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Results for “Microscopy, Scanning Probe”

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PubMed1998

Doxorubicin distribution in multicellular prostate cancer spheroids evaluated by confocal laser scanning microscopy and the "optical probe technique".

Multicell-mediated drug resistance is a major impediment for the effectiveness of chemotherapeutic approaches and has been shown to be a feature of many solid tumors. We used confocal laser scanning microscopy to evaluate the depth distribution of the fluorescent cytostatic drug doxorubicin (Dox) in two size classes of multicellular cancer spheroids (MCS) (psi150+/-50 microm and 350+/-50 microm). MCS (psi150+/-50 microm) solely consist of proliferating cells, whereas in MCS (psi350+/-50 microm) peripheral proliferating cell layers are followed in the depth of the tissue by drug resistant quiescent cell areas. A technique was developed which allows noninvasively to trace fluorescence distributions down to a depth of approximately 180 microm in living MCS. This was achieved by confocal radial recordings of the mean Dox fluorescence in 600 microm2 regions of interest (ROI), equidistantly spaced (10 microm) from the center of MCS towards their periphery. The resulting fluorescence intensity profiles were subsequently corrected for absorbtion and light scattering in the depth of the tissue by a convenient algorithm. A 10 min incubation of MCS (psi150+/-50 microm) with Dox (10 microM) led to a peripheral accumulation, after 2 h Dox was homogeneously distributed within the whole MCS. In contrast, after Dox treatment of MCS (psi350+/-50 microm) for 2 h, the drug was accumulated within the peripheral proliferating cell rim of 78+/-8 microm, whereas deeper, quiescent cell layers remained unstained. When MCS were incubated with verapamil, cyclosporin A, orthovanadate, and quinidine, which are known to reverse P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), Dox accumulated also in deeper cell layers. Genistein and indometacin which reverse multidrug resistance mediated by the multidrug resistance-associated protein (MRP) were without effects. The optical probe technique proved to be well suited to study MDR in a living three dimensional tissue context.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy.

Bacteria of the family Legionellaceae form a monophyletic group within the gamma-subclass of Proteobacteria. Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 16S rRNA characteristic for Legionellaceae. Probe specificities were tested by whole-cell or dot-blot hybridization against 14 serogroups of Legionella pneumophila, 22 different Legionella spp. and 72 non-legionellae reference strains. Using optimized conditions both probes hybridized to all tested strains of L. pneumophila. Probes LEG226 and LEG705 hybridized to 71% and 90% of the Legionella species tested, respectively. With the exception of Methylomonas alba none of the non-target strains showed complete sequence homology within the target molecule. In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay were in good accordance for routine environmental and clinical isolates. L. pneumophila suspended in drinking water at approximately 10(3)-10(4) c.f.u. ml-1 could be rapidly detected by a combination of membrane filtration on polycarbonate filters and whole-cell hybridization. Even after incubation for 1 year a proportion of the released cells was still detectable. In situ hybridization also facilitated visualization of Legionella spp, cells in model biofilms. A combination of in situ hybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L. pneumophila within cells of the ciliated protozoan Tetrahymena pyriformis. Whole-cell probing with 16S rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunofluorescence and PCR in ecological and epidemiological studies of Legionellaceae.

Animals↗
PubMed1998

Scanning tunneling and atomic force microscopy probes of self-assembled, physisorbed monolayers: peeking at the peaks.

The imaging and control of self-assembled, physisorbed monolayers have been the subject of numerous scanning tunneling microscopy and atomic force microscopy investigations. The successful interpretation of the structures observed in scanning probe images of molecules self-assembled at liquid-solid and gas-solid interfaces has benefited greatly from recent experimental and theoretical work. These studies are converging on a general tunneling mechanism that accounts for the images of weakly bound, insulating adsorbates. Experiments in which the dynamical behavior of these monolayers has been monitored as a function of time both statically and after the introduction of an external perturbation are described, and novel studies of the selective control of monolayer structure that make use of internal and external electric fields, photons, and solvent coadsorption are reviewed.

Journal Article↗
PubMedDate not supplied

Coaxial probes for scanning near-field microscopy.

This paper deals with the development of coaxial aperture tips integrated in a cantilever probe for combined scanning near-field infrared microscopy and scanning force microscopy. A fabrication process is introduced that allows the batch fabrication of hollow metal aperture tips integrated on a silicon cantilever. To achieve the coaxial tip arrangement a metal rod is deposited inside the hollow tip using the focused ion beam technique. Theoretical calculations with a finite integration code were performed to study the transmission characteristics of coaxial tips in comparison with conventional aperture probes. In addition, the influence of the geometrical design parameters of the coaxial probe on its optical behaviour is investigated.

