Fluorescence in situ hybridization (FISH) offers an appropriate technique to specifically label any given chromatin region by multi spectrally labeled, specific DNA probes. Using confocal laser scanning microscopy, quantitative measurements on the spatial distribution of labeling sites can be performed in 3D conserved cell nuclei. Recently, 'Spectral Precision Distance Microscopy' has been developed that allows 3D distance measurements between point-like fluorescence objects of different spectral signatures far beyond the diffraction limited resolution. In a well characterized and sequenced DNA region, the Prader- Willi/Angelman region q11-13 on chromosome 15, geometric distances between the fluorescence intensity bary centers of four different 'point-like' labeling sites were measured. More than 300 cell nuclei were evaluated with a 3D resolution equivalent better than 100 nm. The geometric bary center distances in nanometers were compared with the genomic bary center distance in kilobases (kb). A direct correlation, for instance linear correlation between geometric and genomic distances was not observed. From the measured values, a local compaction factor for the high order chromatin folding in the analyzed genome region was calculated. Along the 1000 kb chromatin segment analyzed, which spans nearly the compete Prader-Willi/Angelman region, different compaction factors were found. The compaction factor 40 typical for a straight 30 nm chromatin fiber was not observed. This shows that chromatin folding and compaction in intact nuclei may be more complex. With SPDM, however, a microscopical technique is available that can sensitively analyze chromatin organization in the 100 nm range in 3D conserved cell nuclei.

Scanning near-field optical microscopy (SNOM) can simultaneously map topographic and optical properties of surfaces with a spatial resolution between that of far-field light microscopy and electron microscopy, i.e. in the range of 100 nm. Since commercially available SNOM instruments came on the market, this technique has become interesting for the routine biological research laboratory especially in combination with far-field light imaging. However, due to the usually applied shear-force feedback controlling the SNOM tip, this technique still poses several challenges for biological applications. In our experiments for instance imaging of soft samples, large topographical changes on structurally conserved cell surfaces, and in particular the requirement for completely dried specimen had to be considered. To visualize surfaces of cells fixed on standard glass slides by SNOM, an easy to handle, optimized protocol using dehydration and hexamethyldisilazane exposure before air drying was developed. Using the commercially available instrument SNOM 210 with micro-fabricated silicon nitride tips, it was shown for several cell systems that the cellular morphology and surface structures were well preserved after this procedure of drying.

We describe a novel method of 3D imaging of specific regions of DNA in interphase nuclei and tissues based on multiphoton microscopy and multicolor fluorescence in situ hybridization (M-FISH). Multiphoton Multicolor FISH (MM-FISH) combines the advantages of (i) using a single NIR excitation wavelength for the simultaneous excitation of multiple FISH fluorophores, (ii) absence of fading in out-of-focus regions, (iii) intrinsic 3D imaging capability and (iv) high light penetration depth. Detection of chromosomal aberrations in amniocytes and tumor cells as well as imaging of FISH fluorophores in biopsies using femtosecond laser pulses at 780 nm and 800 nm are described. First two-photon excited fluorescence decay curves of FISH fluorophores are presented. The fluorophores have been excited via non- resonant two-photon excitation with 150 fs pulses of 0.1 to 8 mW mean laser power of a frequency doubled ultra compact 50 MHz fiber laser and with 80 fs pulses of a compact 80 MHz Ti:sapphire laser. MM-FISH may become an interesting tool in preimplantation diagnosis and molecular pathology.

Distances of homologous centromeres and telomeres of human chromosomes were interactively measure din relation to the nuclear diameter. In total about 2000 cell nuclei were acquired by fluorescence microscopy. Here the results are presented for two color fluorescence in situ hybridization (FISH) applied to lymphocyte cell nuclei using commercially available DNA probes for chromosome 3 centromere and 3p- telomere. In 89 cell nuclei (66%) of the homologous centromeres had a distance Dc smaller than 15 percent of the nuclear diameter (dn). For these per definition classified 'paired' centromeres an increased frequency of small distances of homologous telomeres (Dt) was found. Stimulated S-phase cell nuclei were identified by incorporation of bromodeoxyuridine and simultaneous fluorescence labeling by anti-BrdU antibodies. In this case only the centromeres were FISH labeled. Of 301 cell nuclei about 187 (62%) were stimulated and among them 77 (41%) were paired according to the above mentioned criterion (Dc<0,15 dn). These results indicate that proliferating blood lymphocytes show a considerable tendency to centromere pairing. Assuming that the chromosome arm is probably localized between centromere and telomere with a homologous chromatin density, it may be concluded from the data that somatic pairing of whole chromosomes occurs preferentially during S-phase of the cell nucleus.

