Scanning Microscopy System Research Articles

2. Histocytochemistry Series Morphological and Functional Analyses of Cells, Using Confocal Laser Scanning Microscopy System: Example for Morphological and Functional Analyses of Cancer Cells, Using Multi-mode Microscopy System (2)

The confocal laser scanning microscope (CLSM) is a device for obtaining high-resolution optical images of immunofluorescent staining. The CLSM can produce in-focus images of thick specimens, a process known as optical sectioning. The images are reconstructed with a computer, using 3-dimensional image software, allowing 3-dimensional reconstructions of topologically complex objects. On the same tissue sections, the CLSM can obtain the images of differential interference contrast. Recently, a special inverted CLSM-the multimode microscopy system-has been used to examine the morphology and functions of cells. A multimode microscopy system can be used to obtain images of CLSM, total internal reflection fluorescence, time-lapse, and micromanipulation. In the present study, we show images of pancreatic cancer cells as an example.

Fluorescent Liposomes as Contrast Agents for In Vivo Optical Imaging of Edemas in Mice

This study assesses if specially designed fluorescent liposomes can be used as contrast agent for near-infrared fluorescence (NIRF) optical imaging of cultured macrophages in vitro and for NIRF imaging of inflammatory processes, like edema, in an in vivo mouse model. Fluorescent liposomes are prepared by the film hydration and extrusion method using cholesterol, L-phosphatidylcholine, and the NIR fluorescent dye DY-676-C(18) ester. Photon correlation spectroscopy and flow cytometry reveal that fluorescent liposomes are structurally stable for up to 133 days. Distinct uptake/labeling of cultured murine J774 macrophages is demonstrated by confocal laser scanning microscopy (CLSM), flow cytometry, and macroscopic NIRF imaging system at wavelengths >670 nm. Moreover, CLSM analysis reveals fluorescence signals within intracellular compartments. Ear edema is induced in mice (n = 16) by subcutaneous injection of zymosan A. Whole-body NIRF imaging is performed after intravenous injection (0-24 h) of fluorescent liposomes (55 nmol dye per kg body weight). Distinctly higher fluorescence intensities (1613.6 +/- 61.7 a.u.) are detected at inflamed areas of diseased mice as compared to controls (892.8 +/- 19.4 a.u.). Furthermore, cell isolated from ear lavage reveals the presence of labeled F4/80 positive tissue macrophages. Taken together, the results indicate both that mouse macrophages labeled with fluorescent liposomes can be detected in vitro with fluoro-optical methods and that in vivo optical imaging of inflammatory processes with fluorescent liposomes as contrast agent is feasible. Possibly, early stages of other inflammatory diseases could also be detected by the proposed diagnostic tool in the long term.

The Cytoplasmic Region of α-1,6-Mannosyltransferase Mnn9p Is Crucial for Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum in Saccharomyce s cerevisiae

In Saccharomyces cerevisiae, Och1p and Mnn9p mannosyltransferases are localized in the cis-Golgi. Attempts to live image Och1p and Mnn9p tagged with green fluorescent protein or red fluorescent protein, respectively, using a high-performance confocal laser scanning microscope system resulted in simultaneous visualization of the native proteins in a living cell. Our observations revealed that Och1p and Mnn9p are not always colocalized to the same cisternae. The difference in the dynamics of these mannosyltransferases may reflect differences in the mechanisms for their retention in the cis-Golgi, since it has been reported that Mnn9p cycles between the endoplasmic reticulum and the cis-Golgi whereas Och1p does not (Z. Todorow, A. Spang, E. Carmack, J. Yates, and R. Schekman, Proc. Natl. Acad. Sci. USA 97:13643-13648, 2000). We investigated the localization of chimeric proteins of Mnn9p and Och1p in sec12 and erd1 mutant cells. A chimeric protein, M16/O16, which consists of the N-terminal cytoplasmic region of Mnn9p and the transmembrane and luminal region of Och1p, behaved like Mnn9p, suggesting that the N-terminal cytoplasmic region is important for the intracellular dynamics of Mnn9p. This observation is supported by results from subcellular-fractionation experiments. Mutational analysis revealed that two arginine residues in the N-terminal region of Mnn9p are important for the chimeric protein to cycle between the endoplasmic reticulum and the Golgi apparatus.

