Project Summary I plan to add a single hyperspectral detection unit to a new nonlinear vibrational microscope developed in my lab. This detection unit is composed of a spectrograph and an electron-multiplying charge-coupled device (EMCCD) camera. This unit will enable single-molecule/super-resolution vibrational imaging. We will carry out the research in two stages. The first stage is to verify that the new vibrational imaging technique can detect vibrationally encoded photoluminescent signals of single molecules in standard samples. The second stage uses this single-molecule capacity to image biological systems with a sub- diffraction-limit optical resolution of <300 nm. This new development will revolutionize infrared imaging techniques by enabling unprecedented single-molecule sensitivity. Furthermore, this new single-molecule imaging technique can overcome the issue of limited color channels in photoluminescence (PL) based microscopy. Our long-term vision is to enable new hyperspectral imaging techniques for biophysical and biomedical applications, with unprecedented multiplexing color channels and a new approach of super- resolution imaging that can follow the time evolution of molecular functional groups during metabolism and other biological processes.