EMBL Australia Node in Single Molecule Science, and ARC Centre of Excellence in Advanced Molecular Imaging School of Medical Sciences, The University of New South Wales, 2052 Sydney, Australia.
The Garvan Institute of Medical Research, St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW 2010, Australia.
Methods Appl Fluoresc. 2022 Mar 25;10(3). doi: 10.1088/2050-6120/ac5e10.
Stimulated Emission Depletion (STED) microscopy increase spatial image resolution by laterally sharpening the illumination profile of the confocal microscope. However, it remains compromised in axial resolution. To improve axial STED resolution, constructive interference of the STED depletion beam must be formed surrounding the focal plane to turn off the fluorophores beyond the focal plane. For isotropic 3D-STED resolution, this axial STED interference pattern must be overlayed with the doughnut STED beam at nanometer accuracy. Such optical configurations can be challenging in alignment. In this current work, we introduced a straightforward lifetime based axial contrast in STED microscope by imaging the samples on an ITO coated glass coverslip. The STED laser generates surface plasmon resonance on the ITO surface that enhanced the metal induced energy transfer MIET effect on the ITO surface. The enhanced MIET effect established a lifetime gradient with ∼20% dynamic range that extend for mor than 400 nm from the ITO surface. The axial contrast based on the lifetime gradient was directly used for 3D-STED imaging of tubulin fibers inside COS-7 cells, where the vertical displacement of single tubulin fiber was revealed. Lifetime gating could be applied to further improve lateral spatial resolution. Considering that most common implementation of STED microscopes uses pulsed lasers and timing electronics, there is no optical modification of the microscope is required in the current 3D-STED approach.
受激发射耗散（STED）显微镜通过横向锐化共聚焦显微镜的照明轮廓来提高空间图像分辨率。然而，其轴向分辨率仍然受到限制。为了提高轴向 STED 分辨率，必须在焦平面周围形成 STED 耗尽光束的相长干涉，以关闭焦平面以外的荧光团。对于各向同性的 3D-STED 分辨率，该轴向 STED 干涉图案必须以纳米精度与环形 STED 光束叠加。这种光学配置在对准方面可能具有挑战性。在本研究中，我们通过在 ITO 涂层玻璃盖玻片上对样品进行成像，在 STED 显微镜中引入了一种基于寿命的简单轴向对比度。STED 激光在 ITO 表面产生表面等离子体共振，增强了 ITO 表面上的金属诱导能量转移 MIET 效应。增强的 MIET 效应在 ITO 表面建立了一个具有 ∼20%动态范围的寿命梯度，从 ITO 表面延伸超过 400nm。基于寿命梯度的轴向对比度直接用于 COS-7 细胞内微管蛋白纤维的 3D-STED 成像，揭示了单个微管蛋白纤维的垂直位移。寿命门控可用于进一步提高横向空间分辨率。考虑到 STED 显微镜的最常见实现使用脉冲激光器和定时电子设备，当前的 3D-STED 方法不需要对显微镜进行光学修改。
