Berland K M, So P T, Gratton E
Department of Physics, University of Illinois at Urbana-Champaign 61801.
Biophys J. 1995 Feb;68(2):694-701. doi: 10.1016/S0006-3495(95)80230-4.
We report on the application of two photon molecular excitation to fluorescence correlation spectroscopy. We demonstrate the first fluorescence correlation spectroscopy measurements of translational mobility in the cytoplasm of living cells. Two-photon excitation inherently excites small sample volumes in three dimensions, providing depth discrimination similar to confocal microscopy, without emission pinholes. We demonstrated accurate measurements of the diffusion constant, D, for particles of several different known sizes, in bulk solutions of different viscosity. We then showed measurements of translational diffusion for 7- and 15-nm radius latex beads in the cytoplasm of mouse fibroblast cells. We measured time-dependent diffusion coefficients. When first injected in the cells, the spheres moved from two to five times slower than in water, with average rates of 18 x 10(-8) cm2/s for the 7 nm and 5 x 10(-8) cm2/s for the 15 nm radius spheres. After a few hours, spheres stick to the cells, and the motion slows down 10 to 100 times.
我们报告了双光子分子激发在荧光相关光谱学中的应用。我们展示了首次对活细胞细胞质中平移流动性进行的荧光相关光谱测量。双光子激发本质上能在三维空间中激发小的样品体积，提供类似于共聚焦显微镜的深度分辨能力，且无需发射针孔。我们在不同粘度的本体溶液中，对几种不同已知大小的粒子的扩散常数D进行了精确测量。然后我们展示了对小鼠成纤维细胞细胞质中半径为7纳米和15纳米的乳胶珠的平移扩散测量。我们测量了随时间变化的扩散系数。刚注入细胞时，这些球体的移动速度比在水中慢两到五倍，7纳米半径球体的平均速率为18×10⁻⁸平方厘米/秒，15纳米半径球体的平均速率为5×10⁻⁸平方厘米/秒。几小时后，球体附着在细胞上，运动速度减慢10到100倍。