Journal Article↗
PubMed1999

Development of X-ray excitable luminescent probes for scanning X-ray microscopy.

Transmission soft X-ray microscopy is now capable of achieving resolutions that are typically 5 times better than the best-visible light microscopes. With expected improvements in zone plate optics, an additional factor of two may be realized within the next few years. Despite the high resolution now available with X-ray microscopes and the high X-ray contrast provided by biological molecules in the soft X-ray region (lambda = 2-5 nm), molecular probes for localizing specific biological targets have been lacking. To circumvent this problem, X-ray excitable molecular probes are needed that can target unique biological features. In this paper we report our initial results on the development of lanthanide-based fluorescent probes for biological labeling. Using scanning luminescence X-ray microscopy (SLXM, Jacobsen et al., J. Microscopy 172 (1993) 121-129), we show that lanthanide organo-polychelate complexes are sufficiently bright and radiation resistant to be the basis of a new class of X-ray excitable molecular probes capable of providing at least a fivefold improvement in resolution over visible light microscopy. Lanthanide probes, able to bind 80-100 metal ions per molecule, were found to give strong luminescent signals with X-ray doses exceeding 10(8) Gy, and were used to label actin stress fibers and in vitro preparations of polymerized tubulin.

Actins↗
PubMed2005

Scanning electrochemical microscopy. 55. Fabrication and characterization of micropipet probes.

The fabrication and characterization of novel micropipet probes for use in scanning electrochemical microscopy (SECM) are described. These can be used to dispense small (pL) amounts of a solution while monitoring the electrochemical response at a substrate and at a ring electrode tip on the micropipet probe. The probes were constructed by insulating gold-coated borosilicate micropipets with electrophoretic paint and exposing a ring electrode at the tip by heat treatment. Characterization of the probes was performed using scanning electron microscopy, cyclic voltammetry, and SECM approach curve experiments. Routine construction of tips with diameters of the order of 3 microm was possible using this technique. The probes exhibited stable steady-state currents and positive and negative feedback approach curves that agreed with those predicted by theory. Demonstrative SECM imaging experiments were performed using a picodispenser to continuously dispense an electroactive solution (ferrocenemethanol) to the SECM cell while the probe was located within a few micrometers of a Pt substrate surface. Oxidation of the dispensed electroactive solution was performed at the substrate, and feedback currents were measured at the probe tip by holding the gold ring at a reducing potential. This mode of tip-dispensing SECM was used to obtain images of a platinum substrate electrode while monitoring both the substrate current and the feedback current at the probe.

Journal Article↗
PubMed1995

Visualization of silver-enhanced reaction products from protein-and immuno-colloidal gold probes by laser scanning confocal microscopy in reflection mode.

We have employed a laser scanning confocal microscope in reflection mode to directly and indirectly visualize sites of deposition of silver-enhanced reaction products from colloidal gold probes. A direct approach was used for the localization of alpha-fetoprotein receptors in human myoblasts by incubating primary cultures with an alpha-fetoprotein-gold conjugate. For an indirect approach, cultured CEM cells, derived from a human T-lymphoma cell line, were incubated with a mouse monoclonal antibody to mature T-cells, followed by a gold-labelled antibody to mouse immunoglobulins. Multiple optical sections of each sample were collected by reflection laser scanning confocal microscopy and combined into three-dimensional renderings. A (non-confocal) transmission image was generated of each field for comparative purposes. The increasing use of reflection laser scanning confocal microscopy combined with colloidal gold conjugates as biological markers will probably be of considerable advantage in cytochemical analysis.

Antigens, CD↗
PubMedDate not supplied

Optical characterization of probes for photon scanning tunnelling microscopy.

The photon scanning tunnelling microscope is a well-established member of the family of scanning near-field optical microscopes used for optical imaging at the subwavelength scale. The quality of the probes, typically pointed uncoated optical fibres, used is however, difficult to evaluate in a direct manner and has most often been inferred from the apparent quality of recorded optical images. Complicated near-field optical imaging characteristics, together with the possibility of topographically induced artefacts, however, has increased demands for a more reliable probe characterization technique. Here we present experimental results obtained for optical characterization of two different probes by imaging of a well-specified near-field intensity distribution at various spatial frequencies. In particular, we observe that a sharply pointed dielectric probe can be highly suitable for imaging when using p-polarized light for the illumination. We conclude that the proposed scheme can be used directly for probe characterization and, subsequently, for determination of an optical transfer function. which would allow one to deduce from an experimentally obtained image of a weakly scattering sample the field distribution existing near the sample surface in the absence of the probe.

