Measurement of wheat germ agglutinin binding with a fluorescence microscope
Measurement of wheat germ agglutinin binding with a fluorescence microscope
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用荧光显微镜测量小麦胚芽凝集素结合力
DOI：
10.1002/cyto.a.20787
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
G. Fraizer
中科院分区：
文献类型：
--
作者：
M. Model;Jennifer L. Reese;G. Fraizer
荧光显微镜中的信号强度通常相对于任意标准来测量。我们提出了一种校准方法的基础上的解决方案相同的荧光团，其结合到细胞需要进行量化。该方法利用均匀材料的宽视场成像中强度对物距的低灵敏度。通过将球形透镜浸入放置在载玻片上的荧光团液滴中来制备缓慢变化深度的液体层。接触和周围区域的平场校正图像显示灰度与荧光液体深度的线性相关性。这允许将测量的强度转换为每单位面积的分子数量。将该方法应用于通过WGA-Alexa 488和WGA-TRITC染色的不同细胞类型。通过比较显微术与流式细胞术、比较通过不同物镜的成像以及比较不同的WGA缀合物，获得了一致的结果。当使用低放大倍数时，校准的重现性在97%以内。然而，发现游离和结合的WGA的荧光是不同的，因此在这个特定的系统中精确测量细胞结合分子的数量是有问题的。我们的结论是，该方法实现了可靠的测量细胞染色的可溶性荧光团的单位。对于荧光特性不受结合影响的探针，应该可以以真实分子单位定量染色。© 2009国际细胞计数促进学会
Signal intensity in fluorescence microscopy is often measured relative to arbitrary standards. We propose a calibration method based on a solution of the same fluorophore, whose binding to cells needs to be quantified. The method utilizes the low sensitivity of intensity to the object distance in wide‐field imaging of uniform materials. Liquid layers of slowly varying depth were prepared by immersing a spherical lens into a drop of a fluorophore placed on a slide. Flatfield‐corrected images of the contact and surrounding areas showed linear dependence of the gray level on the depth of fluorescent liquid. This allowed conversion of the measured intensity into the number of molecules per unit area. The method was applied to different cell types stained by WGA‐Alexa 488 and WGA‐TRITC. Consistent results were obtained by comparing microscopy with flow cytometry, comparing imaging through different objectives and comparing different WGA conjugates. Reproducibility of calibration was within 97% when low magnification was used. Fluorescence of free and bound WGA was found to be different, however, and therefore precise measurement of the number of cell‐bound molecules was problematic in this particular system. We conclude that the method achieves reliable measurement of cellular staining in the units of soluble fluorophore. For probes whose fluorescent properties are unaffected by binding, quantification of staining in true molecular units should be possible. © 2009 International Society for Advancement of Cytometry
通过基于 GFP 的校准技术确定单个突触的绝对数量
DOI：
--
发表时间：
2005
期刊：
Nature Methods 2
影响因子：
--
作者：
Sugiyama Y.;et. al.
通讯作者：
et. al.