Mitochondrial fluorescence patterns in rhodamine 6G-stained myocardial cells in vitro

Cellular fluorescence in vitro has been studied employing a low light-level video system interfaced with a real-time image array-processing computer system. Changes in cytoplasmic (mitochondrial) fluorescence in myocytes employing the probe rhodamine 6G have been studied over real time with the aid of several computer-based programs. An oscillating pattern of fluorescence is observed that appears to reflect localized variations in mitochondrial activity. The low light level video computer system used in this study compares favorably with laser-stimulated microspot fluorescence photon-counting techniques for the detection of subcellular fluorescence.     

Analysis of rhodamine and fluorescein-labeled F-actin diffusion in vitro by fluorescence photobleaching recovery.

Properties of filamentous acetamidofluorescein-labeled actin and acetamidotetramethylrhodamine-labeled actin (AF and ATR-actin, respectively) were examined to resolve discrepancies in the reported translational diffusion coefficients of F-actin measured in vitro by FPR and other techniques. Using falling-ball viscometry and two independent versions of fluorescence photobleaching recovery (FPR), the present data indicate that several factors are responsible for these discrepancies. Gel filtration chromatography profoundly affects the viscosity of actin solutions and filament diffusion coefficients. ATR-actin and, to a lesser degree, AF-actin show a reduction in viscosity in proportion to the fraction labeled, presumably due to filament shortening. Actin filaments containing AF-actin or ATR-actin are susceptible to photoinduced damage, including a covalent cross-linking of actin protomers within filaments and an apparent cleavage of filaments detected by a decrease of the measured viscosity and an increase in the measured filament diffusion coefficients. Quantum yields of the two photoinduced effects are quite different. Multiple cross-links are produced relative to each photobleaching event, whereas less than 1% filament cleavage occurs. Substantial differences in the filament diffusion coefficients measured by FPR are also the result of differences in illumination geometry and sampling time. However, under controlled conditions, FPR can be used as a quantitative tool for measuring the hydrodynamic properties of actin filaments. Incremented filament shortening caused by photoinduced cleavage or incremental addition of filament capping proteins produces a continuous and approximately linear increase of filament diffusion coefficients, indicating that filaments are not associated in solution. Our results indicate that actin filaments exhibit low mobilities and it is inferred that actin filaments formed in vitro by column-purified actin, under standard conditions, are much longer than has conventionally been presumed.     

Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy.

Whether polarized treadmilling is an intrinsic property of microtubules assembled from pure tubulin has been controversial. We have tested this possibility by imaging the polymerization dynamics of individual microtubules in samples assembled to steady-state in vitro from porcine brain tubulin, using a 2% glycerol buffer to reduce dynamic instability. Fluorescence speckled microtubules were bound to the cover-glass surface by kinesin motors, and the assembly dynamics of plus and minus ends were recorded with a spinning-disk confocal fluorescence microscopy system. At steady-state assembly, 19% of the observed microtubules (n = 89) achieved treadmilling in a plus-to-minus direction, 34% in a minus-to-plus direction, 37% grew at both ends, and 10% just shortened. For the population of measured microtubules, the distribution of lengths remained unchanged while a 20% loss of original and 27% gain of new polymer occurred over the 20-min period of observation. The lack of polarity in the observed treadmilling indicates that stochastic differences in dynamic instability between plus and minus ends are responsible for polymer turnover at steady-state assembly, not unidirectional treadmilling. A Monte Carlo simulation of plus and minus end dynamics using measured dynamic instability parameters reproduces our experimental results and the amount of steady-state polymer turnover reported by previous biochemical assays.     

Phospholipid order in gel- and fluid-phase cell-size liposomes measured by digitized video fluorescence polarization microscopy.

