Detection of doubly stained fluorescent specimens using confocal microscopy

Studies of doubly stained specimens were performed with a confocal scanning microscope. The instrument used provides the possibility of making separate detections of the fluorescent dyes. The optimal choice of excitation wavelengths and optical filters are discussed. The fluorphores that were used are Lucifer Yellow, Texas Red, fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate.     

The use of Lucifer yellow,bodipy, FITC,TRITC, RITC and Texas red for dual immunofluorescence visualized with a confocal scanning laser microscope

A new method of comparing the relative merits of different fluorophores that undergo relatively rapid irreversible photo-inactivation is described. This method showed that the levels of fluorescent emission seen with both fluorescein isothiocyanate (FITC) and bodipy fl conjugated to streptavidin were similar when examined under conditions where they exhibited equal rates of irreversible photo-inactivation. Bodipy fl and FITC give lower levels of cross-talk into images of cells immunofluorescently stained with either rhodamine isothiocyanate (RITC) or tetramethyl rhodamine isothiocyanate (TRITC) than into images of cells stained with Texas red, under conditions where the three red fluorophores exhibited an equal level of sensitivity. Furthermore, bodipy fl gave much lower levels of cross-talk into images of RITC-stained cells than either FITC or Lucifer yellow. TRITC, but not RITC or Texas red, gave significant levels of cross-talk into the green band-pass filters used to visualize FITC and bodipy fl. From these results it seems that a combination of bodipy fl and RITC provides the best contrast when visualizing dual immunofluorescence with a confocal scanning laser microscope if the 488-nm line of an argon ion laser is used as the excitation source.     

Observation of self-assembled fluorescent beads by scanning near-field optical microscopy and atomic force microscopy

Optical response and topography of fluorescent latex beads both on flat self-assembled monolayer and on a micron-patterned surface with poly(dimethylsiloxane) are studied. Scanning near-field optical microscopy and atomic force microscopy were utilized together for detecting fluorescence and imaging topography of the patterned latex beads, respectively. As a result, the micro-patterned latex beads where a specific chemical binding occurred show a strong signal, whereas no signals are observed in the case of nonspecific binding. With fluorescein isothiocyanate (FITC), it is convenient to measure fluorescence signal from the patterned beads allowing us to monitor the small balls of fluorescent latex.     

Method for localization of sialic acid on cell surface and cell interior in peripheral blood: a pilot study

This article presents a method for identification and localization of cell surface and intracellular sialoglycoconjugates of peripheral blood cells. To reveal cell surface conjugates, a sample of peripheral blood was incubated with lectin after centrifugation and rinsing. For intracellular localization in leukocytes, RBCs were lysed and the membranes were permeabilized prior to cytochemical reaction. Fluorescein isothiocyanate conjugated lectins were used for visualization in fluorescence microscope. All lectins bound specifically to the surface of erythrocytes. Confocal microscopy showed surface and intracellular labeling of permeabilized leukocytes. A part of the signal in eosinophils originated from binding of anionic fluorophore to cationic granular proteins.     

Double labeling of antibodies with fluorescein isothiocyanate (FITC) and horseradish peroxidase (HPOD)

This paper presents a method for labeling antibodies with fluorescein isothiocyanate (FITC) and additional with horseradish peroxidase (HPOD). With this double labelled antisera the fluorescence-serologic antibody technique (FAT) as well as the enzyme-serologic antibody technique (EAT) was done bit by bit for the same object. How the presented pictures demonstrate the result of both techniques, FAT and EAT, are the same with the described method. So, it is possible after fluorescence-microscopical exploitation through a following histochemical proof of the HPOD to get permanent preparations with the same biological result.     

固相微萃取气质联用测定百合(Lilium spp.)挥发性成分

运用固相微萃取-气相色谱-质谱法(SPME-GC-MS)测定百合中的挥发性成分。百合中鉴定出31种挥发性成分,主要为苯甲酸甲酯(26.6%)、3,7-二甲基-1,6-辛二烯-3-醇(14.2%)、3,4-二甲氧基甲苯(8.5%)、(Z)-3,7-二甲基-1,3,6-十八烷三烯(4.1%)和2-正戊基呋喃(3.8%)。     

Detection of injected fluorescence-conjugated IgG in living mouse organs using "in vivo cryotechnique" with freeze-substitution

In this experiment, we performed the "in vivo cryotechnique" in tandem with fluorescence microscopy. The fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin (IgG) antibody (FITC-IgG) was directly injected into mouse livers or kidneys, which were then frozen in vivo by pouring an isopentane-propane mixture (-193 degrees C) cooled in liquid nitrogen over these living organs. The organs were subsequently freeze-substituted in acetone containing paraformaldehyde at about -80 degrees C, then gradually brought up to a room temperature, infiltrated with 30% sucrose and refrozen. Some well-frozen areas 300-400 mum below the frozen tissue surface were cryocut into several slices. The slices were observed under the fluorescence microscope. By examining the distribution of FITC-IgG in the frozen livers, some aspects of functional blood circulation in the liver, such as the concept of the liver lobule, were reconfirmed. This also confirmed that the blood flow in the liver after the FITC-IgG injection was normal. The subsequent preparation of the specimens with immunohistochemistry, using the tetramethylrhodamine (TRITC)-conjugated anti-mouse IgG antibody, allowed us to visualize the localizations of both the original mouse IgG and the injected goat IgG in the cryosections with different color images. The experimental protocol presented demonstrates the in situ localization of the various proteins labeled with fluorescent probes, and it can, in conjunction with immunohistochemistry, localize proteins in cells and tissues.     

中药柴胡挥发性成分的静态顶空-气相色谱-质谱分析

运用静态顶空毛细管气相色谱质谱法对6种不同产地柴胡的挥发性成分进行了检测,并运用质谱检索和保留指数相结合的手段对检出的挥发性成分进行定性,6种柴胡中定性相对含量大于0.2%的挥发性成分60种。结果表明:柴胡的挥发性成分中脂肪醛类物质的含量最大,占总挥发性物质的25%～44%;萜类化合物占8.1%～17.3%;萜类衍生物占10.3%～37.0%。分析结果的相对标准偏差(RSD)在1.20%～7.02%。本法可用于柴胡挥发性化学成分的分析。     

沙漠绢蒿和冷蒿挥发油成分的气相色谱-质谱分析

利用水蒸汽蒸馏法提取沙漠绢蒿和冷蒿挥发油成分,采用气相色谱-质谱联用法分析鉴定出沙漠绢蒿和冷蒿植物的36种和54种化合物。沙漠绢蒿挥发油主要成分是醇类(24.71%)和酮类化合物(12.69%),其次是芳香族化合物（8.52%）;冷蒿挥发油主要成分是芳香族化合物(39.04％)、醇类(10.78%)和酮类化合物(6.91%)。     

血橙果实香气成分的气相色谱-质谱分析

采用顶空固相微萃取对血橙的香气成分进行提取,用两个不同极性的色谱柱（HP-5和 DB-WAX）进行分离,利用气相色谱-质谱联用技术对香气成分进行分析。在DB-WAX柱上鉴定出32种组分,HP-5柱共鉴定出45种主要化合物,占总峰面积的71.96%,主要成分为：柠檬烯（56.6%）、乙醇（4.24%）、巴伦西亚桔烯（2.06%）、β-月桂烯（1.73%）、丁酸乙酯（0.92%）、反式-2-己烯醛（0.75%）。HP-5柱更适合血橙香气成分的分离。