IDBR: Instrument Development for In Situ FIONA (Fluorescence Imaging with One Nanometer Accuracy)

该奖项用于在单分子水平上观察生物体中的生物分子马达的仪器，具有1纳米的空间精度和1毫秒的时间分辨率。PI通过观察体外和培养细胞中的分子马达获得了这种能力。这种能力被称为一纳米精度荧光成像（FIONA）。根据目前的合同，PI将扩展这一仪器，使其能够在现场使用，以调查小型蠕虫，即扁虫，地中海Schmidtea mediterranea和线虫C. elegans。FIONA避免了不必要的背景荧光，因为荧光团被激发，但周围没有太多。然而，如果荧光物体不靠近表面，普通的全内反射（TIR）将无法激发荧光团。在这个项目中，FIONA跟踪将应用于荧光标记的神经元和细胞器，这些神经元和细胞器隐藏在活体动物体内深处（半微米到一微米以上）。原位FIONA将通过仪器的三个发展来完成，使蠕虫内部深处的神经元能够兴奋：(a)用2光子激发的Epifluorescence，而不是1光子TIR激发。双光子激发具有巨大的技术优势，在激发荧光粒子的同时保持蠕虫的存活，并聚焦光，因此只有感兴趣的荧光团被激发。特别是，秀丽隐杆线虫在2光子激发下可以无限期地存活，而在1光子激发下，线虫在几分钟内死亡。此外，由于双光子激发固有地只在垂直方向上激发~1微米，因此无论人们在虫体内观察多深（高达~ 250 nm，高数值孔径透镜的工作距离），自身荧光都是最小的。(b)利用逆对称波导（RSW）的一种新的独特形式的TIR将用于激发荧光分子。与使用传统TIR的常规FIONA一样，这将通过通常的单光子激发来完成。另一方面，TIR- rsw将能够在距离表面1微米的范围内选择性激励电机货物，而常规TIR只能在0.1微米范围内进行激励。(c)还将使用另一种独特形式的红外光谱，涉及远程表面等离子激元极化子。这就产生了一个具有相对较大穿透深度（超过一微米）的远程行进光束，它应该消除背景信号，同时仍然允许激发单个荧光团。此外，最近发现LR-SPP具有可调节的穿透深度，这在各种应用中可能很重要。一般来说，LR-SPP是TIR-RSW的替代方案。PI发明的FIONA被《科学》杂志评为2003年十大技术之一，并被许多团体采用。如果将这项技术推广到活的生物体上，成功的话，将会向更多的研究人员开放这项技术，包括秀丽隐杆线虫和涡虫，甚至可能是其他活的生物体。世界各地的合作者都可以使用这种显微镜。这项工作将由研究生和博士后完成，并在会议上传播，这将是对前沿生物物理学的优秀培训。在吸引女性（目前有两名女性在PI小组中）和来自欠发达国家的学生方面，PI有着良好的记录。（该学院目前有六名来自土耳其的学生。）PI在他的小组中还有两名本科生，他们也将参与该仪器的开发。

This award is for an instrument for looking at biological molecular motors in a living organism at the single molecule level, with 1 nm spatial accuracy, and 1 msec temporal resolution. The PI has achieved this ability by looking at molecular motors in vitro and in cultured cells. This ability is called Fluorescence Imaging with One Nanometer Accuracy (FIONA). Under the current award, the PI will extend this instrumentation so that it can be used in situ, to investigate small worms, namely the flatworm planarian, Schmidtea mediterranea, and the roundworm C. elegans. FIONA avoids unwanted background fluorescence because the fluorophore is excited, but not much around it. However, if the fluorescent objects are not in close proximity to the surface, ordinary Total Internal Reflection (TIR) will not be useful in exciting fluorophores. In this project, FIONA tracking will be applied to fluorescently labeled neurons and organelles that are hidden deep (half micron to over one micron deep) inside a live animal. In situ FIONA will be done via three developments in instrumentation, enabling the excitation of neurons deep within the worms: (a) Epifluorescence with 2-photon excitation, instead of 1-photon TIR excitation. 2-photon excitation has the enormous technical advantage of keeping the worms alive while exciting the fluorescent particles, as well as focusing the light so only the fluorophore of interest gets excited. In particular, C. elegans stays alive indefinitely via 2-photon excitation, whereas with epifluorescence, 1- photon excitation, the worm dies within a few minutes. Furthermore, the autofluorescence, no matter how deeply one looks within the worm (up to ~ 250 nm, the working distance of a high-numerical aperture lens), is minimized because 2-photon excitation inherently excites only ~1 micron in the vertical direction. (b) A new and unique form of TIR, which uses Reverse Symmetry Waveguides (RSW), will be used to excite fluorescent molecules. Like regular FIONA, which uses conventional TIR, this will be done by the usual 1-photon excitation. On the other hand, TIR-RSW will enable selective excitation of motor-cargoes within 1 micron of the surface, in contrast to 0.1 micron for regular TIR. (c) Another unique form of TIR involving Long-Range Surface Plasmon Polaritons (LR-SPP) will also be used. This creates a long-range traveling beam which has relatively large penetration depth (more than one micron), which should eliminate the background signal, while still allowing excitation of the single fluorophores. In addition, LR-SPP has recently been found to have adjustable penetration depth, which may be important in various applications. In general, LR-SPP is an alternative to TIR-RSW.The PI invented FIONA, which was rated among the top ten techniques in 2003 by Science magazine and has been adopted by many groups. The extension to living organisms, if successful, should open the technique to many more researchers, in the wide community of C. elegans and planarian, and possibly even among other living organisms. The microscope will be available to collaborators around the world. The work, which will be done by graduate students and post-docs, and disseminated at conferences, will be excellent training in cutting edge biophysics. The PI has an excellent track record in involving women (two currently in the PI's group) and students from less developed nations. (The PI currently has six students from Turkey.) The PI also has two undergraduate students in his group, who will also participate in the development of this instrument.