Xu Fang, Wei Lu, Chen Zhixing, Min Wei
Department of Chemistry, Columbia University, New York, New York 10027, USA.
Opt Express. 2013 Jun 17;21(12):14097-108. doi: 10.1364/OE.21.014097.
Two-photon fluorescence microscopy has become increasingly popular in biomedical research as it allows high-resolution imaging of thick biological specimen with superior contrast and penetration than confocal microscopy. However, two-photon microscopy still faces two fundamental limitations: 1) image-contrast deterioration with imaging depth due to out-of-focus background and 2) diffraction-limited spatial resolution. Herein we propose to create and detect high-order (more than quadratic) nonlinear signals by harnessing the frustrated fluorescence resonance energy transfer (FRET) effect within a specially designed donor-acceptor probe pair. Two distinct techniques are described. In the first method, donor fluorescence generated by a two-photon laser at the focus is preferentially switched on and off by a modulated and focused one-photon laser beam that is able to block FRET via direct acceptor excitation. The resulting image, constructed from the enhanced donor fluorescence signal, turns out to be an overall three-photon process. In the second method, a two-photon laser at a proper wavelength is capable of simultaneously exciting both the donor and the acceptor. By sinusoidally modulating the two-photon excitation laser at a fundamental frequency ω, an overall four-photon signal can be isolated by demodulating the donor fluorescence at the third harmonic frequency 3ω. We show that both the image contrast and the spatial resolution of the standard two-photon fluorescence microscopy can be substantially improved by virtue of the high-order nonlinearity. This frustrated FRET approach represents a strategy that is based on extracting the inherent nonlinear photophysical response of the specially designed imaging probes.
双光子荧光显微镜在生物医学研究中越来越受欢迎，因为它能够对厚生物样本进行高分辨率成像，与共聚焦显微镜相比，具有更高的对比度和穿透深度。然而，双光子显微镜仍然面临两个基本限制：1）由于离焦背景导致成像深度的图像对比度恶化，以及2）衍射极限空间分辨率。在此，我们建议通过利用特殊设计的供体-受体探针对内的受挫荧光共振能量转移（FRET）效应来产生和检测高阶（大于二次）非线性信号。描述了两种不同的技术。在第一种方法中，由聚焦处的双光子激光产生的供体荧光通过调制和聚焦的单光子激光束优先开启和关闭，该单光子激光束能够通过直接受体激发来阻断FRET。由增强的供体荧光信号构建的所得图像实际上是一个整体的三光子过程。在第二种方法中，适当波长的双光子激光能够同时激发供体和受体。通过以基频ω对双光子激发激光进行正弦调制，可以通过解调三次谐波频率3ω处的供体荧光来分离整体的四光子信号。我们表明，借助高阶非线性，可以显著提高标准双光子荧光显微镜的图像对比度和空间分辨率。这种受挫FRET方法代表了一种基于提取特殊设计的成像探针固有非线性光物理响应的策略。
