Kao H P, Verkman A S
Department of Medicine and Physiology, University of California, San Francisco 94143-0521.
Biophys J. 1994 Sep;67(3):1291-300. doi: 10.1016/S0006-3495(94)80601-0.
We present a novel optical technique for three-dimensional tracking of single fluorescent particles using a modified epifluorescence microscope containing a weak cylindrical lens in the detection optics and a microstepper-controlled fine focus. Images of small, fluorescent particles were circular in focus but ellipsoidal above and below focus; the major axis of the ellipsoid shifted by 90 degrees in going through focus. Particle z position was determined from the image shape and orientation by applying a peak detection algorithm to image projections along the x and y axes; x, y position was determined from the centroid of the particle image. Typical spatial resolution was 12 nm along the optical axis and 5 nm in the image plane with a maximum sampling rate of 3-4 Hz. The method was applied to track fluorescent particles in artificial solutions and living cells. In a solution of viscosity 30 cP, the mean squared distance (MSD) traveled by a 264 nm diameter rhodamine-labeled bead was linear with time to 20 s. The measured diffusion coefficient, 0.0558 +/- 0.001 micron2/s (SE, n = 4), agreed with the theoretical value of 0.0556 micron2/s. Statistical variability of MSD curves for a freely diffusing bead was in quantitative agreement with Monte Carlo simulations of three-dimensional random walks. In a porous glass matrix, the MSD data was curvilinear and showed reduced bead diffusion. In cytoplasm of Swiss 3T3 fibroblasts, bead diffusion was restricted. The water permeability in individual Chinese Hamster Ovary cells was measured from the z movement of a fluorescent bead fixed at the cell surface in response osmotic gradients; water permeability was increased by > threefold in cells expressing CHIP28 water channels. The simplicity and precision of this tracking method may be useful to quantify the complex trajectories of fluorescent particles in living cells.
我们提出了一种新颖的光学技术，用于使用改进的落射荧光显微镜对单个荧光粒子进行三维跟踪，该显微镜在检测光学系统中包含一个弱柱面透镜和一个微步进控制的精细聚焦装置。小荧光粒子的图像在聚焦时呈圆形，但在聚焦之上和之下呈椭圆形；椭圆的长轴在通过焦点时会旋转90度。通过对沿x轴和y轴的图像投影应用峰值检测算法，从图像形状和方向确定粒子的z位置；x、y位置则由粒子图像的质心确定。典型的空间分辨率在光轴上为12纳米，在图像平面中为5纳米，最大采样率为3 - 4赫兹。该方法应用于跟踪人工溶液和活细胞中的荧光粒子。在粘度为30厘泊的溶液中，直径为264纳米的罗丹明标记珠子移动的平均平方距离（MSD）在20秒内与时间呈线性关系。测得的扩散系数为0.0558±0.001微米²/秒（标准误差，n = 4），与理论值0.0556微米²/秒相符。自由扩散珠子的MSD曲线的统计变异性与三维随机游走的蒙特卡罗模拟在定量上一致。在多孔玻璃基质中，MSD数据呈曲线状，表明珠子扩散减少。在瑞士3T3成纤维细胞的细胞质中，珠子扩散受到限制。通过固定在细胞表面的荧光珠子响应渗透梯度的z移动来测量单个中国仓鼠卵巢细胞的水渗透性；在表达CHIP28水通道的细胞中，水渗透性增加了三倍以上。这种跟踪方法的简单性和精确性可能有助于量化活细胞中荧光粒子的复杂轨迹。
