ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Department of Physics and Astronomy, Macquarie University, Sydney, NSW, 2109, Australia.
Bioengineering in Reproductive Health Group, Institute for BioEngineering of Catalonia (IBEC), 08028, Barcelona, Spain.
Nat Commun. 2019 Aug 16;10(1):3695. doi: 10.1038/s41467-019-11603-0.
Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF, doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.
亚衍射显微镜能够以前所未有的清晰度进行生物成像。然而，大多数超分辨率方法需要复杂、昂贵的定制系统，涉及图像后处理，并且在 3D 中难以实现亚衍射成像。在这里，我们实现了一种概念上不同的超分辨率方法，该方法规避了这些限制，并能够在传统共聚焦显微镜上实现 3D 亚衍射成像。我们将其称为超线性激发-发射（SEE）显微镜，因为它依赖于具有激发功率超线性发射依赖性的标记。这里提出的超线性标记是掺杂了 20%Yb 和非常规的 8%Tm 的 NaYF 上转换纳米粒子，它们在近红外生物窗口中方便地被激发。我们开发了一种计算框架，用于计算任何可行的扫描光束形状和激发-发射探针轮廓的 3D 分辨率。对神经元细胞内吞的 colominic 酸包覆的上转换纳米粒子进行成像，在侧向和轴向分辨率都比衍射极限好两倍，说明了 SEE 显微镜在亚细胞生物学中的适用性。