A compact device for variable-angle total internal reflection flourescent microscopy was developed. A pulsed Nd:YAG laser operated at 355 nm was adapted using a multimode quartz fiber and collimating optics with a variable angle of incidence between 64 degrees and 72 degrees. Fluorescence spectra of BKEz-7 endothelial cells incubated with the membrane marker 6-dodecanoyl-2- dimethylamino-naphthalene were measured under TIR illumination as a function of the angle of incidence, incubation time and temperature. Emission bands around 440 nm and 490 nm were detected corresponding to laurdan locate within the gel phase or liquid crystalline phase of cellular lipids, respectively. The generalized polarization GP = (I₄₄₀-I₄₉₀)/(I₄₄₀+I₄₉₀) was used as a measure of intracellular temperature with a precision of ±1°C in the physiologically interesting range between 35°C and 38°C. Following pulsed laser excitation, the time delay between excitation and fluorescence detection was varied. A time gate at 10-15 ns after laser excitation revealed to be an optimum for spectral discrimination of the two emission bands. Fluorescence intensity I_F of both bands decreased continuously when the angle of incidence Θ was increased. Between Θ = 68° and 72° the angular dependence corresponded to a fluorophore located within a thin layer (plasma membrane) at 150-200nm distance from the light reflecting surface. Between Θ=65° and 68° additional contributions from intracellular membranes were observed.

The microscopic autofluorescence and images of different tissue layer in normal and cancerous skin tissues were studied under a micro spectrophotometric system when using a laser light at 442 nm. The results show that the autofluorescence images of normal skin tissues are much brighter than the cancerous ones, and the autofluorescence intensity from various tissue layers of normal skin is stronger than that of cancerous skin tissues. It is also found that the dermis emits more intense fluorescence while epidermis fluoresces weakly in the skin tissues. The obtained data on the microscopic fluorescence properties in the skin will be useful to aid in the interpretation of underlying mechanisms by which the laser-induced autofluorescence technique differentiates between normal and cancerous skin tissues.

In this research project a confocal Raman micro spectrometer (CRM) will be designed and incorporated in a scanning electron microscope (SEM). The aim is to develop a new analytical instrumentation to investigate samples on their morphology, atomic composition and molecular composition. Application of the CRM-SEM is in the field of bio-material research where bio-compatibility with an d bio-degeneration by cells and tissues play an important role. The purposes of CRM is for using in medical, biological, chemical and other field science and technology. The energy of these vibration states depends on the molecular structure and environmental condition like PH, temperature. Therefore, the Raman spectrum contains information about the chemical composition of a substance and the structure of molecules. With CRM we can obtain a 3D image of molecular distribution in living cells or composite materials. In this way it provides directly and non-invasively, unique information about the spatial distribution of molecules in inhomogeneous systems. It is important information for such molecules as DNA, protein and other, where a lot of properties their molecules depends on configuration in space. Combining the capacities of the SEM and the CRM will add a powerful device for material investigation.

Scanning Confocal Fluorescence Microscopy is used for single molecule studies on DNA-protein complexes that occur in Nucleotide Excision Repair (NER). During DNA-damage elimination by the NER-pathway, complex protein structures assemble over DNA. It is our aim to resolve the architecture of these DNA-protein complexes and to study dynamic changes that occur in these structures. For this purpose NER- complexes are partly reconstituted onto DNA-substrates using NER-proteins fused to different Green Fluorescent Protein mutants. Here we describe the recombinant production of NER- GFP fusion proteins. Characterization of GFP fluorescence is shown together with results of GFP single molecule imaging. First results with NER-GFP fusion proteins are presented as well.

