荧光显微镜在录井中的应用探讨

荧光显微技术作为把烃类和岩石显微结构构造直接结合观察的手段,可在许多方面解决现场地质录井的疑难问题。文中分析了把这一技术应用于录井工作的必要性和可行性,并简要介绍了该技术在录井工作中的一些应用方法。

Relationship Between Leaf Structure and Aloin Content in Six Species of Aloe L.

The leaf structure, content and the storage location of aloin in the leaves of six species of Aloe L. were studied by means of semi-thin section, high performance liquid chromatography (HPLC) and fluorescent microscope. Results showed that all leaves consisted of epidermis, chlorenchyma, aquiferous tissue and vascular bundles. The leaves had the xeromorphic characteristics, including thickened epidermal cell wall, thickened cuticle, sunken stomata and well-developed aquiferous tissue. With the exception of thus, there were remarkable differences in leaf structure among the six species. The chlorenchyma cells were similar to palisade tissues in Aloe arborescens Mill. and A. mutabilis Pillans, but isodiametric in A. vera L., A. vera L. var. chinensis Berg., A. saponaria Hawer and A. greenii Bali. A. arborescens, A. mutabilis, A. very and A. vera var. chinensis included large parenchymatous cells at the vascular bundles, whereas no such cells were observed at the vascular bundles of A. saponaria and A. greenii. In A. arborescens, A. mutabilis and A. vera, the aquiferous tissue sheaths were present and composed of a layer of small parenchymatous cells without chloroplasts around the aquiferous tissue. While there were no aquiferous tissue sheaths in A. vera var. chinensis, A. saponaria and A. greenii. The HPLC revealed that the content of aloin was high in A. arborescens, low in A. vera, and very low in A. saponaria among the six species. The fluorescent microscopy showed that the yellow-green globule only appeared in the large parenchymatous cells of vascular bundles, vascular bundle sheath and aquiferous tissue sheath, but not in the chlorenchyma and aquiferous tissue. Consequently, the large parenchymatous cells of vascular bundles, vascular bundle sheath and aquiferous tissue sheath were the storage location of aloin. They were positively correlated with the content of aloin.

Observation of Insulin Exocytosis by a Pancreatic β Cell Line with Total Internal Reflection Fluorescence Microscopy

INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.

Chromosome Behavior of Heat Shock Induced Triploid in Fenneropenaeus Chinensis

Triploidy was induced in Chinese shrimp Fenneropenaeus chinensis by 30±0.5℃heat shock treatment (initiated at 20 min after fertilization) for 10 min to inhibit the release of PB2 at 18.0℃. The highest triploid rate obtained was 84.5% in nauplius stage. The effect of heat shock treatment on meiosis and cleavage of eggs was investigated in this work aimed to establish efficient procedures for triploid induction and to gain understanding of the mechanism of triploid production. Three pronuclei that could be observed in the treated eggs under fluorescence microscope developed into triploid embryos. Some abnormal chromosome behavior was observed in heat shocked eggs.

Effects of Res on proliferation and apoptosis of human cervical carcinoma cell lines C33A,SiHa and HeLa

Objective:To explore the effects of Res on the proliferation and apoptosis of human cervical cancer cell lines C33A,SiHa and HeLa.Methods:Inhibition rates by different concentrations of Res were calculated using MTT method.Apoptosis rates and cell cycles were measured and examined by flow cytometry(FCM).Morphological configuration of apoptotic cells were observed under the fluorescence microscope.Results:The growth of cancer cells was inhibited by Res of varied concentrations in a time-and dose-dependent manner(P<0.01).The cells showed characteristic apoptosis morphologic changes under fluorescence microscope.Res exerted no effects on cell cycles.Conclusion:Res inhibits the growth of cervical cell lines C33A,SiHa and HeLa by inducing cell apoptosis in a time-and dose-dependent manner.