Low-light digitized video fluorescence microscopy has been utilized to measure the steady-state polarized fluorescence from the membrane probe diphenylhexatriene (DPH) and its cationic and phosphatidylcholine derivatives 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 2-[3-(diphenylhexatrienyl)propanoyl]-3-palmitoyl-L-alpha-phosphati dylcholine (DPH-PC), respectively, in cell-size (10-70 microns) unilamellar vesicles composed of gel-or fluid-phase phospholipid. Using an inverted microscope with epi-illumination optics and an intensified silicon intensified target camera interfaced to a minicomputer, fluorescence images of single vesicles were obtained at emission polarizer orientations of 0 degrees, 45 degrees, 90 degrees, and 135 degrees relative to the excitation light polarization direction. Fluorescence intensity ratios F90 degrees/F0 degrees (= F perpendicular/F parallel) and F135 degrees/F45 degrees were calculated on a pixel-by-pixel basis from digitized image pairs. Theoretical expressions were derived for collected polarized fluorescence as a function of position on the membrane surface as well as the degree of lipid order, in terms of the fluorophore's maximum angular motional freedom in the bilayer (identical to theta max), using a modification of the method of D. Axelrod (1979. Biophys. J. 26:557-574) together with the "wobbling-in-a-cone" model of probe rotational diffusion. Comparison of experimental polarization ratios with theoretical ratios yielded the following results. In gel-phase dipalmitoyl-phosphatidylcholine, the data for all three probes correspond to a model in which the cone angle theta max = 17 +/- 2 degrees and there exists a collective tilt of the phospholipid acyl chains of 30 degrees relative to the bilayer normal. In addition, approximately 5% of DPH and TMA-DPH molecules are aligned parallel to the plane of the bilayer. In fluid-phase palmitoyloleoyl-phosphatidylcholine, the data are well fit by models in which theta max = 60 +/- 2 degrees for DPH and DPH-PC and 32 +/- 4 degrees for TMA-DPH, with approximately 20% of DPH molecules and 10% of TMA-DPH molecules aligned parallel to the bilayer plane, and a net phospholipid tilt at or near the headgroup region of approximately 30 degrees. The results demonstrate that lipid order can be measured with a spatial resolution of approximately 1 micron2 in cell-size vesicles even with high aperture observation through a microscope.     

Visualizing membrane traffic in vivo by combined video fluorescence and 3D electron microscopy

In studies of dynamic cellular processes, it would be ideal to be able to combine the capability of in vivo fluorescence video microscopy with the power of resolution of electron microscopy (EM). This article describes an approach based on the association of these two techniques, by which an individual intracellular structure can be monitored in vivo, typically through the use of markers fused with green-fluorescent protein, and then analysed by EM and three-dimensional reconstruction methods, resulting in a 'snapshot' of its fine structure at any chosen time in its life cycle. The potential of this approach is discussed in relation to various aspects of cell biology and especially to the question of the morpho-functional organization of the intracellular membrane trafficking pathways.     

Bacterial community structure and colonization patterns of Fagus sylvatica L. ectomycorrhizospheres as determined by fluorescence in situ hybridization and confocal laser scanning microscopy

) grown in natural forest soil in southern Germany was examined by fluorescence in situ hybridization (FISH) using fluorescent oligonucleotide probes, targeting phylogenetic relevant sequences of the 16S and 23S rRNA. Lactarius subdulcis, L. vellereus, L. rubrocinctus and Laccaria amethystina were found to be the prevalent fungi forming ectomycorrhizae with F. sylvatica. For FISH studies using confocal laser scanning microscopy, oligonucleotide probes labeled with carboxymethylindocyanine-succinimidyl ester allowed detection of associated bacteria, because the autofluorescence of ectomycorrhiza samples could be overcome in the infrared. Bacteria of the α-, β and γ-subclasses of the proteobacteriawere detected in high numbers on mantle surfaces, while members of other phylogenetically defined groups were found in smaller numbers. This contrasts with previous published results on the cultivation of mycorrhiza-associated bacteria. Hybridizing bacteria were also found within damaged cells of the hyphal mantle of L. rubrocinctus, as well as on emanating hyphae of L. amethystina. Using a newly developed extraction protocol for bacteria associated with ectomycorhizas, the two most common fungi on F. sylvatica, L. vellereus and L. subdulcis, were mostly associated with members of the α- and β-subclasses of the proteobacteria. The proportion of hybridizing bacteria varied between the two ectomycorrhizae, which were thus host to distinct populations of bacteria.     

Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation.

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from approximately 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.     

Interactions of elastic and rigid vesicles with human skin in vitro: electron microscopy and two-photon excitation microscopy.