In the present work, diamagnetic separation parameters for the porous beads are studied using optical video recording microscopy. The possible direct amount determination of single or double macromolecular layers immobilized in the meshes of the porous beads is demonstrated for the concentrations' range used in heterogenic immunotest and the affinity chromatography, where the direct rapid detection of ligands within sorbent particles is known to be the actual task. A gradient diamagnetic biosensor is described as suitable for rapid quantitative detection of single or double macromolecular layers in porous nonmagnetic beads. Measurements of capture traveling time or accumulation radius in gradient magnetic field have shown that it is possible to determine 0.20 mg/ml of macromolecular amount within several seconds. The portative devices were made on the base of the fabre optic technique to detect accumulation radius of collected beads in two gradient magnetic positions: diamagnetic and paramagnetic zones of magnetized wire with 55 μm in diameter and to registrate with a lot of fabre wires having 30 μm in diameters. The successive procedures of the present method can be described by: the obtaining of agarose immuno-beads, the incubation of beads with the ligand sample or the injection of sample through affinity mini-column, the submerging of the loaded beads into the glass cell containing Ni-wire or the narrow gap of magnetic poles; the computational obtaining of immuno- parameters; binding constants, accumulation radius. Several biotechnological applications of the biosensor are presented on sorbent beads, human lymphocytes.

Spectrally resolved imaging of Green fluorescent protein (GFP) expressed in living COS-7 kidney cells distinguished the subcellular localization and demarcated the processes of protein folding and chromophore formation. COS-7 kidney cells were transfected by a plasmid pEGFP-N1 plasmid followed by incubation for 15 hours for gen expression. At different intervals the cells were examined by fluorescence microscopy and analyzed by a spectral imaging system. After 7 hours, GFP was detected in the cytoplasm, concentrated in a localized form. Spectra of the initial GFP evinced several components that belong both tot he typical fluorescent signal as well as to unspecific fingerprints. At 10 and 15 hours, GFP was seen spread in the cytoplasm as well as in the nucleus, and the specific spectra of the GFP were dominant at the later time. The typical GFP spectral fingerprint is the result of protein folding and chromophore formation following internal oxidation reactions. This folding and chromophore formation process, up to final conformation, was detected by spectral imaging as localized in the nucleus rather than in the cytosol. Thus, the method of fluorescence microscopy combined with multiplex spectral imaging demonstrates distinct biochemical pathways leading to GFP conformation.

Quantitative fluorescence microscopy methods can provide valuable insight into drug delivery and pharmacokinetics. We are investigating the use of single photon fluorescence correlation spectroscopy (FCS) to measure particle concentration and mobility in living tissue. In this study we examined whether a relatively large illumination volume could be used to probe the state of macromolecules in free solution and in tissue. The FCS set-up is based upon an upright research microscope, diode laser with 635 nm wavelength, an avalanche photodiode/single photon counting module, and PC based correlation electronics. Diffusion coefficients were e4xtracted from measured autocorrelation functions. We used fluorescent monodisperse beads with diameter 20 and 200 nm to calibrate the excitation volume. Particle diffusion coefficients measured by FCS were compared with conventional light-scattering measurements. We then applied the technique to measure fluorescently labeled liposome distribution in tissue and tissue models. We found that the difference in quantum brightness and diffusion times of liposomes and free dye may be used to detect changes due to liposome interaction with living cancer cells.

The aim of this work was to develop an efficient fluorescence microscopic technique and selective fluoroprobes for cancer diagnosis in colon tissue sections, as well as to determine the morphological components where selective dye accumulation has occurred. For this purpose a novel fluorescence probe, Rh-B - phenyl boronic acid was synthesized and examined. This derivative distinguished clearly and consistently, healthy form neoplastic human colon tissue sections. Intense accumulation was observed at the amorphous material in the lumen of neoplastic crypts, of the colonic epithelium. To gain insight into the localization pattern of this fluoroprobe and to correlate it with mucin alterations, mucicarmine and the blue-fluorescent conjugate of wheat germ agglutinin with Alexa 350 were used. Alexa 350-WGA reacted primarily with mucin secreted in the malignant crypt lumen suggesting that this materia is rich in cialic acid and N-acetylglucosaminyl residues.

