Project Summary / Abstract (30 line maximum) This research, in response to the PAR-19-253, “Focused Technology Research andDevelopment,” aims to pioneer new advances in biological optical microscopy. Methods such as the development of fluorescent proteins, single molecule fluorescence detection, single molecule fluorescence resonance energy transfer (smFRET) and super-resolution microscopy enabled molecular level study of in vitro and live cells of increasing complexity. The single molecule methods allowed researchers to observe kinetic pathways and transient states unobservable with bulk methods. Despite recent advances, the existing optical probes have limitations. Fluorescent proteins are comparable in size to the proteins they label and photobleach quickly. In situ labeling of cytosol proteins is possible, but in vitro labeling methods are much preferred and there are no reliable methods to introduce these proteins into cytosol of cells. This research will address these grand challenges by fundamentally expanding the toolbox of optical microscopy. Aim 1 will develop new methods to introduce proteins labeled in vitro with organic dyes directly into the cytosol of cells and the insertion of dye-labeled membrane proteins into cell membranes, thereby expanding the application of optical probes to new biological systems. These methods will be used to insert up-converting nanoparticle (UCNP) probes into live cells to allow the long- term tracking of specific individual proteins from minutes to months with nanometer spatial resolution. This technology will also allow the controllable transfection of cells with multiple genes. Aim 2 will fundamentally improve the temporal resolution of smFRET to ≤ 100𝜇𝑠 and develop smFRET methods that can span across cell membranes. Aim 3 will extend biological optical microscopy to access the temporal and spatial scales of molecular motion. Here, UCNPs will be used to measure the continuous transport of cargos by dynein in DRG neurons capable of resolving single molecular steps with one millisecond time resolution over a distance of 900 𝜇𝑚. Using plasmonic optical probes, this work aims to achieve ~ 100 𝑛𝑠 time resolution and < 1 𝑛𝑚 spatial resolution in live cells. By the end of the 4-year funding period, a device will be demonstrated that is able to introduce controllable numbers of nanoparticles, proteins, and multiple genes and promoters into 1000s of cells with high survival rates. The cells will be transferred onto microscope coverslips or microfluidic cells suitable for high-resolution optical microscopy. An instrument capable of 100𝜇𝑠 smFRET will have been used to study the dynamics of G-protein couped receptors (GPCRs). Another instrument will be built to improve the time resolution of sub-nanometer movement to by up to ~ 100 𝑛𝑠. With this instrument, the real-time visualization of the motion of molecular systems may be possible.