Time-resolved two-photon excitation fluorescence spectroscopy and microscopy using a high repetition rate streak camera

We present a time-resolved two-photon excitation fluorescence spectroscopy and a simultaneous time- and spectrum- resolved multifocal multiphoton microscopy system that is based on a high repetition rate picosecond streak camera for providing time- and spectrum- resolved measurement and imaging in biomedicine. The performance of the system is tested and characterized by the fluorescence spectrum and lifetime analysis of several standard fluorescent dyes and their mixtures. Spectrum-resolved fluorescence lifetime images of fluorescence beads are obtained. Potential applications of the system include clinical diagnostics and cell biology etc.

Characterization and recycling of nickel- and chromium-contained pickling sludge generated in production of stainless steel

Pickling sludge generated during the neutralization of pickling wastewater with calcium hydroxide in stainless steel pickling process was characterized using X-ray fluorescence spectrometry, X-ray diffractometry, scanning electron microscopy, thermogravimetry and differential scanning calorimetry, etc. The major compositions of pickling sludge are CaF2, CaSO4, Me（OH）, （M： Fe, Cr, Ni）, and the content of CaF2 is high in the sludge. The melting point of pickling sludge is about 1350℃ and the viscosity is about 0.14 Pa.s at 1450 ℃, which are comparatively lower than those of normal refining slag. After heat treatment, the contents of sulfur and fluorine in the pickling sludge were reduced, confirming the thermal decomposition of sulfate in the sludge. Fluorine in the sludge is reduced by the gaseous SiF4 and A1F3 generated through the reactions of CaF2 with SiO2 and Al2O3. The preliminary results from the reduction test indicate that the sulfur content in the steel is not affected by the presence of sulfur in the sludge. The recovery of nickel is about 40%, and the chromium content changes marginally due to the protective atmosphere under the reduction condition of chromic oxide. The pickling sludge is a potential auxiliary material for the production of stainless steel.

Three-dimensional tracking of GLUT4 vesicles in TIRF microscopy

TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.In this paper,we present a new tracking algorithm combined with the unique features of TIRF.Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time.Mobile granules were then segmented from a time-lapse image stack by an adaptive background subtraction method.Kalman filter was introduced to estimate and track the granules that allowed reducing searching range and hence greater reliability in tracking process.After the tracked granules were located in x-y plane,the z-position was indirectly inferred from the changes in their intensities.In the experiments the algorithm was applied in tracking GLUT4 vesicles in living adipose cells.The results indicate that the algorithm has achieved robust estimation and tracking of the vesicles in three dimensions.

Molecular Determinants Responsible for the Subcellular Localization of HSV-1 UL4 Protein

The function of the herpes simplex virus type 1 （HSV-1） UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study, fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein （EYFP）, the nuclear export signals （NES） of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition, the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattem of UL4. Furthermore, the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1 （CRM1）-dependent manner involving RanGTP hydrolysis

Identification of the Epitopes of Monoclonal Antibodies against P74 of Helicoverpa armigera Nucleopolyhedrovirus

P74 is a per os infectivity factor of baculovirus.Here,we report the production of three monoclonal antibodies (mAbs),denoted as 20D9,20F9 and 21E1,raised against P74 of Helicoverpa armigera nucleopolyhedrovirus (HearNPV),and the identification of their recognition epitopes.The full-length P74,without the transmembrane domains at the C-terminus,was first divided into three segments (N,M and C,respectively),based on the proposed cleavage model for the protein,which were then expressed individually.Western blot analyses revealed specific cross-reactions with the N fragment,for both 20D9 and 21E1.Extensive truncation,followed by prokaryotic expression,of the P74 N fragment was then performed in order to screen for linear epitopes of P74.The recognition regions of 20D9 and 21E1 were revealed to be localized at R144-T153 and T199-C219,respectively.In addition,immunofluorescence microscopy indicated that 20D9 and 20F9 could recognize native P74 in HearNPV-infected cells.These findings will facilitate further investigations of the proteolytic processing of HearNPV P74,and of its involvement in virus-host interactions.

