Building a lightsheet system with dual-color fluorescence anisotropy capability for mapping protein-protein interactions in live cells

Project Summary: protein-protein used combine becoming and designing for adapted used this imaging detector utilized different channel lightsheet pipeline handling fluorescence Fluorescence anisotropy (or polarization) is routinely used for investigating interactions at high resolution in cellular environments. This imaging approach is in onjunction with TIRF, confocal or epi-illumination and lately there have been efforts to i t with lightsheet. Among the available fluorescence imaging modalities, lightsheet is a method of choice for 3D imaging of live samples because of its fast-imaging speed gentleness. We ropose to leverage our experience in building lightsheet systems and polarization optics to advance the field of fluorescence polarization i maging. Our aim this proposal i s to combine these two modalities in a manner such that it can easily be and used for biological applications and quantitative analysis. Our previous research has a specifically designed Wollaston prism for imaging single color fluorescence anisotropy. In application, we propose to build a dual-color optical splitter for fluorescence polarization which will employ the Wollaston prism for polarization separation and a single area such as sCMOS camera (Aim 1a). Fabricated nanostructured pinhole arrays will be to characterize the optical splitter for field dependent distortions, transmission losses in channels (due to various optical components), and to validate the registration of multi- for polarization computation. We will also combine this splitter with the home-built systems for 3D live imaging of cells and embryos (Aim 1b). A tailored image analysis along with a user-friendly graphical user interface (GUI) will be developed for data and quantitative image analysis (Aim 2). Our proposed approach would allow us t o map anisotropy for investigating protein-protein interactions in 3D in live samples. c p