Dental biofilm consists of micro-colonies of bacteria embedded in a matrix of polysaccharides and salivary proteins. pH and oxygen concentration are of great importance in dental biofilm. Both can be measured using fluorescence techniques. The imaging of dental biofilm is complicated by the thickness of the biofilms that can be up to several hundred micrometers thick. Here, we employed a combination of two-photon excitation microscopy with fluorescence lifetime imaging to quantify the oxygen concentration in dental biofilm. Collisional quenching of fluorescent probes by molecular oxygen leads to a reduction of the fluorescence lifetime of the probe. We employed this mechanism to measure the oxygen concentration distribution in dental biofilm by means of fluorescence lifetime imaging. Here, TRIS Ruthenium chloride hydrate was used as an oxygen probe. A calibration procedure on buffers was use to measure the lifetime response of this Ruthenium probe. The results are in agreement with the Stern-Volmer equation. A linear relation was found between the ratio of the unquenched and the quenched lifetime and the oxygen concentration. The biofilm fluorescence lifetime imaging results show a strong oxygen gradient at the buffer - biofilm interface and the average oxygen concentration in the biofilm amounted to 50 μM.

Changes in the autofluorescence at the living eye-ground are assumed as important mark in discovering of the pathomechanism in age-related macular degeneration. The discrimination of fluorophores is required and also the presentation of their 2D distribution. Caused by transmission of ocular media, a differentiation between fluorophores by the spectral excitation and emission range is limited. Using the laser scanner principle, the fluorescence lifetime can be measured in 2D. Keeping the maximal permissible exposure, only a very weak signal is detectable, which is optimal for application of the time- correlated single photon counting (TCSPC). In an experimental set-up, pulses of an active model locked Ar⁺ laser (FWHM = 300 ps, reptition rate = 77.3 MHz, selectable wavelengths: 457.9, 465.8, 472.7, 496.5, 501.7, 514.5 nm)excite the eye-ground during the scanning process. A routing module realizes the synchronization between scanning and TCSPC. Investigation of structured samples of Rhodamin 6G and of Coumarin 522 showed that a mono-exponential decay can be calculated with an error of less than 10 percent using only a few hundred photons. The maximum likelihood algorithm delivers the most correct results. A first in vivo tau-image, exhibit a lifetime of 1.5 ns in the nasal part and 5 ns at large retinal vessels.

We present images of human skin obtained by a two-photon fluorescence microscope, which has been optimized for in- vivo imaging. Using autofluorescence as a contrast mechanism various layers of the skin, e.g., stratum corneum, viable epidermis, basal layer and upper dermis could be clearly distinguished. A comparison between two-photon fluorescence images and reflectance images shows that there is a clear difference in the information content provided by both techniques. In particular, the fluorescence images of the dermis show detail that is absent in the reflectance images.

A prototype of a tomographic microscope has bene realized, which uses phase-shifting holography to sense the wave diffracted by an object. The observed object is successively illuminated by a series of about 1000 plane waves. Each diffracted wave yields a part of the frequency representation of the object, and superposition of these parts yields a 3D frequency representation. An inverse Fourier transform yields the 3D spatial representation, from which sections, projections or stereoscopic images can be extracted. This prototype has been used to image a variety of biological samples. Images show a resolution limit of about a quarter of a wavelength and a depth of field of about 40 microns. Important factors for the understanding of image characteristics include horizontal and vertical resolution, ability to image horizontal and/or vertical surfaces, ability to distinguish variations of refractive index from variations of absorptivity. These and other imaging characteristics of the tomographic microscope are discussed on the basis of a set of images of biological samples. The connection between characteristics of a three-dimensional image and its frequency representation is explained. Influence of an object's characteristics (thickness, refractive index, ...) on image quality is described. Possible improvements and their impact on image quality is also discussed.