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.
