Marriott Gerard, Mao Shu, Sakata Tomoyo, Ran Jing, Jackson David K, Petchprayoon Chutima, Gomez Timothy J, Warp Erica, Tulyathan Orapim, Aaron Holly L, Isacoff Ehud Y, Yan Yuling
Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53705, USA.
Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17789-94. doi: 10.1073/pnas.0808882105. Epub 2008 Nov 12.
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.
在活细胞内对荧光蛋白进行成像的一个限制是，它们通常数量较少，需要在大背景下进行检测。我们已经开发出一种方法，通过对一类称为“光开关”的光学探针的调制荧光进行锁相检测，从背景中分离出特定的荧光信号。这种光学锁相检测（OLID）方法包括通过对探针的荧光和非荧光状态进行确定性光学控制来调制探针的荧光发射，随后应用锁相检测方法从未调制的背景信号中分离出感兴趣的调制信号。互相关分析提供了一种衡量在几个光开关周期内检测到的图像单个像素内的总荧光发射与在相同开关周期内在同一图像中检测到的参考波形之间相关性的方法。这种成像方法提供了一种手段，可在大量传统荧光探针中选择性地检测光开关探针的发射，并且与传统显微镜兼容。使用基于硝基螺苯并吡喃的探针和基因编码的Dronpa荧光蛋白的OLID被证明能够在培养和移植的神经元中的标记细胞、活非洲爪蟾胚胎和斑马鱼幼虫中生成特定结构和蛋白质的高对比度图像。
