Topography of cell-glass apposition revealed by total internal reflection fluorescence of volume markers.
Topography of cell-glass apposition revealed by total internal reflection fluorescence of volume markers.
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DOI：
10.1083/jcb.100.4.1334
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发表时间：
1985-04
期刊：
影响因子：
--
通讯作者：
Bailey J
中科院分区：
文献类型：
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作者：
Gingell D;Todd I;Bailey J
我们已经开发了一种基于全内反射荧光的新方法来绘制玻璃和散布在玻璃上的活细胞的下表面之间区域的形状。荧光标记的非吸附体积标记分子不能渗透到细胞中，局部刺激使它们仅在玻璃/介质界面附近发出荧光。在细胞下任意点的总荧光强度取决于细胞与玻璃的分离。由于染料排斥，焦点接触出现为暗区域，而当间隙超过约150 nm时，荧光渐近线到明亮的背景水平。我们的技术提供了比干涉反射显微镜更大的对比度，并且没有由于细胞质厚度和细胞质内含物引起的折射率不均匀而产生的误差。我们已经证明，足够大的分子在小分子可进入的区域遭受空间排斥，这为相对区域的侧向穿透性提供了新的信息。
We have developed a new method based on total internal reflection fluorescence to map the shape of the region between glass and the lower surface of a living cell spread upon it. Fluorescently labeled nonadsorbing volume marker molecules that cannot penetrate into the cell are locally stimulated so that they fluoresce only very near the glass/medium interface. The total fluorescence intensity at any point beneath the cell depends on the cell-to-glass separation. Focal contacts appear as dark areas owing to dye exclusion, whereas when the gap exceeds approximately 150 nm, fluorescence asymptotes to the bright background level. Our technique provides greater contrast than does interference reflection microscopy and is free from errors due to cytoplasmic thickness and refractive index inhomogeneities arising from cytoplasmic inclusions. We have shown that sufficiently large molecules suffer steric exclusion from regions accessible to small molecules, which gives new information about lateral penetrability in the apposition region.