Journal Article↗
PubMed1986

Silver-enhanced colloidal gold probes as markers for scanning electron microscopy.

Silver enlargement of small colloidal gold particles has been extensively used for the light microscopical visualization of gold probes. Very recently, a few investigators have employed physical developers in electron microscopy (both pre-embedding and on-grid staining methods). We now demonstrate that physical development of small colloidal gold particles advantageously can be exploited for labelling biological surfaces in scanning electron microscopy. This novel application of silver enhancement of colloidal gold particles is characterized by a high detection efficiency. Thus, specimens are labelled with small gold probes affording high immunocytochemical efficiency but being impossible to detect with the present scanning microscopes. These particles are subsequently scanning electronmicroscopically visualized by silver enhancement.

Animals↗
PubMedDate not supplied

Diode laser contact transscleral cyclophotocoagulation: getting the most from the G-probe.

BACKGROUND AND OBJECTIVE: To determine the multi-use behavior of the G-probe that is traditionally marketed as a single-use cyclophotocoagulation instrument. MATERIALS AND METHODS: A diode laser equipped with a G-probe was used to perform cyclophotocoagulation of the ciliary body in 4 human cadaver eyes and 15 porcine eyes (1,750 mW x 2 seconds). A determination of G-probe effectiveness was made following measurements of G-probe energy output, scanning electron microscopy examination of probe tips, and histologic examination of treated tissue. RESULTS: The mean energy output of 12 of the 15 G-probes remained constant at 3.10 J (range, 3.04 to 3.14 J) for 20 treatment cycles of 20 applications each. Scanning electron microscopy demonstrated minimal surface change. Three G-probes suffered a significant drop-off in energy output during the experiment, and scanning electron microscopy showed significant surface change. Histologic examination of human eyes treated with laser revealed disruption of the ciliary body stroma and separation of pigmented and nonpigmented layers of the ciliary epithelium. More pronounced tissue necrosis and disruption was observed in eyes treated with newer G-probes. Milder coagulative damage with cellular vacuolization was observed in G-probes used for higher numbers of treatment cycles. CONCLUSION: Every G-probe tested delivered more than 100 consecutive applications of laser energy before any loss of efficiency was noted. This suggests that in the absence of obvious structural damage, a G-probe may be safely used for treatment of at least 5 eyes.

Aged↗
PubMed2000

Scanning Near-Field Optical Microscopy and Spectroscopy as a Tool for Chemical Analysis.

Research into the nanometer-scale region is currently of relevance in many branches of modern science and engineering, such as in microelectronics, supramolecular chemistry, and in a biological context. A great deal of attention is given to the design of molecular devices, usually towards understanding the function of existing "molecular machines". Central to this task are powerful diagnostic tools capable of recording chemical information with spatial resolution in the nanometer range. While elemental analysis of surfaces with a lateral resolution of a few dozen nanometers is almost routine, analysis of molecular species with a resolution of <1 µm is very difficult. Scanning-tunneling and atomic-force microscopies usually do not give any chemical information. By combining scanning near-field optical microscopy (SNOM)-the "optical member" of the family of scanning-probe microscopies-with optical spectroscopy, it is possible to obtain molecular information from sample areas as little as 50 nm in diameter. In SNOM, a light source is scanned above the object of interest at a distance of a few nanometers. In the optical near field, the illuminated area is not subject to the Abbé diffraction limit, but merely by the size of the illuminating source. High quality SNOM probes can be reproducibly prepared by a chemical-etching method. These etched probes have an optical transmission up to 1000-fold higher than commercial (pulled) SNOM tips and can withstand higher laser power. This last advantage allows not only high resolution optical imaging, but also localized spectroscopic investigations of surfaces and even optical "nanosampling" by pulsed-laser ablation. The ablated material can be transported over a considerable distance, which opens the possibility for its subsequent analysis with a complementary, highly sensitive analytical method, such as mass spectrometry.

Journal Article↗
PubMed2002

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.

Animals↗
PubMed2006

Neutral red as a probe for confocal laser scanning microscopy studies of plant roots.

