Gould Travis J, Myers Jordan R, Bewersdorf Joerg
Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, USA.
Opt Express. 2011 Jul 4;19(14):13351-7. doi: 10.1364/OE.19.013351.
Stimulated emission depletion (STED) microscopy achieves diffraction-unlimited resolution in far-field fluorescence microscopy well below 100 nm. As common for (single-lens) far-field microscopy techniques, the lateral resolution is better than the axial sectioning capabilities. Here we present the first implementation of total internal reflection (TIR) illumination into STED microscopy which limits fluorophore excitation to ~70 nm in the vicinity of the cover slip while simultaneously providing ~50 nm lateral resolution. We demonstrate the performance of this new microscope technique with fluorescent bead test samples as well as immuno-stained microtubules. Total internal reflection STED microscopy provides superior axial sectioning capabilities with the potential to reduce photo-bleaching and photo-damage in live cell imaging.
受激辐射损耗（STED）显微镜在远场荧光显微镜中实现了远低于100纳米的衍射极限分辨率。与（单透镜）远场显微镜技术一样，横向分辨率优于轴向切片能力。在此，我们展示了全内反射（TIR）照明首次应用于STED显微镜，它将荧光团激发限制在盖玻片附近约70纳米范围内，同时提供约50纳米的横向分辨率。我们用荧光微珠测试样品以及免疫染色的微管展示了这种新显微镜技术的性能。全内反射STED显微镜具有卓越的轴向切片能力，有望减少活细胞成像中的光漂白和光损伤。