Experimental Mechanical Stress Characterization of Micro-Electro-Mechanical-Systems Device Using Confocal Laser Scanning Microscope Combined with Raman Spectrometer System

We developed a confocal laser scanning microscopy (CLSM) system combined with a Raman spectrometer system. The potential of this system for characterizing micro-electro-mechanical-system (MEMS) devices was demonstrated by analyzing a Si membrane of a pressure sensor device. A deflection profile and a Raman frequency shift were simultaneously measured. The relationship between the mechanical stress and the deflection of the Si membrane was clearly observed. The mechanical stresses obtained by Raman measurements and deflection measurements were in good agreement.

MAPK-activated Protein Kinase-2 (MK2)-mediated Formation and Phosphorylation-regulated Dissociation of the Signal Complex Consisting of p38, MK2, Akt, and Hsp27

The p38 MAPK and heat shock protein 27 (hsp27) form a signaling complex with serine/threonine kinase Akt and MAPK-activated protein kinase-2 (MK2), which plays an important role in controlling stress-induced apoptosis and reorganizing actin cytoskeleton. However, regulation of the complex is poorly understood. In this study, the interaction between p38 and hsp27 was visualized in single living L929 cells using fluorescence resonance energy transfer technology, while their association with Akt was examined by immunoprecipitation analysis. Under normal growth conditions, p38 kinase constitutively interacts with hsp27. When cells were exposed to H(2)O(2) or stimulated by arachidonic acid, this interaction was disrupted. However, inhibition of the activation of p38 and Akt by selective inhibitors or overexpression of the kinase-dead mutant of p38 diminished such effects. Furthermore, mutation of phosphorylation sites of hsp27 renders the interaction resistant to H(2)O(2) and arachidonic acid. It was interesting to find that the interaction disappeared in the cells from MK2-knock-out mice or the cells treated with lemptomycin B that blocks export of MK2 from nucleus to cytosol. However, MK2 is not required for the association of hsp27 with Akt. This study suggests that MK2 mediates the incorporation of p38 into the pre-existing complex of hsp27 with Akt. Phosphorylation of hsp27 finally breaks the signaling complex.

Turbulence of glomerular hemodynamics involved in progressive glomerulosclerosis

There is increasing evidence that changes of glomerular hemodynamics or glomerular growth responses may promote the development of glomerulosclerosis. Major problems retarding research progress include lack of suitable experimental animal models, with the exception of the ablation model, and the need for in vivo real-time analysis of glomerular hemodynamics. This study examined the sequence of pathological changes from the viewpoints of microcirculation and histopathology, from the acute stage to the chronic course and the final stage of glomerulosclerosis, using the confocal laser scanning microscope system. There is a marked difference in prognosis between sham-operated (two-kidney) and nephrectomized (one-kidney) rats after injection with anti-Thy-1 antibody. The former reversibly returns to normal and the latter irreversibly go to progressive sclerosis, respectively. The turning point determining the progression of glomerulosclerosis in both groups seemed to be the period from 7 to 14 days after disease induction, when disturbance of local intraglomerular blood flow continued in the one-kidney groups. In conclusion, this study provides the first hemodynamic-based evidence showing that disturbance of intraglomerular microcirculation is a critical marker for progressive glomerulosclerosis.

Novel Multiwavelength Microscopic Scanner for Mouse Imaging

Real-time in vivo imaging of molecular targets at (sub)cellular resolution is essential in better understanding complex biology. Confocal microscopy and multiphoton microscopy have been used in the past to achieve this goal, but their true capabilities have often been limited by bulky optics and difficult experimental set-ups requiring exteriorized organs. We describe here the development and validation of a unique nearinfrared laser scanning microscope system that uses novel optics with a millimeter footprint. Optimized for use in the far red and near-infrared ranges, the system allows an imaging depth that extends up to 500 Mm from a 1.3-mm-diameter stick objective, which is up to 2 cm in length. We show exceptionally high spatial, temporal, and multiwavelength resolutions of the system and show that it can be applied to virtually any internal organ through a keyhole surgical access. We demonstrate that, when combined with novel far red imaging probes, it is possible to image the cellular details of many organs and disease processes. The new optics, coupled with the use of near-infrared probes, should prove immensely valuable for in vivo cancer imaging.

