High-resolution infrared imaging of biological samples with third-order sum-frequency generation microscopy.

We studied the use of vibrationally resonant, third-order sum-frequency generation (TSFG) for imaging of biological samples. We found that laser-scanning TSFG provides vibrationally sensitive imaging capabilities of lipid droplets and structures in sectioned tissue samples. Although the contrast is based on the infrared-activity of molecular modes, TSFG images exhibit a high lateral resolution of 0.5 µm or better. We observed that the imaging properties of TSFG resemble the imaging properties of coherent anti-Stokes Raman scattering (CARS) microscopy, offering a nonlinear infrared alternative to coherent Raman methods. TSFG microscopy holds promise as a high-resolution imaging technique in the fingerprint region where coherent Raman techniques often provide insufficient sensitivity.