Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy
Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy
复制标题
DOI：
10.1016/s0006-3495(97)78312-7
复制
发表时间：
1997-11-01
影响因子：
3.4
通讯作者：
Verkman, AS
中科院分区：
文献类型：
--
作者：
Olveczky, BP;Periasamy, N;Verkman, AS
在介电界面外，消失场强度的衰减取决于光束入射角，因此可以从多个入射角下获得的全内反射荧光显微镜（TIRFM）图像中推断出荧光团的三维分布。采用直角F2玻璃棱镜（n(r) 1.632）创建介电界面，构建了计算机自动化多角度TIRFM （MA-TIRFM）仪器。激光束（488 nm）经声光调制器衰减后照射到棱镜表面的指定点上。光束入射角由三个微步进电机控制两个可旋转反射镜和一个可旋转光学鼠来设定，从临界角开始，以> - 15的入射角以类似0.5度的步长获取TIRFM图像。对于细胞研究，将细胞直接生长在玻璃棱镜上（不含折射率匹配液）并放置在光路中，从多个角度获取样品图像。并使用用荧光团溶液代替样品获得的“参考”图像来校正与角度相关的消失场强。通过角分辨强度函数的拉普拉斯逆变换，建立了计算荧光团z分布的理论。该理论包括分析多层不同折射率的细胞研究，以及介电界面附近荧光团的各向异性发射，通过绘制F2玻璃棱镜和钢琴凸硅透镜（半径458 mm， n(r) 1.463）之间的二己烷碳蓝膜在DMSO/水中的厚度（n(r) 1.463）来验证仪器性能；MA-TIRFM图精确地再现了透镜球面。MA-TIRFM用于比较bcecf标记的3T3成纤维细胞与MDCK上皮细胞的细胞-底物接触几何形状的纳米z分辨率，这些研究建立了MA-TIRFM用于测量荧光探针与细胞膜之间的亚微观距离。
The decay of evanescent field intensity beyond a dielectric interface depends upon beam incident angle, enabling the 3-d distribution of fluorophores to be deduced from total internal reflection fluorescence microscopy (TIRFM) images obtained at multiple incident angles, Instrumentation was constructed for computer-automated multiple angle-TIRFM (MA-TIRFM) using a right angle F2 glass prism (n(r) 1.632) to create the dielectric interface. A laser beam (488 nm) was attenuated by an acoustooptic modulator and directed onto a specified spot on the prism surface. Beam incident angle was set using three microstepper motors controlling two rotatable mirrors and a rotatable optical Rat, TIRFM images were acquired by a cooled CCD camera in similar to 0.5 degree steps for >15 incident angles starting from the critical angle, For cell studies, cells were grown directly on the glass prisms (without refractive index-matching fluid) and positioned in the optical path, Images of the samples were acquired at multiple angles, and corrected for angle-dependent evanescent field intensity using ''reference'' images acquired with a fluorophore solution replacing the sample, A theory was developed to compute fluorophore z-distribution by inverse Laplace transform of angle-resolved intensity functions. The theory included analysis of multiple layers of different refractive index for cell studies, and the anisotropic emission from fluorophores near a dielectric interface, Instrument performance was validated by mapping the thickness of a film of dihexyloxacarbocyanine in DMSO/water (n(r) 1.463) between the F2 glass prism and a piano-convex silica lens (458 mm radius, n(r) 1.463); the MA-TIRFM map accurately reproduced the lens spherical surface. MA-TIRFM was used to compare with nanometer z-resolution the geometry of cell-substrate contact for BCECF-labeled 3T3 fibroblasts versus MDCK epithelial cells, These studies establish MA-TIRFM for measurement of submicroscopic distances between fluorescent probes and cell membranes.