Sun Wei, Xu Aoshuang, Marchuk Kyle, Wang Gufeng, Fang Ning
Ames Laboratory, U.S. Department of Energy, Iowa State University, Ames, IA 50011, USA.
J Lab Autom. 2011 Aug;16(4):255-62. doi: 10.1016/j.jala.2010.10.001. Epub 2011 Apr 1.
An automatic calibration and angle-scanning prism-type total internal reflection fluorescence microscope (TIRFM) was modified to function in both TIRFM and pseudo-TIRFM modes. When the incident angle of the excitation laser beam was controlled to be larger than the critical angle, the instrument served as a variable-angle TIRFM. A homemade computer program automatically calibrates the laser illumination spot in the sample to overlap with the center of the microscope's field of view. Then, by measuring the fluorescence intensities at different incident angles, the z-positions of fluorescent nanospheres close to the cell basolateral membrane can be extracted. When the incident angle is reduced to be in the subcritical range, the instrument works as a pseudo-TIRFM. The whole cell body from bottom to top can be imaged in a vertical scan process. Furthermore, the illumination field depth in the pseudo-TIRFM can be controlled by changing the incident angle or the horizontal position of the laser spot.
一台自动校准和角度扫描棱镜型全内反射荧光显微镜（TIRFM）被改装为可在TIRFM和伪TIRFM模式下工作。当激发激光束的入射角被控制为大于临界角时，该仪器用作可变角度TIRFM。一个自制的计算机程序会自动校准样品中的激光照射光斑，使其与显微镜视野中心重叠。然后，通过测量不同入射角下的荧光强度，可以提取靠近细胞基底外侧膜的荧光纳米球的z位置。当入射角减小到亚临界范围内时，该仪器作为伪TIRFM工作。在垂直扫描过程中可以对整个细胞体从底部到顶部进行成像。此外，伪TIRFM中的照明场深度可以通过改变入射角或激光光斑的水平位置来控制。