Cxcl16 Interact With SARS-CoV N Protein In and Out Cell

Our study investigated the host cell protein which can interact with SARS-CoV N protein, and explored the functional connections. The eukaryotic expression vectors pEGFP-N1/SARS-CoVN and pdsRed2-N1/ CXCL16 were constructed and used to co-transfect HEK293FT cells by the calcium phosphate method. The HIS-tagged fusion protein SARS-CoVN-GFP was then built and purified for the binding assay in vitro. The co-localization of SARS-CoVN and CXCL16 in the cytoplasm of HEK293FT cells was also shown using confocal laser scanning microscopy. It is suggested that their interaction might be through direct combination. Under a fluorescence microscope, it was observed that the purified fusion protein SARS-CoVN-GFP was attached to the cell membrane of CXCL16-transfected cells, indicating that SARS-CoVN and CXCL16 can be mutually combined.

Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

Study of the imaging property of a fluorescent confocal microscopy with a phase-only filter in an extended source

The phase information of an enlarged source is reconstructed with an annular two-zone phase-only filter in a fluorescent confocal scanning optical microscope for resolution improvement. The dependences of its resolution on the source size and on the phase transmission of the outer annular zone of the falter are investigated theoretically by use of its three-dimensional optical transfer function （3D OTF ）. The increased source size and the required phase value of the outer annular zone of the phase-only falter for an optimal 3D OTF of the optical system are presented.

Antinecrotic and Antiapoptotic Effects of Different Concentrations of Copaiferalangsdorffii Essential Oil in Macrophages and Lymphocytes of Mice

Purpose： to evaluate the in vitro influence of Copaiferalangsdorffii essential oil on the antinecrotic activity in macrophages, and on the antiapoptotic activity in macrophages and lymphocytes of Swiss mice. Methods： lymphocytes and macrophages were obtained from ten 60-day-old male mice weighing 42-45 g, and then cultured in 24-well plates. Cells were treated with concentrations of 2%, 5%, 10% and 20% of oil from Copaiferalangsdorffii before or after induction of apoptosis with ultraviolet light. We used positive and negative controls. Hoechst staining was performed, and then images of cells under fluorescence microscope were captured and digitalized. We used Image J_1.46o to quantify the fluorescence intensity/brightness of condensation of the chromatin ofapoptotic cells. We used one-way ANOVA with Bonferroni＇s and Tukey＇s multiple comparison tests. Results are expressed as the means of relative densities after adjustment and 95% confidence intervals; P 〈 0.05 was considered statistically significant. Results： oil from Copaiferalangsdorffii at different concentrations has decreased necrosis and apoptosis of both lymphocytes and macrophages from mice, when compared to C＋ （all P 〈 0.05）. Discussion： Copaiferalangsdorffii is typically Brazilian and found in the Amazon region, with anti-inflammatory, antioxidant and antineoplastic effects due to its oil compounds, such as xyloglucans and kaurenoic acid. Its potential therapeutical usage was demonstrated in a national study. The present study showed that its oil has significant in vitro antiapoptotic and antinecrotic activity and it should be tested experimentally in vivo.

Intrahepatic transplantation of hepatic oval cells for fulminant hepatic failure in rats

AIM： To evaluate the effect of intrahepatic transplantation of hepatic oval cells （HOC） on fulminant hepatic failure （FHF） in rats.METHODS： HOC obtained from rats were labeled with green fluocescent protein （GFP） or 5, 6- carboxyfluorescein diacetate succinmidyl ester （CFDASE）. Cell fluorescence was observed under fluorescent microscope at 6, 24, 48 and 72 h after labeling. CFDA- SE labeled HOC （5 × 10^6 cells each rat） were injected into livers of rats with FHF induced by D-galactosamine. Serum albumin （ALB）, alanine aminotransferase （ALT）, aspartate aminotransferase （AST） and total bilirubin （TBil） levels were measured at different time points. Liver function of rats was examined on days 3, 7, 14 and 21 after HOC transplantation.RESULTS： The positive rate of GFP and CFDA-SE labeled HOC was 10% and 90%, respectively, with no significant change in cell viabilities. The survival rate was higher in HOC transplantation group than in control group, especially 48 （9/15 vs 6/15） and 72 h （9/15 vs 4/15） after HOC transplantation. The serum ALT, AST and TBil levels were decreased while the serum AIb level was increased after HOC transplantation. Fluorescence became faded and diffused in liver tissues, suggesting that proliferation and differentiation occur in transplanted HOC.CONCLUSION： CFDA-SE is superior to GFP in labeling HOC, although fluorescence intensity is decreased progressively with cell division. HOC transplantation can improve the liver function and increase the survival rate of recipients.