Interactions between vesicle formulations and human skin were studied, in vitro, in relation to their composition and elasticity. The skin ultrastructure was investigated using transmission electron microscopy (TEM), freeze-fracture electron microscopy (FFEM) and two-photon fluorescence microscopy (TPE). The main difference between the vesicle formulations was their elasticity. Elastic vesicle formulations contained bilayer forming surfactants/lipids and single-chain surfactant octaoxyethylenelaurate-ester (PEG-8-L), whereas rigid vesicles contained bilayer surfactants in combination with cholesterol. TEM results showed three types of interactions after non-occlusive application of elastic PEG-8-L containing vesicle formulations on human skin: (1) the presence of spherical lipid structures containing or surrounded by electron dense spots; (2) oligolamellar vesicles were observed between the corneocytes in the upper part of the stratum corneum; and (3) large areas containing lipids, surfactants and electron dense spots were observed deeper down into the stratum corneum. Furthermore, after treatment with vesicles containing PEG-8-L and a saturated C12-chain surfactant, small stacks of bilayers were found in intercellular spaces of the stratum corneum. Rigid vesicles affected only the most apical corneocytes to some extent. FFEM observations supported the TEM findings. Major morphological changes in the intercellular lipid bilayer structure were only observed after treatment with PEG-8-L containing elastic vesicles. TPE showed a distinct difference in penetration pathways after non-occlusive application of elastic or rigid vesicles. After treatment with elastic vesicles, thread-like channels were formed within the entire stratum corneum and the polygonal cell shape of corneocytes could not be distinguished. Fluorescent label incorporated in rigid vesicles was confined to the intercellular spaces of the upper 2-5 micrometer of the stratum corneum and the cell contours could still be distinguished.     

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).     

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.     

Analysis of deoxyribonucleic acid replication in human X chromosomes by fluorescence microscopy.

The genetically inactive, late-replicating human female X chromosome can be effectively distinguished from its more active, earlier-replicating homologue, when cells are grown according to the appropriate BrdU-33258 Hoechst protocol. Results obtained from a fluorescence analysis of DNA replication in X chromosomes are consistent with those from previous autoradiographic studies, but reflect additional sensitivity and resolution offered by the BrdU-Hoechst methodology. Both qualitative and quantitative differences in 33258 Hoechst fluorescence intensity, reflecting alterations in replication kinetics, can be detected between the two X chromosomes in female cells. The pattern of replication in the single X chromosome in male cells is indistinguishable from that of the early female X. Intercellular fluctuations in the distribution of regions replicating early or late in S phase, particularly with reference to the late female X, can be localized to structural bands, suggesting multifocal control of DNA synthesis in X chromosomes.     

Isolation of spectrin subunits and reassociation in vitro. Analysis by fluorescence polarization

Fluorescence polarization has been used to probe the exposure of tryptophan residues of erythrocyte spectrin. A significant decrease in anisotropy occurred when spectrin was heated at temperatures ranging from 38 to 48 degrees C. At low concentrations of urea, these anisotropy changes shifted to lower temperatures and were minimal in concentrations of urea 3 M or greater. These findings were attributed to the stepwise unfolding of the subdomain structure of spectrin under these conditions and eventual dissociation of oligomeric spectrin to the monomer state. DEAE-cellulose column chromatography in the presence of 3 M urea confirmed this prediction and permitted isolation of pure alpha and beta subunits of spectrin in good yields. The isolated subunits were soluble in neutral salt solutions and were readily reconstituted into high molecular weight forms that displayed "native" tryptophan fluorescence anisotropy changes and migrated as discrete oligomeric species when analyzed by nondenaturing acrylamide gel electrophoresis. The reconstituted complexes were indistinguishable from native spectrin molecules when examined by low angle shadowing and electron microscopy.     

Efficiency of cytoplasmic delivery by non-cationic liposomes to cells in vitro: A confocal laser scanning microscopy study

It is necessary to understand liposomal uptake mechanisms and intracellular distribution in order to design more efficient gene (drug) carrier systems. Until now, a few studies have been carried out using confocal laser scanning microscopy (CLSM) to investigate the cellular uptake and transfection mediated with liposomes. So, by CLSM, we demonstrated that artificial virus-like envelope (AVE) vesicles labeled with rhodamine-PE (Rh-PE), carbocyanine (DiI) and carboxyfluorescein (CF) were investigated into the cytoplasm of two human cell lines, Mewo (human melanoma cell line) and HepG2 (human hepatoma cell line) cells grown in DMEM medium supplemented with different percentages (0%, 30%, and 100%) fetal calf serum (FCS). The liposome uptake was dependent on the cell line, in view that the whole process of liposomes associated with cells (uptake) is a two-step process involving binding and endocytosis. Based upon the various assays used to measure cellular uptake of liposomes, we conclude the efficacy of cytoplasmic delivery by AVE-liposomes to cells in culture.     