Mitochondrial Behaviour in the Early Stages of ROS Stress Leading to Cell Death in Arabidopsis thaliana

Reactive oxygen species (ROS) are involved in triggering cell death. To visualize mitochondrial behaviour under ROS stress, transgenic arabidopsis plants possessing mitochondrial-targeted GFP (S65T) were studied. Arabidopsis leaves were treated with ROS and ROS-inducing chemicals such as hydrogen peroxide, paraquat and menadione. Microscopic observations were carried out using a confocal laser scanning microscope system, and electrolyte leakage was also monitored. After treatment, mitochondria showed morphological changes from a bacillus-like to a round shape. The size of mitochondria treated with H(2)O(2) decreased by half compared with controls. Concurrently, cytoplasmic streaming was blocked and mitochondria eventually swelled. Treatment of leaves with butanedione monoxime, an inhibitor of myosin ATPase, resulted in similar behaviour of mitochondria to that under ROS stress. The results indicate that morphological changes of mitochondria and cessation of cytoplasmic streaming may interact, and this phenomenon is one of the features of ROS stress-induced cell death.

Changes of the Collagen composition in the heart caused by microcurrent application. An explanation for the improvement of cardiac function by bi-ventricular pacing?

Objective Improvement of cardiac function by the unloading effect of a cardiac assist device mainly depends on the duration of heart failure (HF). Patients with a short history of HF ( 5 years) do not show significant cardiac function improvement is that the collagen composition of the extracellular matrix is irreversible. It is successful clinical practice to apply microcurrent in patients with bone fractures and wound healing disturbances in order to improve the healing process by modulation of the collagen synthesis. In order to examine whether microcurrent can also influence the collagen synthesis in the myocardium, the effect of microcurrent application on collagen synthesis of adult cardiomyocytes was investigated. Methods Adult cardiomyocytes were isolated and cultivated in 24 well cell culture plates. Current of different magnitudes (0, 20, 40, 60, 80, 100 :A) was applied via platinum electrodes by a special custom-made device. The whole equipment was incubated under cell culture conditions (+37°C, 5% CO2) over a period of 7 days. Changes of the collagen type I and type III synthesis were analyzed using immunohistochemical staining methods. Collagen type I and type III content was quantified using a special fluorescence confocal laser scanning microscopy system including special analysis software. Results Compared to cardiomyocytes exposed to 0 :A (control cells), collagen type I synthesis of cardiomyocytes showed no significant change after exposure to a moderate current magnitude(40, 60 :A) but a highly significant mean decrease (20.6 %) if exposed to high current (80, 100 :A). Collagen type III revealed a mean increase at moderate current of 29.7 % and a decrease of 25.2 % at high current exposure. As a side effect, we detected an increase in the cell proliferation rate at moderate and high current. Conclusion The results obtained in cell culture suggest that the application of micro-current is able to modulate the synthesis of collagen. In particular, in dependency of the current magnitude collagen type I can be up- or down-regulated. Collagen type I is responsible for the stiffness and the degree of dilatation of the heart. Therefore it can be envisaged that this method -if applied clinically - may help to improve cardiac function, as it helps to heal bone fractures.

Simple optimization procedure for objective lens correction collar setting

We present a new method for setting a coverglass correction collar on an objective lens. Axial scans across the interface between the specimen volume and the slide are used together with a quantitative function of merit to determine the optimum setting of the correction collar. The method, which simplifies the adjustment for the user and reduces photobleaching, was implemented within the software environment of a scanning microscope system.

Blockade of Highly Expressed CD55 Related to Tumor Bulkiness by RNA Interference Overcomes Resistance to CDC with Rituximab.

[Background] Recently, We have presented that sensitivity to anti-CD20 antibody-mediated CDC was different during cell growth dependent on expression of CD55 (ASH 2003, San Diego). In some cases with malignant lymphoma after the treatment of anti-CD20 antibody, lymphoma cells get resistance to the treatment of anti-CD20 antibody. Moreover, bulky disease of malignant lymphoma like 7.0 cm of tumor gets resistance to rituximab. One of some regulators in complement system, CD55, is a major regulator of the alternative and classical pathways of complement activation and is expressed on all serum-exposed cells. Here, we have found the relationship among extirpated lymph nodes, sensitivity to CDC, and CD55 expression, and small interference of RNA (siRNA) for CD55 is a useful tool for blockade of CD55 in monoclonal antibody therapy.[Methods] Human B cell line, Daudi and Rajii cells were used as sensitive and insensitive controls in complement-mediated cytotoxycity (CDC) with anti-CD20 antibody. Lymphoma cells were collected from extirpated lymph nodes of the patients with lymphoma. CDC assay with anti-CD20 antibody was performed with FACscan or under confocal laser scanning microscopy system. Three parts of siRNAs for CD55 (CD55-N, -M, and -C) were designed, and transfected to the cells using lipofectin[Results] The relationship between sensitivity to CDC and size of extirpated tumor showed negative correlation. The formula's intercept was 7.775 cm, suggesting that lymphoma with more than 7 cm of size is not effective in rituximab therapy as Coiffier's report. The level of CD55 expression on lymphoma cells was correlated with size of its lymph node, and in some patient with lymphoma, level of CD55 expression on Lymphoma cells and susceptibility to CDC were different among before, after relapse and after partial remission, indicating that susceptibility to CDC depends on CD55 expression. This suggests that increased cell numbers contribute to higher or enhanced CD55 expression and resistance to CDC with rituximab. Lymphoma cells from all five cases with recurrent lymphomas strongly expressed CD55 molecules under laser scanning confocal microscopy. Percentages of dead cells showed no significant differences among transfection with and without CD55-N, CD55-M and CD55-C before CDC assay. Percentage of PI-positive cells in transfection with CD55-N significantly increased from 7.1% to 67.9%. These indicate that the siRNA against CD55 (1–380 nucleotides), CD55-N could efficiently knock down the expression of CD55 on freshly isolated lymphoma cells from recurrent lymphomas, and that it could induce cell death in freshly isolated lymphoma cells from recurrent lymphomas by CDC.[Conclusion] This siRNA for CD55 could be a good tool for blockade of CD55 in resistance and bulkiness related with highly expressed CD55 with monoclonal antibody therapy.

