Yu Bin, Yu Jie, Li Weihai, Cao Bo, Li Heng, Chen Danni, Niu Hanben
Appl Opt. 2016 Jan 20;55(3):449-53. doi: 10.1364/AO.55.000449.
The double-helix point spread function (DH-PSF) microscopy has become an essential tool for nanoscale three-dimensional (3D) localization and tracking of single molecules in living cells. However, its localization precision is limited by fluorescent contrast in thick samples because the signal-to-noise ratio of the system is low due to the inherent low transfer function efficiency and background fluorescence. Here we combine DH-PSF microscopy with light-sheet illumination to eliminate out-of-focus background fluorescence for high-precision 3D single particle tracking. To demonstrate the capability of the method, we obtain the single fluorescent bead image with light-sheet illumination, with three-dimensional localization accuracy better than that of epi-illumination. We also show that the single fluorescent beads in agarose solution can be tracked, which demonstrates the possibility of our method for the study of dynamic processes in complex biological specimens.
双螺旋点扩散函数（DH-PSF）显微镜已成为活细胞中单个分子纳米级三维（3D）定位和跟踪的重要工具。然而，其定位精度受到厚样品中荧光对比度的限制，因为由于固有的低传递函数效率和背景荧光，系统的信噪比很低。在这里，我们将DH-PSF显微镜与光片照明相结合，以消除离焦背景荧光，实现高精度的3D单粒子跟踪。为了证明该方法的能力，我们通过光片照明获得了单荧光珠图像，其三维定位精度优于落射照明。我们还表明，可以跟踪琼脂糖溶液中的单荧光珠，这证明了我们的方法用于研究复杂生物样本中动态过程的可能性。
