Xiao Chao, Zhang Chen, Zhu Jie, Zhao Wei, Bai Jintao, He Qingli, Wang Kaige
Appl Opt. 2019 Mar 10;58(8):2112-2120. doi: 10.1364/AO.58.002112.
Probing depth and system aberrations have direct impacts on the spatial resolution of stimulated emission depletion (STED) microscopes. Based on the vectorial diffraction theory, the influence of coma and astigmatism on the focal patterns of STED microscopes in probing stratified mediums with discontinuous refractive indices (e.g., glass cover slip, solution, and biological samples, etc.) have been illustrated in detail. The spatial resolution of the STED system has been discussed by analyzing the full width at half-maximum size of the fluorescence spots. It is found that, while probing in stratified media with discontinuous refractive indices, the spatial resolution of a STED microscope can be very sensitive to the existence of aberrations, e.g., coma and astigmatism, at different probing depths, as a result of mismatched axial positions of the excitation and depletion patterns. The spatial resolution of STED can be degraded up to 1.87- and 1.95-fold compared to that without aberrations. Therefore, a careful evaluation of the influence of aberration and discontinuous refractive indices should be taken into account when applying a STED microscope to realize super-resolution images.
探测深度和系统像差对受激辐射损耗（STED）显微镜的空间分辨率有直接影响。基于矢量衍射理论，详细阐述了彗差和像散对STED显微镜在探测具有不连续折射率的分层介质（如玻璃盖玻片、溶液和生物样品等）时焦斑图案的影响。通过分析荧光斑点的半高宽大小来讨论STED系统的空间分辨率。研究发现，在探测具有不连续折射率的分层介质时，由于激发和损耗图案的轴向位置不匹配，STED显微镜的空间分辨率在不同探测深度下可能对彗差和像散等像差的存在非常敏感。与无像差时相比，STED的空间分辨率可能会降低至1.87倍和1.95倍。因此，在应用STED显微镜以实现超分辨率图像时，应仔细评估像差和不连续折射率的影响。
