3D单分子荧光成像系统-SAFe 360

3D单分子荧光成像系统-SAFe 360


SAFe 360是法国abbelight公司推出的一款基于单分子定位的显微成像(SMLM)的新型3D单分子成像系统,它独有的DAISY技术整合了散光技术和超临界角光技术,能够提高定位精度,xyz三轴定位精度高达15nm,可以提供高清晰三维亚细胞结构图像,支持同时四色成像,可以用于细胞纳米三维成像,观测高清晰亚细胞器结构,实时研究不同的结构功能蛋白的共定位信息,在单分子水平研究分子动力学反应以及细胞间的相互作用等。


加装
TIRF
PALM
STORM
SPT

smFRET

...... 


兼容
Confocal
Spinning-Desk
Widefield
SIM

STED

......


先进的DAISY技术具备高性能、低漂移、四色同时成像等优点#提供STORM、PLAM、PAINT、SPT、smFRET成像模式#

设备参数


+ 成像模式:PALM、STORM、PAINT、smFRET 、SPT

+ 光源模式:Epi、TIRF、HILO

+ 高分辨率:15 nm的XYZ轴分辨率

+ 超大视野:200 × 200 μm2的视野

+  一次可同时采集1.2 μm深度图像信息

+  高图像深度:10 μm

+  实时漂移矫正

+  高四色同时成像

+  活细胞成像模式


■  细胞质膜上的内陷结构与功能研究


真核细胞的细胞质膜是一个不均一体系,包含大量脂类组成和生物物理特性不同的区域。这些蛋白脂类区域使质膜产生内陷,而内陷在胞吞过程中起重要作用。在这些质膜区域中,网格蛋白包被的小窝(clathin coated pits, CCP)和胞膜窖(caveolae)由于表面有明显的蛋白包被,很容易用电镜成像等方法观测到。CCP呈80-120 nm的球形结构,包被有网格蛋白和衔接蛋白。网格蛋白调控的内吞作用通路(CME)通过被膜小窝实现胞吞。Caveolae是50-80 nm的内陷结构,在信号转导,膜运输,胆固醇运输,机械传感等过程中起作用,在受到机械应力作用的细胞,如脂肪细胞,内皮细胞和肌细胞中含量较高。有研究发现了小凹的产生及与胞内体的融合,但Caveolae是否在胞吞中起作用尚无定论。

在下图的实例中,研究人员使用了Abbelight的成像3D单分子荧光成像系统-SAFe 360分辨出了内皮细胞中的Clathrin和Caveolae。结果显示了清晰、无重叠的荧光分布,与电镜成像结果一致。

图1. Abbelight SAFe360系统重现质膜上蛋白脂类区域的蛋白分布


图2. 通过SAFe360的同时多色成像功能可以清晰、无重叠地区分Clathrin 和Caveolae,进一步研究Clathrin 和Caveolae的内吞作用通路的相互关系。



■  大肠杆菌RNA聚合酶的空间分布与动力学研究


大肠杆菌中的RNA聚合酶(RNAP)空间上呈拟核形状分布,是核糖体RNA(rRNA)的转录中心。下方实例使用Abbelight SAFe 360系统通过PALM超分辨成像与单分子追踪(sptPALM)解析大肠杆菌RNAP的纳米尺度空间分布与动力学,进而研究转录调控机制。


图1. (A)Denndra2标记RNAP在大肠杆菌中分布定位(定位精度约为15 nm);(B)通过DBSCAN算法对大肠杆菌中RNA聚合酶分布进行团簇分析。


  

图2. 在活细胞中以200 FPS(5 ms/帧)的采集速度对大肠杆菌中Dendra2标记的RNAP分子进行单分子追踪以研究单个RNAP分子运动轨迹,按照运动状态分为静态(红色,即转录中)或动态(紫色,扩散运动)。

 


3D线粒体结构


核孔复合物


老鼠海马神经元


微管蛋白网络




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