Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells
Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells
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DOI：
10.1021/acs.analchem.9b01813
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发表时间：
2019-09-03
影响因子：
7.4
通讯作者：
Vukojevic, Vladana
中科院分区：
文献类型：
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作者：
Krmpot, Aleksandar J.;Nikolic, Stanko N.;Vukojevic, Vladana
功能荧光显微镜成像 (fFMI) 是一种无需扫描的时间分辨（21 μ s/帧）共焦荧光显微镜成像技术，旨在定量表征溶液和活细胞中的快速反应传输过程。该方法基于大规模并行荧光相关光谱（FCS）。使用衍射光学元件 (DOE) 可以同时激发焦平面上多个点上的荧光分子。由 DOE 生成的 1024 个照明点发出的荧光由包含 32 x 32 单光子雪崩光电二极管 (SPAD) 的匹配矩阵检测器以共焦排列进行检测。使用图形处理单元 (GPU) 通过并行信号处理进行数据采集和快速自相关和互相关分析的软件可在 4 秒内实现图像帧中所有像素的时间自相关，并在 45 秒内实现一阶和二阶相邻像素之间的互相关。我们在此提出这种具有单分子灵敏度的定量、时间分辨成像方法，并证明其对于绘制不同亚细胞区室中分子浓度和平移扩散的活细胞位置特异性差异的有用性。特别是，我们发现没有特定生物功能的分子，例如增强型绿色荧光蛋白（eGFP），表现出均匀的扩散。相比之下，执行专门生物功能并与其分子靶标特异性结合的分子在浓度和扩散方面表现出位置特异性差异，这里以两种转录因子分子为例，即核易位前后的糖皮质激素受体（GR）和果蝇离体唾液腺中的性梳减少（Scr）转录因子。
Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 mu s/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 x 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and cross-correlation between first- and second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.