Torsten Porwol

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

Two‐photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580–1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two‐photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two‐photon and the one‐photon emission spectra. However, a blue‐shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non‐linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.

Cytochromes and oxygen radicals as putative members of the oxygen sensing pathway

This study applies biophysical methods like light absorption spectrophotometry of cytochromes, determination of NAD(P)H-dependent superoxide anion (O2-) formation and localisation of hydroxyl radicals (*OH) by 3-dimensional (3D) confocal laser scanning microscopy to reveal in human cells putative members of the oxygen sensing signal pathway leading to enhanced gene expression under hypoxia. A cell membrane localised non-mitochondrial cytochrome b558 seems to be involved as an oxygen sensor in the hepatoma cell line HepG2 in cooperation with the mitochondrial cytochrome b563 probably probing additionally metabolic changes. *OH the putative second messenger of the oxygen sensing pathway generated by a Fenton reaction could be visualized in the perinuclear space of the three human cell lines used. Substances like cobalt or the iron chelator desferrioxamine, which have been applied in HepG2 cells to mimic hypoxia induced gene expression, interact on various sides of the oxygen sensing pathway confirming the importance of b-type cytochromes and the Fenton reaction.

Endostatin reduces vascularization, blood flow, and growth in a rat gliosarcoma

Endostatin, the 20-kDa C-terminal fragment of collagen XVIII, has previously been shown to inhibit growth and induce regression of different experimental tumors in rodents. In this study, we show that recombinant murine and human endostatin, produced in 293 EBNA cells and yeast, respectively, inhibit ectotopic as well as orthotopic growing BT4Cn gliosarcomas in BD-IX rats. In rats in which s.c. gliomas were grown for a total of 29 days, systemic treatment with recombinant murine endostatin induced about 50% reduction of intratumoral blood flow and tumor size after only 10 days of therapy. In contrast, the blood flow to irrelevant organs was unaffected by endostatin, indicating its specificity of action. Tumors were not observed to increase in size or regrow after cessation of therapy. Furthermore, endostatin-treated rats with i.c. tumors had significantly longer survival time than did untreated controls. In the treated rats, endostatin therapy resulted in a reduced tumor blood vessel volume and an increased tumor cell density with an increased apoptotic index within a given tumor volume, as verified by flow cytometry and by staining with deoxynucleotidyltransferase-mediated dUTP nick-end labeling. This work verifies the general anti-angiogenic and antitumor effects of endostatin and indicates that the protein may also be considered as a treatment strategy for malignant brain tumors.

Tissue oxygen sensor function of NADPH oxidase isoforms, an unusual cytochrome aa3 and reactive oxygen species

NADPH oxidase isoforms with different gp91phox subunits as well as an unusual cytochrome aa3 with a heme a/a3 relationship of 9:91 are discussed as putative oxygen sensor proteins influencing gene expression and ion channel conductivity. Reactive oxygen species (ROS) are important second messengers of the oxygen sensing signal cascade determining the stability of transcription factors or the gating of ion channels. The formation of ROS by a perinuclear Fenton reaction is imaged by 1 and 2 photon confocal microscopy revealing mitochondrial and non-mitochondrial generation.

Two-photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP

Two‐photon (TP) excitation (820–1150 nm) and emission (280–700 nm) spectra for the fluorescent proteins (FPs) ECFP 3 , EGFP 3 and EYFP 3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one‐photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue‐shifted and emission red‐shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out‐of‐maximum excitation, common practice in TP excitation microscopy, can be used for routine application.

Oxygen tension modulates beta-globin switching in embryoid bodies.

Little is known about the factors influencing the hemoglobin switch in vertebrates during development. Inasmuch as the mammalian conceptus is exposed to changing oxygen tensions in utero, we examined the effect of different oxygen concentrations on β‐globin switching. We used an in vitro model of mouse embryogenesis based on the differentiation of blastocyst‐derived embryonic stem cells to embryoid bodies (EBs). Cultivation of EBs at increasing oxygen concentrations (starting at 1% O2) did not influence the temporal expression pattern of embryonic (βH1) globin compared to the normoxic controls (20% O2). In contrast, when compared to normoxically grown EBs, expression of fetal/adult (βmaj) globin in EBs cultured at varying oxygen concentrations was delayed by about 2 days and persisted throughout differentiation. Quantitation of hemoglobin in EBs using a 2,7‐diaminofluorene‐based colorimetric assay revealed the appearence of hemoglobin in two waves, an early and a late one. This observation was verified by spectrophotometric analysis of hemoglobin within single EBs. These two waves might reflect the switch of erythropoiesis from yolk sac to fetal liver. Reduced oxygenation is known to activate the hypoxia‐inducible factor‐1 (HIF‐1), which in turn specifically induces expression of a variety of genes among them erythropoietin (EPO). Although EBs increased EPO expression upon hypoxic exposure, the altered β‐globin appearance was not related to EPO levels as determined in EBs overexpressing EPO. Since mRNA from both mouse HIF‐1α isoforms was detected in all EBs tested at different differentiation stages, we propose that HIF‐1 modulates β‐globin expression during development.—Bichet, S., Wenger, R. H., Camenisch, G., Rolfs, A., Ehleben, W., Porwol, T., Acker, H., Fandrey, J., Bauer, C., Gassmann, M. Oxygen tension modulates β‐globin switching in embryoid bodies. FASEB J. 13, 285–295 (1999)

Laminin expression by glial fibrillary acidic protein positive cells in human gliomas.