BACKGROUND AND AIMS: Neutral red (NR), a lipophilic phenazine dye, has been widely used in various biological systems as a vital stain for bright-field microscopy. In its unprotonated form it penetrates the plasma membrane and tonoplast of viable plant cells, then due to protonation it becomes trapped in acidic compartments. The possible applications of NR for confocal laser scanning microscopy (CLSM) studies were examined in various aspects of plant root biology. METHODS: NR was used as a fluorochrome for living roots of Phaseolus vulgaris, Allium cepa, A. porrum and Arabidopsis thaliana (wild-type and transgenic GFP-carrying lines). The tissues were visualized using CLSM. The effect of NR on the integrity of the cytoskeleton and the growth rate of arabidopsis primary roots was analysed to judge potential toxic effects of the dye. KEY RESULTS: The main advantages of the use of NR are related to the fact that NR rapidly penetrates root tissues, has affinity to suberin and lignin, and accumulates in the vacuoles. It is shown that NR is a suitable probe for visualization of proto- and metaxylem elements, Casparian bands in the endodermis, and vacuoles in cells of living roots. The actin cytoskeleton and the microtubule system of the cells, as well as the dynamics of root growth, remain unchanged after short-term application of NR, indicating a relatively low toxicity of this chemical. It was also found that NR is a useful probe for the observation of the internal structures of root nodules and of fungal hyphae in vesicular-arbuscular mycorrhizas. CONCLUSIONS: Ease, low cost and absence of tissue processing make NR a useful probe for structural, developmental and vacuole-biogenetic studies of plant roots with CLSM.

Microscopy, Confocal↗
PubMed1999

The transport kinetics of lanthanide species in a single erythrocyte probed by confocal laser scanning microscopy.

A novel method has been developed to visualize and follow the temporal course of lanthanide transport across the membrane into a single living erythrocyte. By means of confocal scanning microscopy and the optical section technique, the entry of lanthanide ions was followed by the fluorescence quenching of fluorescein isothiocyanate (FITC)-labeled membrane and cytosol. From the difference of the quenching kinetics of the whole section and the central area, the time for diffusion through the membrane and the diffusion in the extracellular and intracellular media can be deduced. To clarify the mechanism of lanthanide-induced fluorescence quenching of FITC-labeled erythrocytes and to ensure that this reaction can be used in this method, the reaction was investigated by steady-state fluorescence techniques. The results showed that the lanthanides strongly quenched the florescence emitted by FITC covalently bound to membrane proteins and cytosolic proteins. The static quenching mechanism is responsible for the fluorescence quenching of FITC-labeled proteins by Ln species. The quenching mechanism is discussed on the basis of complex formation. The dependence of fluorescence quenching on both ion size and the total orbital angular momentum L supports the complexation mechanism. The transport time across the membrane is strikingly correlated with Ln species and extracellular concentration. For a given concentration, the transport time of [Ln(cit)2]3- is much shorter than that of Ln3+, since they enter the cells via the anion channel. This is supported by the inhibition effect of 4,4'-diisothiocyanato-2,2'-stilbenendisulfonate on the transport of [Ln(cit)2]3-. On the other hand, the transport of free Ln3+ might be attributed to the enhanced permeability of erythrocytes owing to Ln3+ binding. These findings strongly demonstrate the existence of the non-internalization mechanism of Ln species uptake by erythrocytes.

Animals↗
PubMed2004

Probing the limits of the Derjaguin approximation with scanning force microscopy.

We have measured the interaction force between a silicon nitride scanning force microscopy (SFM) probe and the basal plane of highly oriented pyrolitic graphite as a function of pH and ionic concentration in aqueous solutions. Forces in the range +/- 50 pN were reconstructed from measured signals using dynamical analysis of the cantilever. We modeled the force-separation data using a flat plate electric double-layer interaction and assumed the Derjaguin approximation to adapt the flat plate geometry for the SFM probe shape. Measured forces were well modeled by the theory at high ionic concentrations (10 and 100 mM), where Debye lengths were 3.0 and 0.96 nm, respectively. The theory failed to model forces at a lower ionic concentration (1 mM), where the Debye length was 9.6 nm. To investigate this, we calibrated the SFM probe geometry using blind reconstruction and obtained an apex radius of 7 nm. This value suggested that failure of the theory was due to an invalidation of the Derjaguin approximation at long Debye lengths, where the characteristic length scale for the interaction was larger than the size of the SFM probe. The errors were reduced by replacing the Derjaguin approximation with a surface element integration. The result experimentally demonstrates the limitations of the Derjaguin approximation for predicting interactions of nanoscale colloids.

Graphite↗
PubMed1999

Differentiation of methanosaeta concilii and methanosarcina barkeri in anaerobic mesophilic granular sludge by fluorescent In situ hybridization and confocal scanning laser microscopy

Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe. Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcina species. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of all mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.

Journal Article↗