Jayakumar Nikhil, Villegas-Hernández Luis E, Zhao Weisong, Mao Hong, Dullo Firehun T, Tinguely Jean-Claude, Sagini Krizia, Llorente Alicia, Ahluwalia Balpreet Singh
Department of Physics and Technology, UiT The Arctic University of Norway, Tromsø, 9037, Norway.
Innovation Photonics and Imaging Center, State Key Laboratory of Space Environment Interation with Matters, Key Laboratory of Ultra-Precision Intelligent Instrumentation of Ministry of Industry and Information Technology, School of Instrumentation Science and Engineering, Harbin Institute of Technology, Harbin, 150080, China.
Light Sci Appl. 2025 Aug 4;14(1):259. doi: 10.1038/s41377-025-01914-x.
The photo-kinetics of fluorescent molecules have enabled the circumvention of the far-field optical diffraction limit. Despite its enormous potential, the necessity to label the sample may adversely influence the delicate biology under investigation. Thus, continued development efforts are needed to surpass the far-field label-free diffraction barrier. The statistical similarity or finite coherence of the scattered light off the sample in label-free mode hinders the application of existing super-resolution methods based on incoherent fluorescence imaging. In this article, we present physics and propose a methodology to circumvent this challenge by exploiting the photoluminescence (PL) of silicon nitride waveguides for near-field illumination of unlabeled samples. The technique is abbreviated EPSLON, Evanescently decaying Photoluminescence Scattering enables Label-free Optical Nanoscopy. We demonstrate that such an illumination has properties that mimic the photo-kinetics of nano-sized fluorescent molecules, i.e., such an illumination permits incoherence between the scattered fields from various locations on the sample plane. Thus, the illumination scheme enables the development of a far-field label-free incoherent imaging system that is linear in intensity and stable over time, thereby permitting the application of techniques like structured illumination microscopy (SIM) and intensity-fluctuation-based optical nanoscopy (IFON) in label-free mode to circumvent the diffraction limit. In this proof-of-concept work, we observed a two-point resolution of 180 nm on super-resolved nanobeads and resolution improvements between 1.9× to 2.8× over the diffraction limit, as quantified using Fourier Ring Correlation (FRC), on various biological samples. We believe EPSLON is a step forward within the field of incoherent far-field label-free super-resolution microscopy that holds a key to investigating biological systems in their natural state without the need for exogenous labels.
荧光分子的光动力学能够突破远场光学衍射极限。尽管具有巨大潜力，但标记样品的必要性可能会对所研究的微妙生物学过程产生不利影响。因此，需要持续开展研发工作以突破远场无标记衍射障碍。在无标记模式下，样品散射光的统计相似性或有限相干性阻碍了基于非相干荧光成像的现有超分辨率方法的应用。在本文中，我们阐述了相关物理原理，并提出了一种方法来应对这一挑战，即利用氮化硅波导的光致发光（PL）对未标记样品进行近场照明。该技术简称为EPSLON，即倏逝衰减光致发光散射实现无标记光学纳米显微镜。我们证明，这种照明具有类似于纳米级荧光分子光动力学的特性，也就是说，这种照明允许样品平面上不同位置的散射场之间存在非相干性。因此，这种照明方案能够开发出一种强度呈线性且随时间稳定的远场无标记非相干成像系统，从而允许在无标记模式下应用诸如结构照明显微镜（SIM）和基于强度波动的光学纳米显微镜（IFON）等技术来突破衍射极限。在这项概念验证工作中，我们在超分辨纳米珠上观察到了180 nm的两点分辨率，并且在各种生物样品上，通过傅里叶环相关（FRC）量化，分辨率比衍射极限提高了1.9倍至2.8倍。我们相信，EPSLON是在非相干远场无标记超分辨率显微镜领域向前迈出的一步，它为在无需外源性标记的情况下研究自然状态下的生物系统提供了关键。
