Ultrasound-enabled two-photon FRET microscopy

描述（申请人提供）：荧光共振能量转移（FRET）可以作为纳米尺度的分子标尺。当用于成像应用时，它是一个高度敏感的供体-受体分子构型报告器。在大多数情况下，FRET是利用标准的单光子激发。将FRET扩展到双光子激发通常受到供体和受体流血问题的阻碍，这就要求技术上复杂的光谱分解算法或荧光寿命测量。我们建议用双光子激发大大简化FRET的检测。我们的目标是建立利用超声波调制FRET信号的可行性。由于FRET在纳米尺度上对供体-受体对之间的距离很敏感，声波产生的机械压缩和张力可能会调制FRET信号，作为一个有用的特征来区分FRET与供体和受体的穿透，这是多光子FRET的一个特殊问题。特别是，我们建议进行初步实验，以建立超声调制FRET的前提。我们将研究两种类型的样品：组织（或类组织环境）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秀丽隐杆线虫体内成像中的应用。FRET是一个功能成像的强大工具。我们相信，我们的混合超声光学技术将显著提高FRET信号的隔离，因此有利于任何基于FRET的功能成像应用。此外，当与双光子激发相结合时，我们的技术应该很好地适应于厚组织成像，从而使体内成像界受益。

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.