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.
Cryopreservatives like dimethyl sulfoxide and glycerol are common agents that prevent cellular damage upon freezing of tissues or entire organisms. Although the cryopreservation capabilities of these compounds have been known empirically for years, much is unknown about the actual perfusion and distribution of the agents within cells on the microscopic scale. In this contribution, we report on studies that aim to uncover the dynamic distribution of cryopreservatives in the tissue with the aid of stimulated Raman scattering microscopy, enabling a direct and real-time view of the cellular loading and accumulation dynamics of these agents at the micrometer scale.
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.
In coherent Raman scattering microscopy, the sample is examined through the Raman-active modes. Although many molecular modes display a nonzero Raman cross-section, some modes are intrinsically weak and are better viewed through their dipole-allowed (IR-active) transition. A more complete spectroscopic assessment of the sample would thus include a mechanism to prove both Raman and IR-active modes. In this contribution, we combine coherent Raman microscopy with second- and third-order sum-frequency generation microscopy, providing sensitivity to all optically accessible vibrational modes. All techniques can be carried out on the same laser-scanning imaging platform, generating images at fast acquisition rates and with high spatial resolution. We show examples where the combination of these techniques reveal new information about the tissue.