Merging Vibrational Spectroscopy with Fluorescence Microscopy: Combining the Best of Two Worlds

Vibrational spectroscopy and fluorescence spectroscopy have historically been two established but separate fields of molecular spectroscopy. While vibrational spectroscopy provides exquisite chemical information, fluorescence spectroscopy often offers orders of magnitude higher detection sensitivity. However, they each lack the advantages of each other. In recent years, a series of novel nonlinear optical spectroscopy studies have been developed that merge both spectroscopies into a single double-resonance process. These techniques combine the chemical specificity of Raman or infrared (IR) spectroscopy with the superb detection sensitivity and spatial resolution of fluorescence microscopy. Many facets have been explored, including Raman transition versus IR transition, time domain versus frequency domain, and spectroscopy versus microscopy. Notably, single-molecule vibrational spectroscopy has been achieved at room temperature without the need for plasmonics. Even superresolution vibrational imaging beyond the diffraction limit was demonstrated. This review summarizes the growing field of vibrational-encoded fluorescence microscopy, including key technical developments, emerging applications, and future prospects.