Multiplexed bioluminescence microscopy via phasor analysis.
Multiplexed bioluminescence microscopy via phasor analysis.
复制标题
通过相量分析的多重生物发光显微镜。
DOI：
10.1038/s41592-022-01529-9
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发表时间：
2022
期刊：
影响因子：
48
通讯作者：
Prescher,JenniferA
中科院分区：
文献类型：
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作者：
Yao,Zi;Brennan,CarolineK;Scipioni,Lorenzo;Chen,Hongtao;Ng,KevinK;Tedeschi,Giulia;Parag-Sharma,Kshitij;Amelio,AntonioL;Gratton,Enrico;Digman,MichelleA;Prescher,JenniferA
生物发光成像与双酶-荧光素酶是一种成熟的技术，用于可视化跨组织和整个生物体的生物过程。相比之下，由于缺乏检测平台和易于分辨的探针，微尺度的应用受到了阻碍。我们通过将生物发光与相量分析相结合来解决这一限制，相量分析是一种常用的区分光谱相似的荧光团的方法。我们建立了一个基于相机的显微镜，配备了特殊的光学过滤器，直接分配相量的位置，以独特的双螺旋桨。六种生物发光报告基因在活细胞中很容易分辨，并且读数是定量和瞬时的。还在延长的时间段内进行多路复用成像。生物发光相量进一步提供了单细胞中共振能量转移的直接测量，为细胞和分子特征的动态测量奠定了基础。生物发光与相量分析的合并填补了成像能力的长期空白，并将支持未来在真实的时间和多个长度尺度上可视化生物事件的努力。
Bioluminescence imaging with luciferase–luciferin pairs is a well-established technique for visualizing biological processes across tissues and whole organisms. Applications at the microscale, by contrast, have been hindered by a lack of detection platforms and easily resolved probes. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We built a camera-based microscope equipped with special optical filters to directly assign phasor locations to unique luciferase–luciferin pairs. Six bioluminescent reporters were easily resolved in live cells, and the readouts were quantitative and instantaneous. Multiplexed imaging was also performed over extended time periods. Bioluminescent phasor further provided direct measures of resonance energy transfer in single cells, setting the stage for dynamic measures of cellular and molecular features. The merger of bioluminescence with phasor analysis fills a long-standing void in imaging capabilities, and will bolster future efforts to visualize biological events in real time and over multiple length scales.