Extracellular matrix components are regarded as important substrates for invasive tumor cells. The present work focuses on the expression of laminin in the brain in response to invading brain tumors. Biopsies obtained from tissue macroscopically evaluated as the border zone between tumor and normal brain, in 5 patients undergoing surgery for glioblastoma multiforme, were examined by immunocytochemistry and scanning confocal microscopy for the expression of laminin and glial fibrillary acidic protein. Laminin was mainly found in all the specimens associated with the basal lamina of blood vessels, but a variable degree of punctate laminin deposits were also observed in the parenchyma not associated with blood vessels. In the specimens with substantial deposits, scanning confocal microscopy showed that some of the laminin co‐localized with intracellular glial fibrillary acidic protein.

Tomography of cells by confocal laser scanning microscopy and computer-assisted three-dimensional image reconstruction: Localization of cathepsin B in tumor cells penetrating collagen gels in vitro

We used the nondestructive procedures of confocal laser scanning microscopy in combination with computer-assisted methods to visualize tumor cells in the process of penetrating collagen gels. Three independent sets of images were collected. The image information of all data sets was combined into one image, giving a three-dimensional (3D) impression at high light microscopic resolution and sensitivity. We collected information about the extracellular matrix using the reflection mode, the cell surface/morphology by staining with the fluorescent dye DiOC6(3), and the distribution of cathepsin B by Cy-3-labeled immunolocalization. The specific aim of our study was visualization of the spatial relationship of cell organelles as far as they contain the enzyme cathepsin B to cell morphology and motility in a 3D model of extracellular matrix. The majority of the enzyme was localized pericellularly, with no visible relationship to the direction of movement. However, substantial amounts also appeared in intramatrix pseudopodia and associated with the extracellular face of the plasma membane, which may be indicative either of secretion and/or epicellular activity. Our approach has general applicability to study of the spatial relationships of cell compartments and their possible reorganization over time. This could open new horizons in understanding cell structure and function.

Three-Dimensional Organization of Microtubules in Tumor Cells Studied by Confocal Laser Scanning Microscopy and Computer-Assisted Deconvolution and Image Reconstruction

Confocal microscopy, image deconvolution and computer-assisted methods have been used to reconstruct the three-dimensional (3-D) distribution of tubulin in cells. The techniques were applied to tumor cells growing under regular culture conditions (planar cultivation) and those penetrating into reconstituted collagen matrices (spatial cultivation). As expected the application of deconvolution algorithms enhanced the resolution of results. The deconvolution using the maximum likelihood estimation included the measurement of the point spread function of the optical setup. The data visualization of the resulting data sets uses volume as well as surface rendering approaches. The 3-D reconstruction gives a clear image of the spatial arrangement of tubulin fibers in relation to cell shape and position of other cellular organelles, particularly the nucleus. The tubulin forms an intricate network of fibers of variable thickness. The highest tubulin concentrations appear in the cell periphery and particularly in pseudopodia/invadopodia. This is indicative of an enhanced transport of intracellular material facilitating cell movement and lysis of the extracellular matrix. The investigation is assumed to stimulate further experiments using a variety of techniques leading to the complete understanding of the spatial architecture of living cells.

Three-Dimensional Imaging of Rhodamine 123 Fluorescence Distribution in Human Melanoma Cells by Means of Confocal Laser Scanning Microscopy

Three-dimensional (3D) imaging of intracellular rhodamine 123 fluorescence distribution was performed by means of confocal laser scanning microscopy (CLSM). Human IGR melanoma cells grown in monolayer or multicellular spheroid culture were studied for elucidating mitochondrial membrane potential characteristics, and cell and nucleus volume dimensions. Microspheres 6 microns in diameter loaded with rhodamine B were used to calibrate our instruments for performing 3D imaging of optical sections as obtained by CLSM. Accurate optical slicing is only possible taking into consideration the physical characteristics of the objectives used like chromatic and spherical aberrations, depth discrimination or cover slip correction and the temperature dependence of the immersion medium. While 3D imaging of optical slices can be carried out showing the original shape of the object being tested without physical distortion, 3D images of microspheres show well-reproducible structures of rhodamine B fluorescence. These can be explained by a superposition of two effects, namely scattering of the fluorescence light and a gradient of the electromagnetic field strength of the laser beam due to the shape of the object. 3D imaging of optical slices of IGR cells in monolayer or multicellular spheroid culture, which have been loaded with rhodamine 123, show the location of the dye predominantly within the cytoplasm of the cells with a remarkable heterogeneity of fluorescence intensity within and between single cells, indicating differences in the mitochondrial membrane potential and thus in the metabolic activity. Due to the heterogeneity of the cell shape the cell nucleus occupies between 4 and 14% of the total cell volume. These data reveal calibrated 3D imaging as a valuable noninvasive tool to visualize the heterogeneity of cell parameters under different cell culture conditions.