TRACKING OF SINGLE FLUORESCENT PARTICLES IN 3 DIMENSIONS - USE OF CYLINDRICAL OPTICS TO ENCODE PARTICLE POSITION
TRACKING OF SINGLE FLUORESCENT PARTICLES IN 3 DIMENSIONS - USE OF CYLINDRICAL OPTICS TO ENCODE PARTICLE POSITION
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DOI：
10.1016/s0006-3495(94)80601-0
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发表时间：
1994-09-01
影响因子：
3.4
通讯作者：
VERKMAN, AS
中科院分区：
文献类型：
--
作者：
KAO, HP;VERKMAN, AS
我们提出了一种新颖的光学技术，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器件中包含弱柱面透镜和微步进控制的精细焦点。小荧光颗粒的图像在焦点处呈圆形，但在焦点上方和下方呈椭圆形；椭球体的长轴在通过焦点时移动了 90 度。通过将峰值检测算法应用于沿 x 和 y 轴的图像投影，根据图像形状和方向确定粒子 z 位置； x、y 位置由粒子图像的质心确定。典型的空间分辨率沿光轴为 12 nm，在图像平面中为 5 nm，最大采样率为 3-4 Hz。该方法用于追踪人工溶液和活细胞中的荧光颗粒。在粘度为 30 cP 的溶液中，直径为 264 nm 的罗丹明标记珠移动的均方距离 (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 μ m(2)/s（SE，n = 4），与理论值 0.0556 μ m(2)/s 一致。自由扩散珠的 MSD 曲线的统计变异性与三维随机游走的 Monte Carte 模拟定量一致。在多孔玻璃基质中，MSD 数据呈曲线并显示珠扩散减少。在 Swiss 3T3 成纤维细胞的细胞质中，珠扩散受到限制。通过固定在细胞表面的荧光珠响应渗透梯度的 z 轴运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。
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 mu m(2)/s (SE, n = 4), agreed with the theoretical value of 0.0556 mu m(2)/s. Statistical variability of MSD curves for a freely diffusing bead was in quantitative agreement with Monte Carte 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.