Distribution of the Golgi apparatus in the mitosis of cultured tobacco cells as revealed by DiOC6 fluorescence microscopy

Summary We developed a new method for distinguishing the Golgi apparatus from the other membranous organelles which contain DNA, such as mitochondria and chloroplasts, under a fluorescence microscope. Thin sections of cells embedded in Technovit 8100 resin were stained with both 3,3-dihexyloxacarbocyanine iodide (DiOC₆) and 4,6-diamidino-3-phenylindole (DAPI), and those three membranous organelles were observed under an epifluorescence microscope. The Golgi apparatus, which do not contain DNA, were easily recognized when the two images stained with DiOC₆ and DAPI were superimposed using an image processor. Using this method, we investigated the dynamics of cellular membranes and organelles during the mitotic cycle of synchronized cultured tobacco cells BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2). The Golgi apparatus did not accumulate in the rim of the formating early cell plate at anaphase, while it accumulated near the maturing cell plate at telophase, and this accumulation seemed to be related to the maturation of cell plates. To confirm this hypothesis, synchronized BY-2 cells were treated with caffeine, which is known to inhibit the cell plate formation. Most of the cells treated with caffeine remained in a phase in which Golgi vesicles were accumulated at the equatorial plate, but the cell plate was only partially maturing. The Golgi apparatus accumulated only near the partially maturing cell plate, but not by the equatorial plate where the Golgi vesicles had accumulated.Abbreviations DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DAPI 4,6-diamidino-3-phenylindole - LSD a modified Linsmaier and Skoog's medium containing 2,4-D     

Real-time analysis of intracellular glucose and calcium in pancreatic beta cells by fluorescence microscopy

Glucose is the physiological stimulus for insulin secretion in pancreatic beta cells. The uptake and phosphorylation of glucose initiate and control downstream pathways, resulting in insulin secretion. However, the temporal coordination of these events in beta cells is not fully understood. The recent development of the FLII(12)Pglu-700μ-δ6 glucose nanosensor facilitates real-time analysis of intracellular glucose within a broad concentration range. Using this fluorescence-based technique, we show the shift in intracellular glucose concentration upon external supply and removal in primary mouse beta cells with high resolution. Glucose influx, efflux, and metabolism rates were calculated from the time-dependent plots. Comparison of insulin-producing cells with different expression levels of glucose transporters and phosphorylating enzymes showed that a high glucose influx rate correlated with GLUT2 expression, but was largely also sustainable by high GLUT1 expression. In contrast, in cells not expressing the glucose sensor enzyme glucokinase glucose metabolism was slow. We found no evidence of oscillations of the intracellular glucose concentration in beta cells. Concomitant real-time analysis of glucose and calcium dynamics using FLII(12)Pglu-700μ-δ6 and fura-2-acetoxymethyl-ester determined a glucose threshold of 4mM for the [Ca(2+)](i) increase in beta cells. Indeed, a glucose concentration of 7mM had to be reached to evoke large amplitude [Ca(2+)](i) oscillations. The K(ATP) channel closing agent glibenclamide was not able to induce large amplitude [Ca(2+)](i) oscillations in the absence of glucose. Our findings suggest that glucose has to reach a threshold to evoke the [Ca(2+)](i) increase and subsequently initiate [Ca(2+)](i) oscillations in a K(ATP) channel independent manner.     

Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy.

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml. h(-1) with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 microm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.     

Three-dimensional imaging and quantification of real-time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy

The development of a minimally invasive, robust, and inexpensive technique that permits real-time monitoring of cell responses on biomaterial scaffolds can improve the eventual outcomes of scaffold-based tissue engineering strategies. Towards establishing correlations between in situ biological activity and cell fate, we have developed a comprehensive workflow for real-time volumetric imaging of spatiotemporally varying cytosolic calcium oscillations in pure microglial cells cultured on electrospun meshes. Live HMC3 cells on randomly oriented electrospun fibers were stained with a fluorescent dye and imaged using a laser scanning confocal microscope. Resonance scanning provided high-resolution in obtaining the time-course of intracellular calcium levels without compromising spatial and temporal resolution. Three-dimensional reconstruction and depth-coding enabled the visualization of cell location and intracellular calcium levels as a function of sample thickness. Importantly, changes in cell morphology and in situ calcium spiking were quantified in response to a soluble biochemical cue and varying matrix architectures (i.e., randomly oriented and aligned fibers). Importantly, raster plots generated from spiking data revealed calcium signatures specific to culture conditions. In the future, our approach can be used to elucidate correlations between calcium signatures and cell phenotype/activation, and facilitate the rational design of scaffolds for biomedical applications.