First tantalum zone plates for the TWINMIC X-ray microscope project

We report the fabrication of X-ray zone plates made of tantalum, designed for use in the TWINMIC microscope project at photon energies between 0.25 and 2.5 keV. The zone plates have an outermost zone width of 80 nm, Ta thicknesses between 200 and 300 nm, and diameters between 250 and 500 μm. Tantalum layers were sputtered onto silicon nitride membranes. The zone-plate pattern was generated with electron-beam lithography at an electron energy of 2.5 keV, and transferred into tantalum by reactive ion etching. Central stops were deposited on some zone plates. Performance tests at the European synchrotron radiation facility (ESRF) with 2.5-keV X rays show that the spatial resolution of the zone plates is 100 nm or better when used in a scanning microscopy system.

Swelling-induced modulation of static and dynamic fluctuations in polyacrylamide gels observed by scanning microscopic light scattering.

The nature of inhomogeneities in vinylpolymer gels has hardly been clarified yet. Inhomogeneities on submicron and nanometer scales in polyacrylamide gels have been investigated by using a scanning microscopic light-scattering system and applying a general formula for an ensemble-averaged correlation function. The network structure of the gels is modified by varying the preparation conditions and can be roughly divided into two types. Swelling-induced modulation of inhomogeneities depends on the type of the network structure. At low monomer concentrations in preparation, both submicron- and nanometer-scale inhomogeneities increase with swelling. At high monomer concentrations in preparation, submicron-scale inhomogeneities increase with swelling, but nanometer-scale inhomogeneities decrease anomalously. This behavior is explained by a model of inhomogeneous network structure of vinylpolymer gels, where macrogel is formed from a large number of microgel particles.

Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection.

We present a transmission-mode confocal laser scanning microscope system based on the use of second-harmonic generation (SHG) for signal detection. Our method exploits the quadratic intensity dependence of SHG to preferentially reveal unscattered signal light and reject out-of-focus scattered background. The SHG crystal acts as a virtual pinhole that remains self-aligned without the need for descanning.

Super-resolution Fluorescence Microscopy in Nano-meter Scale Region Using Two-color Laser Beams

A super-resolution fluorescence microscopy using two-color laser beams was proposed. The microscopy is based on the combination of two-color fluorescence dip spectroscopy and a phase modulation technique for the laser beam. By applying the proposed technique to a laser-scanning microscope, a fluorescence image of a sample can be observed with a spatial resolution overcoming the optical diffraction limit. To demonstrate validity of the microscopy, we constructed a scanning microscope system using commercial nano-second pulse lasers. An image of micro beads containing dye molecules was observed by the microscopy. We succeeded in obtaining the image with a resolution overcoming the diffraction limit in nano-meter scale region. The experimental data showed that the resolution was improved three times at least. The microscopy is expected to be an appropriate analysis method for the samples with nano-meter scale structures.

Noninvasive imaging of signals in digital circuits

In this article we describe the construction and use of a noninvasive (noncontact) electric potential probe to measure time delays of signals propagating through digital circuits. As we show, by incorporating such probes into a scanning microscope system we have been able to create time delay images of these signals. We suggest that future developments of this technique may lead to real time, high resolution imaging of digital pulses across complex very large scale integrated circuits.

