Ultrasound-enabled two-photon FRET microscopy

说明（申请人提供）：荧光共振能量转移（FRET）可作为纳米级分子标尺。当用于成像应用时，它是供体-受体分子构型的高灵敏度报告物。在大多数情况下，FRET与标准单光子激发一起使用。FRET扩展到双光子激发通常受到供体和受体渗漏问题的阻碍，这要求技术上复杂的光谱解混算法或荧光寿命测量。我们建议大大简化检测FRET与双光子激发。 我们的目标是建立使用超声波调制FRET信号的可行性。由于FRET对纳米级供体-受体对之间的距离敏感，因此由声波产生的机械压缩和张力可能调制FRET信号，作为区分FRET与供体和受体渗透的有用特征，这是多光子FRET中的一个特殊问题。 特别是，我们建议进行初步的实验，以建立的前提下，超声调制FRET。我们将研究两种类型的样品：组织（或组织样环境）中的基因编码FRET探针和溶液中的标记微泡。在前一种情况下，我们将研究我们的技术在C. Elegans体内成像的应用。 FRET是功能成像的有力工具。我们相信，我们的混合超声光学技术将显着提高FRET信号的隔离，因此有利于任何FRET为基础的功能成像应用。此外，当与双光子激发耦合时，如这里所提出的，我们的技术应该很好地适应厚组织成像，从而有利于体内成像界。 公共卫生相关性：荧光共振能量转移（FRET）是一种强大的研究技术，可以成像组织中的细胞功能。我们建议使用超声波来改善FRET信号的产生。这将有利于任何基于FRET的成像应用，并将特别适用于厚组织中的体内显微镜检查。

DESCRIPTION (provided by applicant): Fluorescence resonance energy transfer (FRET) can serve as a nanoscale molecular ruler. When used in imaging applications, it is a highly sensitive reporter of donor-acceptor molecular configuration. In most cases, FRET is utilized with standard one-photon excitation. Extensions of FRET to two-photon excitation are generally hampered by the problem of donor and acceptor bleed through, which imposes the requirement of technically complicated spectral unmixing algorithms or fluorescence lifetime measurements. We propose to considerably simplify the detection of FRET with two-photon excitation. Our goal is to establish the feasibility of modulating FRET signal using ultrasonic waves. Since FRET is sensitive to distance between donor-acceptor pairs at the nanoscale, mechanical compression and tension produced by sound waves are likely to modulate the FRET signal, serving as a useful signature to distinguish FRET from donor and acceptor bleed-through, which is a particular problem in multiphoton FRET. In particular, we propose to conduct preliminary experiments to establish the premise that ultrasound modulates FRET. We will investigate two types of samples: genetically encoded FRET probes in tissue (or tissue-like environments), and labeled microbubbles in solution. In the former case, we will investigate the application of our technique to in-vivo imaging in C.Elegans. FRET is a powerful tool for functional imaging. We believe that our hybrid ultrasound-optical technique will significantly improve the isolation of FRET signals and therefore be of benefit to any FRET-based functional imaging application. Moreover, when coupled with two-photon excitation, as proposed here, our technique should be well adapted to thick tissue imaging, thereby benefiting the in-vivo imaging community. PUBLIC HEALTH RELEVANCE: Fluorescence resonance energy transfer (FRET) is a powerful research technique to image cellular function in tissue. We propose to improve FRET signal generation with the use of ultrasound. This will benefit any FRET-based imaging applications, and will be specifically adapted to in-vivo microscopy in thick tissue.