Ultrasound-enabled two-photon FRET microscopy

描述(申请人提供)：荧光共振能量转移(FRET)可用作纳米尺度的分子标尺。当用于成像应用时，它是供体-受体分子构型的高度灵敏的报告。在大多数情况下，FRET用于标准的单光子激发。FRET到双光子激发的扩展通常受到施主和受主渗漏问题的阻碍，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来大大简化FRET的检测。我们的目标是确定使用超声波调制FRET信号的可行性。由于FRET在纳米尺度上对供体-受体之间的距离很敏感，声波产生的机械压缩和张力很可能调制FRET信号，作为区分FRET与供体和受体出血的有用信号，这是多光子FRET中的一个特殊问题。特别是，我们建议进行初步实验，以建立超声波调节FRET的前提。我们将研究两种类型的样本：在组织(或类似组织的环境)中遗传编码的FRET探针，以及在溶液中标记的微泡。在前一种情况下，我们将研究我们的技术在线虫体内成像中的应用。FRET是一种强大的功能成像工具。我们相信，我们的超声-光学混合技术将显著改善FRET信号的隔离，因此对任何基于FRET的功能成像应用都是有益的。此外，当结合这里提出的双光子激发时，我们的技术应该能够很好地适应厚组织成像，从而使体内成像界受益。 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的活体显微镜。

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.