Infrared (IR) absorption microscopy is a general method for generating images with vibrational spectroscopic contrast. IR microscopy has been used in a myriad of applications, including imaging of excised tissue samples. One of the major drawbacks of the technique is the limited spatial resolution, which is too low to resolve intracellular details. We developed a new nonlinear optical imaging technique, called third-order sum-frequency generation (TSFG), which addresses this issue. This approach uses an infrared IR pulse to excite fundamental molecular vibrations and a near-infrared (NIR) pulse for two-photon upconversion, producing a visible signal. TSFG is sensitive to the same molecular modes as probed in IR absorption microscopy, but offers a spatial resolution that is one order of magnitude better and enables straightforward detection in the visible range of the spectrum. TSFG is a third-order optical imaging technique, and can be regarded as the IR analogue of coherent anti-Stokes Raman scattering (CARS). We show that TSFG enables fast laser-scanning microscopy of biological samples with a resolution of 0.5 micron or better.
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