Optically modulated fluorescence bioimaging: visualizing obscured fluorophores in high background.
Optically modulated fluorescence bioimaging: visualizing obscured fluorophores in high background.
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DOI：
10.1021/ar400325y
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发表时间：
2014-05-20
影响因子：
18.3
通讯作者：
Dickson, Robert M.
中科院分区：
文献类型：
--
作者：
Hsiang, Jung-Cheng;Jablonski, Amy E.;Dickson, Robert M.
荧光显微镜和检测对于理解生物系统的组织和动态已变得不可或缺。具有改进的亮度、光稳定性和生物相容性的新型荧光团继续推动进一步的进步，但通常依赖于最小的背景。在非常高的生物背景下相互作用的可视化，特别是对于拷贝数非常低的蛋白质或结合复合物，仍然是一个主要挑战。我们没有关注荧光团的分子亮度，而是采用高灵敏度吸收光谱的原理来提高荧光生物成像的灵敏度和信号辨别力。利用动力学捕获暗态的长波长瞬态吸收，我们采用不同时调制背景荧光的分子调制方案。与基于高能光开关的恢复方案相比，这提高了灵敏度和易于实施，因为不需要内部染料参考或基于纳米颗粒的荧光团来将所需信号与背景分离。在本报告中，我们描述了荧光团的选择过程和识别过程，这些荧光团能够通过光学调制荧光来减少模糊背景。与使用较高能量二次激光器的热稳定光电开关不同，非常低能量的线圈照明会减少瞬态暗态，动态改变荧光并为每个可调制发射器提供特征调制时间尺度。这个过程被称为同步放大荧光图像恢复（SAFIRe）显微镜。通过理解和光学控制染料光物理学，我们可以独立于所有自发荧光背景选择性地调节所需的荧光团信号。由于没有收集背景信号，这将感兴趣的荧光转移到独特的检测频率，检测几乎受到散粒噪声限制。尽管荧光亮度略有提高，但 SAFIRe 通过基本上消除模糊的、未调制的背景，将信号可见度提高了 100 倍 (; , , )。虽然 SAFIRe 表现出宽广的线性动态范围，但我们已经证明了隐藏在 200 nM 模糊染料内的单分子信号恢复。除了通过背景减少实现信号恢复之外，每种染料还表现出指示其光物理动力学的特征调制频率。因此，这些特征时间尺度不仅提供了通过使用暗态寿命来扩展荧光成像维度的机会，而且还提供了基于光物理与扩散时间尺度来区分亚群动态的机会，甚至在可调节群体内也是如此。信号恢复和生物动力学观察调制的持续发展为研究自然环境中的一系列瞬态生物现象带来了巨大希望。通过开发在稳态照明下表现出显着暗态群体的各种荧光蛋白、有机染料和无机发射体，我们可以极大地扩展荧光成像的适用性，以探测低丰度复合物及其动力学。
Fluorescence microscopy and detection have become indispensible for understanding organization and dynamics in biological systems. Novel fluorophores with improved brightness, photostability, and biocompatibility continue to fuel further advances but often rely on having minimal background. The visualization of interactions in very high biological background, especially for proteins or bound complexes at very low copy numbers, remains a primary challenge. Instead of focusing on molecular brightness of fluorophores, we have adapted the principles of high-sensitivity absorption spectroscopy to improve the sensitivity and signal discrimination in fluorescence bioimaging. Utilizing very long wavelength transient absorptions of kinetically trapped dark states, we employ molecular modulation schemes that do not simultaneously modulate the background fluorescence. This improves the sensitivity and ease of implementation over high-energy photoswitch-based recovery schemes, as no internal dye reference or nanoparticle-based fluorophores are needed to separate the desired signals from background. In this Account, we describe the selection process for and identification of fluorophores that enable optically modulated fluorescence to decrease obscuring background. Differing from thermally stable photoswitches using higher-energy secondary lasers, coillumination at very low energies depopulates transient dark states, dynamically altering the fluorescence and giving characteristic modulation time scales for each modulatable emitter. This process is termed synchronously amplified fluorescence image recovery (SAFIRe) microscopy. By understanding and optically controlling the dye photophysics, we selectively modulate desired fluorophore signals independent of all autofluorescent background. This shifts the fluorescence of interest to unique detection frequencies with nearly shot-noise-limited detection, as no background signals are collected. Although the fluorescence brightness is improved slightly, SAFIRe yields up to 100-fold improved signal visibility by essentially removing obscuring, unmodulated background (; , , ). While SAFIRe exhibits a wide, linear dynamic range, we have demonstrated single-molecule signal recovery buried within 200 nM obscuring dye. In addition to enabling signal recovery through background reduction, each dye exhibits a characteristic modulation frequency indicative of its photophysical dynamics. Thus, these characteristic time scales offer opportunities not only to expand the dimensionality of fluorescence imaging by using dark-state lifetimes but also to distinguish the dynamics of subpopulations on the basis of photophysical versus diffusional time scales, even within modulatable populations. The continued development of modulation for signal recovery and observation of biological dynamics holds great promise for studying a range of transient biological phenomena in natural environments. Through the development of a wide range of fluorescent proteins, organic dyes, and inorganic emitters that exhibit significant dark-state populations under steady-state illumination, we can drastically expand the applicability of fluorescence imaging to probe lower-abundance complexes and their dynamics.
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影响因子：
15
作者：
Richards CI;Hsiang JC;Senapati D;Patel S;Yu J;Vosch T;Dickson RM
通讯作者：
Dickson RM
DOI：
10.1073/pnas.0808882105
发表时间：
2008-11-18
影响因子：
11.1
作者：
Marriott, Gerard;Mao, Shu;Yan, Yuling
通讯作者：
Yan, Yuling
影响因子：
64.8
作者：
Dickson, RM;Cubitt, AB;Moerner, WE
通讯作者：
Moerner, WE
影响因子：
2.3
作者：
Gratz, H;Penzkofer, A
通讯作者：
Penzkofer, A
影响因子：
15
作者：
Richards, Chris I.;Hsiang, Jung-Cheng;Khalil, Andrew M.;Hull, Nathan P.;Dickson, Robert M.
通讯作者：
Dickson, Robert M.