Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells
Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells
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DOI：
10.1073/pnas.0808882105
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发表时间：
2008-11-18
影响因子：
11.1
通讯作者：
Yan, Yuling
中科院分区：
文献类型：
--
作者：
Marriott, Gerard;Mao, Shu;Yan, Yuling
在活细胞内对荧光蛋白进行成像的局限性之一是，它们通常以少量存在，需要在大背景下才能检测到。我们已经开发了一种方法，通过锁定检测一类被称为“光开关”的光学探针的调制荧光来将特定的荧光信号与背景分离。这种光学锁定检测(OLID)方法包括通过对其荧光和非荧光状态的确定性光学控制来调制探针的荧光发射，并且随后应用锁定检测方法以将感兴趣的调制信号与非调制背景信号分离。互相关分析提供了在几个光学切换周期内检测到的图像的单个像素内的总荧光发射与在相同切换周期内在同一图像内检测到的参考波形之间的相关性的测量。这种成像方法提供了一种在较大数量的传统荧光探针中选择性地检测来自光开关探针的发射的方法，并且与传统显微镜兼容。使用基于亚硝基螺苯并吡喃的探针和基因编码的Dronpa荧光蛋白的OLID被证明在培养和移植的神经元以及活的非洲爪哇胚胎和斑马鱼幼体的标记细胞中产生特定结构和蛋白质的高对比度图像。
One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed "optical switches." This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.