Cathrin Dressler

Heat Stress Induced Redistribution of Fluorescent Quantum Dots in Breast Tumor Cells

The probing of living cells in different colors over extended periods of time can be used to see the complicated processes that take place during carcinogenesis or heat stress, for example. Since most therapeutic laser tissue interactions are based on thermal effects a detailed characterization of thermal tissue damages in the cellular and sub-cellular levels is important. In order to study such microdosimetry laser-induced fluorescences of Quantum dots provide a suitable approach. Streptavidin conjugated Qdot™ 605 (Quantum Dot Corp., USA) were used in combination with the concanavalin A-biotin labeling system (Molecular Probes, NL) to observe membrane associated thermal lesions. Fluorescent Qdot conjugates are a promising alternative to organic dyes. The extinction coefficient of Qdot™ 605 streptavidin conjugate is 650,000 M−1 cm−1 at 600 nm. Red fluorescent Qdots™ 605 were selected because autofluorescence of cells in the red spectral range is not relevant. Fluorescence detection was performed with a confocal laser scan microscope LSM410 (Carl Zeiss, Germany). Breast cancer cells were used in the thermal stressing experiments performed at 40°C, 42°C, 45°C, 50°C or 56°C for 30 min, each. In this methodical approach Qdot mediated labeling of heat stressed cells were demonstrated to show alterations of plasma membrane organizations and integrities, respectively.

Fluorescence Imaging of Heat-Stress Induced Mitochondrial Long-Term Depolarization in Breast Cancer Cells

Various thermotherapies are based on the induction of lethal heat in target tissues. Spatial and temporal instabilities of elevated temperatures induced in therapy targets require optimized treatment protocols and reliable temperature control methods during thermotherapies. Heat-stress induced effects on mitochondrial transmembrane potentials were analyzed in breast cancer cells, species MX1, using the potential sensor JC-1 (Molecular Probes, Invitrogen, Germany). Potential dependant labeling of heat-stressed cells was imaged and evaluated by fluorescence microscopy and compared with control cells. JC-1 stains mitochondria in cells with high mitochondrial potentials by forming orange-red fluorescent J-aggregates while in cells with depolarized or damaged mitochondria the sensor dye exists as green fluorescent monomers. In MX1 cells orange-red and green fluorescence intensities were correlated with each other after various heat-stress treatments and states of mitochondrial membrane potentials were deduced from the image data. With increasing stress temperatures the intensity of red fluorescent J-aggregates decreased while the green fluorescence intensity of JC-1 monomers increased. This heat-stress response happened in a nonlinear manner with increasing temperatures resulting in a nonlinear increase of red/green fluorescence ratios. These data indicated that mitochondria in MX1 cells were increasingly depolarized in response to increasing ambient temperatures.

Microscopical heat stress investigations under application of quantum dots

Heat stress responses are analyzed in cancer cells by applying different microscopy techniques for targeting various fluorescently labeled or native structures. Thermotreatments are performed at 40, 45, 50, and 56 degrees C, respectively, for 30 min each, while controls were kept at 37 degrees C. Actin cytoskeletons labeled with Alexa Fluor 488-conjugated phalloidin are imaged by wide-field fluorescence microscopy (WFFM). Structural plasma membrane stabilities are labeled with fluorescent quantum dots and analyzed by laser scanning microscopy (LSM). High-resolution atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to study morphological features and surface structures. Fluorescence images reveal F-actin to be a comparatively thermolabile cell component showing distinctive alteration after heat treatment at 40 degrees C. Destabilization of actin cytoskeletons proceed with increasing stress temperatures. Active reorganization of plasma membranes coincidental to heat-induced shrinkage and rounding of cell shapes, and loosening of monolayered tissue are observed after treatment at 45 or 50 degrees C. Active stress response is inhibited by stress at 56 degrees C, because actin cytoskeletons as well as plasma membranes are destroyed, resulting in necrotic cell phenotypes. Comparing data measured with the same microscopic technique and comparing the different datasets with each other reveal that heat stress response in MX1 cells results from the overlap of different heat-induced subcellular defects.

Evaluation of Raman spectroscopic macro raster scans of native cervical cone biopsies using histopathological mapping

Abstract. Raman spectroscopy based discrimination of cervical precancer and normal tissue has been shown previously in vivo with fiber probe based measurements of colposcopically selected sites. With a view to developing in vivo large area imaging, macro raster scans of native cervical cone biopsies with an average of 200 spectra per sample are implemented (n=16). The diagnostic performance is evaluated using histopathological mapping of the cervix surface. Different data reduction and classification methods (principal component analysis, wavelets, k-nearest neighbors, logistic regression, partial least squares discriminant analysis) are compared. Using bootstrapping to estimate confidence intervals for sensitivity and specificity, it is concluded that differences among different spectra classification procedures are not significant. The classification performance is evaluated depending on the tissue pathologies included in the analysis using the average performance of different classification procedures. The highest sensitivity (91%) and specificity (81%) is obtained for the discrimination of normal squamous epithelium and high-grade precancer. When other non-high-grade tissue sites, such as columnar epithelium, metaplasia, and inflammation, are included, the diagnostic performance decreases.

