Stimulation of fluorescence in a small contact region between rat basophil leukemia cells and planar lipid membrane targets by coherent evanescent radiation.
Stimulation of fluorescence in a small contact region between rat basophil leukemia cells and planar lipid membrane targets by coherent evanescent radiation.
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通过相干瞬逝辐射刺激大鼠嗜碱性粒细胞白血病细胞和平面脂质膜靶标之间的小接触区域中的荧光。
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发表时间：
1982
期刊：
影响因子：
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通讯作者：
H. Mcconnell
中科院分区：
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作者：
R. Weis;K. Balakrishnan;B. Smith;H. Mcconnell
我们已经包括了脂质半抗原的脂质单层烷基化石英显微镜载玻片上涂覆作为特定的抗体依赖性大鼠嗜碱性白血病细胞的目标。在室温下，仅当单层含有脂质半抗原且仅在Mg 2+和/或Ca 2+存在下，装备有特异性单克隆IgE抗体的嗜碱性粒细胞才紧紧粘附于单层。嗜碱性粒细胞在这些条件下发生特异性IgE和半抗原依赖性免疫脱颗粒。当在石英载玻片内经历全内反射的激光辐射激发时，与嗜碱性粒细胞上的Fc受体结合并位于单层靶的~100 nm内的标记IgE抗体发荧光。由在石英载玻片内具有全内反射的两个聚焦的相交相干激光束产生的光学干涉图案用作控制，以确保从IgE分子发射的所有荧光辐射是由于倏逝辐射而不是散射辐射的刺激。干涉图案还用于图案光漂白实验，以确定在没有细胞的情况下与单层结合的IgE分子的侧向扩散速率。嗜碱性粒细胞对单层靶的特异性抗体依赖性粘附导致倏逝波场中IgE荧光显著增强。在嗜碱性粒细胞膜-单层膜接触区域的荧光IgE分子的荧光显微镜检查揭示了一种极其复杂的模式，推测是由于细胞丝状伪足样结构通过同时结合到Fc受体和脂质半抗原的IgE分子锚定到靶膜。这些结构是不可观察到的与传统的落射荧光显微镜由于荧光的IgE分子结合的嗜碱性粒细胞的区域以外的界面之间的单层和嗜碱性细胞膜。接触区中IgE分子的浓度似乎很高，可能导致Fc-Fc受体复合物形成或Fc受体的其他再分布，为嗜碱性粒细胞脱粒提供信号。
We have included a lipid hapten in lipid monolayers coated on alkylated quartz microscope slides to serve as specific antibody-dependent targets for rat basophil leukemia cells. At room temperature basophils armed with specific monoclonal IgE antibodies adhere tightly to the monolayer only when the monolayers contain lipid hapten and only in the presence of Mg2+ and/or Ca2+. The basophils undergo specific IgEand haptendependent immunologic degranulation under these conditions. Fluoresceinated IgE antibodies bound to Fc receptors on the basophil and located within -100 nm of the monolayer target fluoresce when excited by laser radiation undergoing total internal reflection within the quartz slide. An optical interference pattern produced by two focused intersecting coherent laser beams having total internal reflection within the quartz slide serves as a control to ensure that all of the emitted fluorescence radiation from the IgE molecules is due to stimulation by evanescent radiation and not by scattered radiation. The interference pattern is also used for pattern photobleaching experiments to determine the rates of lateral diffusion of IgE molecules bound to the monolayers, in the absence of cells. Specific antibody-dependent adherence of basophils to the monolayer target results in a marked enhancement of IgE fluorescence in the field of the evanescent wave. Microscopic examination of the fluorescence of the fluoresceinated IgE molecules in the region of basophil membrane-monolayer membrane contact reveals an extremely complex pattern, presumably due to cellular filopodia-like structures that are anchored to the target membrane via IgE molecules bound simultaneously to Fc receptors and lipid haptens. These structures are not observable with conventional epifluorescence microscopy due to the fluorescence of IgE molecules bound to the basophils in regions other than the interface between the monolayer and basophil membranes. The concentration of IgE molecules in the contact region appears to be high and could give rise to Fc-Fc receptor complex formation or other redistributions of Fc receptors that provide signals for basophil degranulation.