基于荧光涨落的亚细胞同步多色超分辨成像

It is of vital importance to unveil the inherent behaviors and laws of life by investigating biological subcellular structures. Multicolor super-resolution optical microscopy employs fluorophores with different wavelengths to label various subcellular structures, enabling the observation on the distributions of multiple subcellular units. It is an important technical method to investigate complicated intracellular structures and functions. Existing multicolor super-resolution methods generally occupy multiple spectral channels or utilize a single channel for time-divided collection during the image acquisition process, which leads to the mutual restriction between the reduction of the spatial wavelength-divided acquisition and the elimination of the channel delay in synchronous observation of multiple subcellular structures. Therefore, in this research, we propose a method based on fluorescence fluctuation to achieve synchronous multicolor super-resolution imaging with zero time delay. The proposed method requires only two spectral channels and a single image detector for acquisition. It is able to achieve synchronous multicolor super-resolution imaging with zero time delay when imaging various subcellular structures labeled with multiple fluorophores. This method is expected to effectively eliminate the channel delay in multicolor imaging from two collected spectral channels under spectral crosstalk condition. The signal-to-noise ratio, image field-of-view and spatial resolution can also be improved. We will perform synchronous multicolor imaging and monitoring on multiple subcellular structures and their dynamic interactions. The proposed method will provide a new pathway for the research on the synchronous observation of various subcellular structures.

研究生物亚细胞结构对于揭示生命体的内在行为和规律至关重要，多色超分辨光学显微成像使用不同波长的荧光分子标记多种亚细胞结构，能够观察到多种亚细胞单元的分布特征，是研究细胞内部复杂结构和功能的重要技术方法。现有的多色超分辨方法在图像采集过程中，通常需要占用多个波长采集通道或者使用单通道分时段延时采集，在应用于同步观测多种亚细胞结构时，存在减少空间分波长采集与消除通道延时二者之间相互制约的问题。因此，本项目提出基于荧光涨落的零延时同步多色超分辨成像的方法，仅需单个图像探测器和两个光谱通道进行采集，实现对多种荧光标记的亚细胞结构进行零延时同步多色超分辨成像。本方法能够在通道串色的情况下，仅通过两个光谱采集通道消除多色成像的通道延时，并且提升同步多色成像的信噪比、图像视场和空间分辨率，拟对多种亚细胞结构及其动态相互作用进行同步多色成像和监测。本项目的方法将为同步观测多种亚细胞结构的研究提供新途径。

本项目首先从理论上构建了同步多色荧光超分辨显微成像模型框架，基于MATLAB软件自主开发了多色超分辨光学显微成像算法，自主设计了Sigmoid函数透过率分布的二向色镜多色分离方案，通过数值仿真模拟实现了多种亚细胞尺度精细结构的多色分离和超分辨率图像重建，基于宽场荧光显微系统搭建了同步多色荧光超分辨显微成像系统并完成了多色图像序列的采集，通过实验采集的亚细胞精细结构图像实现了基于荧光涨落的亚细胞多色超分辨成像。本研究进一步通过结合SOFI与SRRF超分辨方法，提出荧光涨落累积量分析的径向度超分辨显微成像（CERN）的新方法，联合扩增显微技术（ExM），在实验上对细胞骨架蛋白和环形网格蛋白等亚细胞精细结构实现了多色荧光超分辨成像，能够有效分辨空间上相距50nm左右的亚细胞微管结构，CERN结合扩增显微ExM方法能够达到10nm左右的空间定位精度，并且实现对RNA转录因子进行高密度的超分辨成像。本研究同时开发了多模态荧光涨落超分辨及同步多色显微成像集成软件系统平台，集成了多种荧光涨落超分辨算法实现多算法与同步多色超分辨图像重建、对比分析和图像质量评价与优化，为研究多种细胞内部亚细胞精细结构和功能提供了新型的技术方法和分析平台。