School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, P. R. China.
Acc Chem Res. 2013 Feb 19;46(2):269-79. doi: 10.1021/ar300108d. Epub 2012 Oct 24.
Because of its ultrasensitivity, fluorescence offers a noninvasive means to investigate biomolecular mechanisms, pathways, and regulations in living cells, tissues, and animals. However, real-world applications of fluorescence technologies encounter many practical challenges. For example, the intrinsic heterogeneity of biological samples always generates optical interferences. High background such as autofluorescence can often obscure the desired signals. Finally, the wave properties of light limit the spatial resolution of optical microscopy. The key to solving these problems involves using chemical structures that can modulate the fluorescence output. Photoswitchable fluorescent molecules that alternate their emissions between two colors or between bright-and-dark states in response to external light stimulation form the core of these technologies. For example, molecular fluorescence modulation can switch fluorophores on and off. This feature supports super-resolution, which enhances resolution by an order of magnitude greater than the longstanding diffraction-limit barrier. The reversible modulation of such probes at a particular frequency significantly amplifies the frequency-bearing target signal while suppressing interferences and autofluorescence. In this Account, we outline the fundamental connection between constant excitation and oscillating fluorescence. To create molecules that will convert a constant excitation into oscillating emission, we have synthesized photoswitchable probes and demonstrated them as proofs of concept in super-resolution imaging and frequency-domain imaging. First, we introduce the design of molecules that can convert constant excitation into oscillating emission, the key step in fluorescence modulation. Then we discuss various technologies that use fluorescence modulation: super-resolution imaging, dual-color imaging, phase-sensitive lock-in detection, and frequency-domain imaging. Finally, we present two biological applications to demonstrate the power of photoswitching-enabled fluorescence imaging. Because synthetic photoswitchable probes can be much smaller, more versatile, and more efficient at high-performance modulation experiments, they provide a complement to photoswitchable fluorescent proteins. Although new challenges remain, we foresee a bright future for photoswitching-enabled imaging and detection.
由于其超高的灵敏度，荧光技术提供了一种非侵入式的手段，可用于研究活细胞、组织和动物中的生物分子机制、途径和调控。然而，荧光技术的实际应用面临着许多挑战。例如，生物样本的固有异质性总是会产生光学干扰。自发荧光等强背景往往会掩盖所需的信号。最后，光的波动性质限制了光学显微镜的空间分辨率。解决这些问题的关键在于使用可以调节荧光输出的化学结构。光致变色荧光分子是这些技术的核心，它们可以对外界光刺激做出响应，在两种颜色或亮暗状态之间交替发射。例如，分子荧光调制可以开启和关闭荧光团。这一特性支持超分辨率，其分辨率比长期存在的衍射极限障碍提高了一个数量级。以特定频率对这些探针进行可逆调制，可以极大地放大带有频率的目标信号，同时抑制干扰和自发荧光。本综述概述了恒定激发与振荡荧光之间的基本联系。为了创造将恒定激发转换为振荡发射的分子，我们合成了光致变色探针，并在超分辨率成像和频域成像中证明了它们的概念验证。首先，我们介绍了将恒定激发转换为振荡发射的分子设计，这是荧光调制的关键步骤。然后我们讨论了使用荧光调制的各种技术：超分辨率成像、双色成像、相敏锁定检测和频域成像。最后，我们提出了两个生物学应用，以展示光致变色荧光成像的强大功能。由于合成的光致变色探针可以更小、更通用且在高性能调制实验中更高效，因此它们为光致变色荧光蛋白提供了补充。尽管仍然存在新的挑战，但我们预计光致变色成像和检测将有一个光明的未来。