Mitosis and microtubule organizational changes in rice root-tip cells

The pattern of change of the microtubule cytoskele-ton of the root-tip cells of rice during mitosis was studied using immunofluorescence technic and confocal laser scanning microscopy. All the major stages of cell division including preprophase, prophase, metaphase, anaphase and telophase were observed. The most significant finding was that in the preprophase cells microtubules radiating from the nuclear surface to the cortex were frequently seen. During development these microtubules became closely associated with the preprophase band and prophase spindle indicating that the microtubules radiating from the nuclear surface, the preprophase band and the prophase spindle were structurally and functionally closely related to each other. Granule-like anchorage sites for the radiating microtubules at the nuclear surface were often seen and the possibility that these granule-like anchorage sites might represent the microtubule organizing centres was discussed.

Localization and Functional Analysis of SeMNPV IE1 in Mammalian Cells

In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus （SeMNPV）, belonging to group II nucleopolyhedrovirus, was studied in mammalian cells We amplified the SeMNPV iel gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn＇t influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.

Liquidus and phase equilibria in CaO-SiO2-5%MgO-20%Al2O3-TiO2 system

The single hot thermocouple technique （SHTT） and high temperature equilibrium technique were combined to investigate the phase diagram of the CaO-SiO2-5%MgO-20%AlzO3-TiO2 system. The 1300 ℃ to 1500 ℃ liquidus lines are calculated according to the thermodynamic equations based on the pseudo-melting temperatures measured by the single hot thermocouple technique. The phase equilibria relationships are experimentally determined at 1400 ℃ using the high temperature equilibria technique followed by X-ray fluorescence （XRF）, X-ray diffraction（XRD）, scanning electron microscopy （SEM） and energy dispersive X-ray spectroscopy （EDX） analysis. The liquid phase（L）, melilite solid solution phase （（C2MSz,C2AS）ss）, diopside phase（CMS2） and perovskite phase （CaO·TiO2） are found. Coupled with the liquidus lines and equilibria results, the phase diagram is constructed for the specified region of the CaO-SiO2-5%MgO-20%Al2O3-TiO2 system.

Three-Dimensional Sub-100 nm Super-Resolution Imaging of Biological Samples Using a Phase Ramp in the Objective Pupil

Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional （3D） localisation microscopy by engineering of the point-spread function （PSF） of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions （standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively）. The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications.

Adiponectin levels are associated with the number and activity of circulating endothelial progenitor cells in patients with coronary artery disease

Objective:To study the relationship between plasma adiponectin concentration and the functional activities of circulating endothelial progenitor cells(EPCs) in patients with coronary artery disease(CAD).Methods:Circulating EPCs were enumerated as AC133+/KDR+ cells via flow cytometry and identified by co-staining with DiI-acLDL and fluorescein isothiocy-anate(FITC)-conjugated lectin under a fluorescent microscope.The migratory capacity of EPCs was measured by modified Boyden chamber assay.Adhesion capacity was performed to count adherent cells after replating EPCs on six-well culture dishes coated with fibronectin.Results:The number of circulating EPCs(AC133+/KDR+ cells) decreased significantly in CAD patients,compared with control subjects [(74.2±12.3) vs(83.5±12.9) cells/ml blood,P<0.01].In addition,the number of EPCs also decreased in CAD patients after ex vivo cultivation [(54.4±8.6) vs(71.9±11.6) EPCs/field,P<0.01].Both circulating EPCs and differentiated EPCs were positively correlated with plasma adiponectin concentration.The functional activities of EPCs from CAD patients,such as migratory and adherent capacities,were also impaired,compared with control subjects,and positively correlated with plasma adiponectin concentration.Conclusion:The study demonstrates that the impairment of the number and functional activities of EPCs in CAD patients is correlated with their lower plasma adiponectin concentrations.