Neupane Bhanu, Chen Fang, Sun Wei, Chiu Daniel T, Wang Gufeng
Chemistry Department, North Carolina State University, Raleigh, North Carolina 27695, USA.
Rev Sci Instrum. 2013 Apr;84(4):043701. doi: 10.1063/1.4799665.
In stimulated emission depletion (STED)-based or up-conversion depletion-based super-resolution optical microscopy, the donut-shaped depletion beam profile is of critical importance to its resolution. In this study, we investigate the transformation of the donut-shaped depletion beam focused by a high numerical aperture (NA) microscope objective, and model STED point spread function (PSF) as a function of donut beam profile. We show experimentally that the intensity profile of the dark kernel of the donut can be approximated as a parabolic function, whose slope is determined by the donut beam size before the objective back aperture, or the effective NA. Based on this, we derive the mathematical expression for continuous wave (CW) STED PSF as a function of focal plane donut and excitation beam profiles, as well as dye properties. We find that the effective NA and the residual intensity at the center are critical factors for STED imaging quality and the resolution. The effective NA is critical for STED resolution in that it not only determines the donut shape but also the area the depletion laser power is dispersed. An improperly expanded depletion beam will have negligible improvement in resolution. The polarization of the depletion beam also plays an important role as it affects the residual intensity in the center of the donut. Finally, we construct a CW STED microscope operating at 488 nm excitation and 592 nm depletion with a resolution of 70 nm. Our study provides detailed insight to the property of donut beam, and parameters that are important for the optimal performance of STED microscopes. This paper will provide a useful guide for the construction and future development of STED microscopes.
在基于受激发射损耗（STED）或基于上转换损耗的超分辨率光学显微镜中，环形损耗光束轮廓对其分辨率至关重要。在本研究中，我们研究了由高数值孔径（NA）显微镜物镜聚焦的环形损耗光束的变换，并将STED点扩散函数（PSF）建模为环形光束轮廓的函数。我们通过实验表明，环形暗核的强度轮廓可以近似为抛物线函数，其斜率由物镜后孔径之前的环形光束尺寸或有效NA决定。基于此，我们推导了连续波（CW）STED PSF作为焦平面环形光束、激发光束轮廓以及染料特性的函数的数学表达式。我们发现有效NA和中心处的残余强度是影响STED成像质量和分辨率的关键因素。有效NA对STED分辨率至关重要，因为它不仅决定环形形状，还决定损耗激光功率分散的区域。损耗光束扩展不当对分辨率的提高可忽略不计。损耗光束的偏振也起着重要作用，因为它会影响环形中心的残余强度。最后，我们构建了一台在488 nm激发和592 nm损耗下工作、分辨率为70 nm的连续波STED显微镜。我们的研究详细深入地了解了环形光束的特性以及对STED显微镜最佳性能很重要的参数。本文将为STED显微镜的构建和未来发展提供有用的指导。
