Electronic-Resonance Coherent Anti-Stokes Raman Scattering Spectroscopy and Microscopy

Abstract

Advanced optical microscopy techniques, such as fluorescence and vibrational imaging, play a key role in science and technology. Both the sensitivity and the chemical selectivity are important for applications of these methods in biomedical research. However, there are few bioimaging techniques that could offer vibrational sensitivity and selectivity simultaneously. Here, we demonstrate electronic-resonance coherent anti-Stokes Raman scattering (ER-CARS) spectroscopy and microscopy with high sensitivity and high selectivity by using lock-in detection. With this ER-CARS strategy, we observed the low-wavenumber Raman spectra of various infrared dyes in solution at ultralow vibrational frequencies (from ∼20 to 330 cm–1) using a sharp edge filter and impulsive CARS excitation at low input power. We demonstrate low-frequency ER-CARS imaging of cells stained with infrared dyes using only 200 mW of input power, fundamentally mitigating photobleaching issues. We also show the application of ER-CARS in the detection of nonfluorescent molecules. Finally, we demonstrate the chemically selective capabilities of ER-CARS microscopy by imaging tissues stained with two different infrared dyes. These ER-CARS spectroscopy and microscopy results pave the way toward ultrasensitive Raman imaging of biological systems and present a pathway toward highly multiplexed selective imaging.