Scanning near-field ellipsometric microscope-imaging ellipsometry with a lateral resolution in nanometer range

An apertureless optical near-field scanning microscope system has been created by combining a commercially available atomic force microscope and an ellipsometer without any prior changes in design of the respective devices. In preliminary experiments, an optical resolution of about 20 nm (λ/32) has been achieved using the combined microscope. The intensity of the measured optical signal has been found to be a periodic function of the thickness of the sample. Moreover, the period of this function is dependent upon the local optical properties of the sample material.

Effects of electroacupuncture on blood-brain barrier after cerebral ischemia-reperfusion in rat.

The effect of electroacupuncture (EA) on the blood-brain barrier (BBB) after cerebral ischemia-reperfusion (I-R) was investigated in rats. The dye Evans Blue (EB) was used as a tracer for assessing the disruption of BBB. Fluorescence quantification of EB was performed to explore the temporal pattern of permeability of BBB after the cerebral I-R with a fluorescence spectrophotometer (HITACHI 650-60). Furthermore, the morphology of BBB opening was detected under confocal laser scanning microscopy system. It was found that the BBB opening after cerebral I-R was biphasic. A rare scattered extravasation of EB was detected 2 hours after cerebral I-R. The EB extravasation reached its first peak at 6 h then decreased at 24 h and increased again at the time-point of 48h after cerebral I-R. EA can attenuate the disruption of BBB after cerebral I-R. EA could not only limit the area of extravasation of EB, but also reduce the concentration of extravasation of EB in the rat brain after cerebral I-R. The results indicated that one of the mechanisms of curative effect of EA on the cerebral ischemia might be due to its function of protecting the integrity of BBB.

Transepithelial migration of activated eosinophils induces a decrease of E-cadherin expression in cultured human nasal epithelial cells.

The damage of respiratory epithelium in allergic diseases has a close correlation with the extent of eosinophil infiltration. It seems to be a good possibility that eosinophil infiltration could induce the changes in the expression of the epithelial cell adhesion molecules, which play a key role in the maintenance of structural and functional rigidity of epithelium. We observed the expression of E-cadherin in cultured human nasal epithelial cells (HNECs) to study whether could it be affected by transepithelial migration of inflammatory cells, especially eosinophils. In vitro study of the transmigration assay was designed using various types of inflammatory cells and HNEC monolayers. Various assays of each experimental group were done under the stimulation of interleukin-5 (IL-5) and/or platelet activating factor (PAF). Subsequently immunohistochemistry for E-cadherin was performed in the HNECs. The intensity of immunofluorescence of E-cadherin was quantified using confocal laser scanning microscopy (CLSM) system and compared before and after the transmigration. The mean intensity of immunofluorescence for E-cadherin decreased significantly after the transmigration of any types of inflammatory cells. Above all, the migration of eosinophils treated with IL-5 and PAF had an eminent effect on the decrease, whereas the degranulation extracts derived from eosinophils activated by IL-5 and secretory IgA (sIgA) did not affect the intensity. This work suggests that transepithelial migration of inflammatory cells can directly induce the decrease in epithelial E-cadherin expression. Furthermore, the most prominent change was induced by transmigration of activated eosinophils, which might be caused by some mechanisms independent of the eosinophil contents. The decrease in E-cadherin expression may trigger the damage of epithelial barrier, which contributes to the pathogenesis of allergic diseases.

Low Level Measurement of Plutonium Content in Bioassay Using SSNTD Alpha-Spectrometry

Solid state nuclear track detectors (SSNTD) find wide application in registration of different types of ionization irradiation due to excellent features: low cost, compactness, passivity and possibility of long term exposure for low activity measurements. One of the most shortcomings is in complex processing of the detector surface and following image analysis. However, present methods which allow reconstruction of track ranges and particle energies are intensively being developed. A developing trend in SSNTD alpha-spectrometry uses automated scanning microscope systems and specially designed mathematical software. Several parameters obtained from the analysis of etched tracks indi information about alpha-particle ranges in detector. The possibility to use some parameters (minor axis m and track end diameter d) for the spectrometry has been reported earlier. Intercomparison of SSNTD with semiconductor detectors (SCD) for 238,239Pu content in bioassay within the interval 10-3 to 1 Bq has been carried out. This comparison has demonstrated a good coincidence between these independent radiometric methods. A perspective of SSNTD use for spectrometric determination of low level activity is discussed. The background level playing a main role in low level detection has been also estimated. Influence of radon and thoron has been found as insignificant.