First Results with Ablating Superficial Cell Layers of the Porcine Vocal Fold Using a Femtosecond Laser

Summary Most of benign, precancerous and malignant epithelial lesions in the human endolarynx, especially the vocal fold require a highly precise and controllable therapy, providing an effective ablation of injured tissue under functional preservation of adjacent structures. The dominant instrument currently used in clinical laser surgery is the CO 2 laser. However, CO 2 laser therapies in the endolarynx may result in severe postoperative risks like impairment of the vocal fold and voice quality. The femtosecond (fs) lasers available today are mainly characterized by extreme short pulses ranging from about 10 fs (10 −14 s) to 1 ps (10 −12 s) and single pulse powers in the range of gigawatts (10 9 W) to terawatts (10 12 W). Thus a fs laser enables the exact ablation of cells or superficial cell layers in target tissue by inducing microplasms during nonlinear absorption processes directly on the target. This process minimizes thermal and mechanical stress into surrounding tissues (“cold ablation”). In this basic study we tried to ablate small volumes of cells from a highly sensitive tissue using an experimental fs laser system without producing secondary damages in the periphery of the lased area. The vocal folds of a porcine larynx were used as a tissue model. First results showed that minimal ablation depths below 200 μm were achieved when a 350 μJ pulse energy and a scan rate of 1mm/s were applied. No defects were observed in the structures beneath the ablated area. Therefore, fs lasers may be suitable for the therapy of superficial small-volume lesions in the endolarynx. This has to be validated in further preclinical studies.

Research on human carcinoma cells in different physiological states using laser phase microscopy

In this study we investigate the potentials of imaging the morphological and physiological features of cells by means of Laser Phase Microscopy (LPM). Since LPM is a relatively new microscopical device it has only been used in a few experimental laboratories so far. Human carcinoma cells were treated with the chemical stress factor ethanol and the patterns of the phase shift distributions were observed for single cells.

Fluorescence Imaging of Calcium Loading and Mitochondrial Depolarization in Cancer Cells Exposed to Heat Stress

One main issue of thermotherapy is the stress response of mitochondria to heat. Thermotherapies function by inducing lethal heat inside target tissues. Actually spatial and temporal instabilities of temperature distributions inside target volumes require optimized treatment protocols and reliable temperature-control methods during thermotherapies. Since solid tumors present predominant targets to thermotherapy, on the one hand, the hyperthermic stress-induced effects on mitochondrial transmembrane potentials in breast cancer cells (MX1) were analyzed. On the other hand, the intracellular Ca2+ fluctuations in different cell types responding to heat stress were investigated.

Isolation of Renal Glomeruli Specific Cell Material Using an Experimental NIR-Laser Microdissection Setup

Summary The requirement for a laser microdissection (LMD) device resulted from research activities aiming at the identification of defective genes correlated with arterial hypertension or diabetes mellitus type II accompanied by renal dysfunctions. In recent years, molecular genetic methods like polymerase chain reaction (PCR) developed significantly in terms of biochemical agents and automation. Therefore these techniques now are highly sensitive and require cell specific homogeneous biomaterials in order to produce correct results. We have developed a low cost experimental LMD device by combining a NIR diode laser with a conventional inverse microscope via coordinate fiber optics. This LMD setup was functionally tested by isolating single glomeruli from rat kidney tissue sections. The dissected material then was checked for the molecular genetic quality in a PCR procedure using two DNA primers specific for a 128bp fragment of a household gene coding for the rat leptin receptor. Amplification of this DNA fragment in the glomeruli samples was detected by gel electrophoresis. With this study we could show, that our experimental LMD-setup allows easy isolation of PCR suitable cell material. Therefore devices like ours are valuable complements in molecular genetic laboratories, which could be built at reasonable costs.

Early responses of human cancer cells upon photodynamic treatment monitored by laser phase microscopy

Photodynamic treatment of cancer cells is known to eventually cause cell death in most cases. The precise pathways and the time course seem to vary among different cell types and modes of photodynamic treatment. In this contribution, the focus was put on the responses of human colon carcinoma cells HCT-116 within the first 15 minutes after laser irradiation in the presence of Photofrin® II (PII). To monitor the cell response in this early time period laser phase microscopic imaging was used, a method sensitive to changes in overall cell shape and intracellular structures, mediated by changes in the local refractive index. Laser irradiation of cells loaded with PII induced a significant reduction of the phase shifts, which probably reflects the induced damage to the different cellular membrane structures. The data suggest that even within the first 30 s after the onset of laser illumination, a significant reduction of the phase shifts can be detected. These results underline that laser phase microscopy is a suitable diagnostic tool for cellular research, also in the early time domain.

Laser phase microscopy and functional imaging of living human cancer cells during the cell cycle

The purpose of the investigation was to elaborate a new method of functional imaging of living tumor cells. Human colon carcinoma cells HCT116 were investigated with a conventional light microscope, confocal laser scanning microscope and with a laser phase microscope (LPM). The LPM is a functional imaging technique providing information about cell morphology which is imposed by the physiological inhomogeneity of the refractive index. The phase of the light wave passing through an object contains quantitative information about the object thickness, the shape, and the spatial distribution of the refractive index varying with morphology and chemical composition inhomogeneity inside the object. The new method of investigation of the cells in different stages of the cell cycle is developed. Every phase image of the investigated cells has been compared with conventional light microscopic and confocal microscopic images of the same cell. the relation between the cell state, their morphological peculiarities and the phase characteristics of the measured cell is determined. Data thus acquired, quantitatively characterizing intra- and intercellular processes during the cell cycle, and the method of measurements can be used to investigate with high optic resolution the mechanisms of different physical, chemical and biomolecular interactions with the tumor cells.