal microscopy images to extract information on collagen matrix orientation and on skin microrelief images for the calculation of the primary direction of microglyphics.

We exploited ultraprecision milling to fabricate three dimensional microfluidic structures incorporating cascaded hydrodynamic focusing. Stable operation when varying the sample flow over three orders of magnitude was demonstrated. Impedance detection or forward light scatter served to detect polystyrene microspheres and to differentiate red blood cells and blood platelets.

Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

We describe a novel label-free method to analyse protein interactions on microarrays as well as in solution. By this technique the time resolved native protein fluorescence in the UV is probed. The method is based on alterations of the protein upon ligand binding, and, as a consequence, of alterations of the environment of the proteins' aromatic amino acids. These amino acids act as internal probes, and as a result, the fluorescence lifetime of the proteins change due to binding to a ligand partner such as another protein. We were able to demonstrate the feasibility of the method with many compounds, including protein-protein, protein-antibody, protein-nucleic acid and protein-small ligand pairs. Unlike to many other label-free techniques, the sensitivity of the method does not depend on the size of the counterbinding ligand and therefore is particularly suitable for drug monitoring, when small molecules are involved.

In this work, the analysis of human and rat red blood cells (RBC) deformability, internal viscosity and yield stress of RBC in norm and ischemia was performed by means of laser diffractometry - a modern technique allowing for measuring the flexibility of RBC, which determines the blood flow parameters in vessels. Ischemic diseases of people and animals are accompanied with deterioration of microrheologic properties of their blood, in particular, with impairing the RBC deformability. Human RBCs were obtained from the blood of healthy individuals and from patients suffering from ischemic diseases. The RBC deformability indices from both groups of individuals were measured. Rat RBCs were obtained from a control group of animals and from a group with experimentally induced ischemia (EII). This animal model is frequently used for studying the response of an organism to ischemia. The effect of semax, a medication that is frequently used for therapeutic treatments of human brain diseases in clinical practice, on RBC deformability was studied with its application in vitro and in vivo. It is shown that in human ischemic patients, the deformability index of RBC was lower than that from healthy individuals. Both in vivo and in vitro applied semax positively influences the impaired deformability properties of RBCs of ischemic rats.

Chemical approaches allow for the synthesis of highly defined metal heterostructures, such as core-shell nanoparticles. As the material of metal nanoparticles determines the plasmon resonance-induced absorption band, the control of particle composition results in control of the absorption maximum position. Metal deposition on gold or silver nanoparticles was used to prepare core-shell particles with modified optical properties with respect to monometal nanoparticles. UV-vis spectroscopy on solution-grown and immobilized particles was conducted as ensemble measurements, complemented by single particle spectroscopy of selected structures. Increasing layers of a second metal, connected to a dominant contribution of the shell material to the extinction spectrum, lead to a shift in the absorption band. The extent of shell growth could be controlled by reaction time or the concentration of either the metal salt or the reducing agent. Additional to the optical characterization, the utilization of AFM, SEM and TEM yielded important information about the ultrastructure of the nanoparticle complexes.

Novel optical labels based on nanophosphor materials like LaPO₄:Ce,Tb and CePO₄:Tb-LaPO₄ core-shell nanophosphors were presented. Core particles could be synthesised smaller than 10nm and stabilized in aqueous media. Polymer coatings of individual nanoparticles increased long term stability and introduced functional groups of interest for bioconjugation chemistry. A new strategy for conjugation of bioligands via His-tags was given. In feasibility studies for in-vitro diagnostic applications these dnanophosphors featured their advantage over organic dyes. As an example the detection of hybridization and thermal induced denaturation events of DNA strands read out by FRET processes was presented.

We present single-molecule fluorescence studies of σ⁵⁴-dependent gene-transcription complexes using singlemolecule fluorescence resonance energy transfer (smFRET) and alternating-laser excitation (ALEX) spectroscopy. The ability to study one biomolecule at the time allowed us to resolve and analyze sample heterogeneities and extract structural information on subpopulations and transient intermediates of transcription; such information is hidden in bulk experiments. Using site-specifically labeled σ⁵⁴ derivatives and site-specifically labeled promoter-DNA fragments, we demonstrate that we can observe single diffusing σ⁵⁴-DNA and transcription-initiation RNA polymerase-σ⁵⁴- DNA complexes, and that we can measure distances within such complexes; the identity of the complexes has been confirmed using electrophoretic-mobility-shift assays. Our studies pave the way for understanding the mechanism of abortive initiation and promoter escape in σ⁵⁴-dependent transcription.