基于荧光辐射时序特性的非线性频移超分辨显微成像

Super resolution microscopy with large field of view, fast imaging speed, ultra-high resolution and low damage is quite significant to the development of biology, medicine and so on. Spatial frequency shift microscopy has shown big potential on achieving large field of view and fast imaging speed, and thus attracted research interest worldwide. However, the main concern of spatial frequency shift microscopy is the weak super resolvability and the difficulty to induce nonlinear effect of high order stably at low excitation intensity. In order to improve its resolvability, novel nonlinear effect with low excitation intensity and good stability should be explored. This project proposes a novel nonlinear spatial frequency shift super resolution microscopy based on the temporal properties of fluorescence. The spatial distribution of excitation intensity will be temporally modulated to cause variation of the intensity of fluorescence. During the non-steady state of fluorescence, the spatial modulation to the fluorescence could depend nonlinearly on the spatial distribution of excitation, and consequently, nonlinear spatial frequency shift microscopy can be achieved at low excitation intensity. A super resolution microscopy system will be established through studying the temporal fluorescence model at non-steady state, designing the relative intensity and temporal parameters of excitation, extracting signals at non-steady state simultaneously, and exploring the reconstruction algorithm. This project proposes a novel method to achieve nonlinear effect in space, pave a new way to enhance the resolution in wide-field microscopy, and could achieve important progress on high-resolution, low-damage, large-field-of-view and fast super resolution microscopy.

大视场、快速、超高分辨率与低光损伤的超分辨显微技术对生物、医学等领域的发展至关重要。频移显微成像由于在大视场快速成像上具有显著优势，引起了广泛关注，但目前分辨率不够高，遇到了无法于低激发功率下稳定地引入高阶非线性响应的限制，需要探索新型非线性频移机制来提升分辨率。本项目提出基于荧光辐射时序特性展开新型非线性频移超分辨显微成像研究。将对空间激发光进行时序调控，改变荧光辐射强度，基于在荧光辐射非稳态时间段内，荧光强度的空间调制可与激发光强的空间分布之间呈现非线性对应关系，实现低激发功率下的非线性频移效应。项目将通过建立荧光辐射动态模型，设计照明光的相对强度和时序参数，同步提取荧光辐射非稳态时间段的信号，开发信号解调与频谱重构算法，形成一套完备的超分辨显微成像系统。该研究提出在空间建立非线性响应的新机理，可为增强宽场显微成像分辨率提供新途径，以实现高分辨率、低光损伤、大视场快速超分辨显微成像。

大视场、快速、超高分辨率与低光损伤的超分辨显微技术对生物、医学等领域的发展至关重要。移频显微成像在大视场、快速成像上具有显著优势，引起了广泛关注，而分辨率不够高（100nm以上）；或需要依赖强激发下的非线性辐射来提取更高频信息是其面临的限制。本项目深入研究纳米结构的非线性辐射新机理和可调深移频新效应，探索超高频探测新机制，实现无缺失的超宽频谱成像，为高分辨、低损伤、大视场快速超分辨显微成像提供有效途径。. 取得的重要结果、关键数据如下：（1）基于硅纳米盘内Anapole模式转换，实现低功率下（~MW/cm^2）的非线性远场辐射和分辨率~40nm的远场超分辨定位成像。（2）提出与光子芯片兼容的多级调频机理，结合高折射率GaP波导设计，即可实现照明空间频率从零频至超高频的主动调节，无需高激发功率，可实现样品无缺失超宽频谱探测与成像，成像分辨率优于65nm，视场达到10^4 um^2量级；将该机理用于高折射率超材料波导，可实现30nm分辨率。（3）提出将饱和非线性辐射用于纵向调频，可实现精度3nm的纵向层析成像。（4）提出基于Gouy相位梯度调节的入瞳优化机制，可实现照明焦斑在纵向具有远大于介质波矢的局域空间频率，首次在纵向上发现类似超振荡现象。