Advances in Vibrationally Resonant Sum-Frequency Generation Microscopy

Vibrationally resonant sum-frequency generation microscopy is a member of the family of nonlinear optical microscopy techniques. It enables chemically selective imaging of non-centrosymmetric structures. We present the latest advances in this field. OCIS codes: 180.4315, 190.4223 1. The emerging field of sum-frequency generation microscopy Second-harmonic generation (SHG) is a second-order optical effect, which is sensitive to the presence of noncentrosymmetrically arranged molecular structures. Since the 1990s, SHG microscopy rapidly ascended as a method for visualizing key structural protein complexes in tissues and cells [1, 2] Facilitated by the production of commercial SHG imaging modalities, the technique has become a staple of the nonlinear optical imaging approach and has found its way into many research laboratories worldwide. Applications range from the visualization of collagen in numerous tissues such as skin, bone, tendon, lung, liver and kidneys, to the imaging of myosin complexes in muscles and microtubules in individual cells. Yet, the contrast in standard SHG microscopy is not spectroscopic in nature. Virtually all SHG imaging studies are based on the use of a near-infrared light source such as a Ti:sapphire laser, which produces a nonresonant SHG signal in biological materials. Under these conditions, the signal contains information about the spatial organization of the structures, but its nonresonant character is unspecific about which part of the molecule contributes to the detected signal. In the case of collagen I, many studies have been devoted to connect the polarization properties of the SHG signal to the orientation of particular chemical groups. Despite the numerous publications on SHG imaging of collagen and other structures, a direct relation between the nonlinear signal and the molecular structure remains obscure, because SHG is not chemically selective. Sum-frequency generation (SFG), is a technique related to SHG with the important exception that SFG can be operated under vibrationally resonant conditions. SFG spectroscopy employs two laser beams, one in the visible/NIR range (ωvis), and another in the mid-IR range (ωIR). Vibrational resonance is achieved when ωIR is close to the frequency of a molecular mode with the proper symmetry. The signal is detected at ωSFG = ωIR+ωvis, which is typically in the visible range of the spectrum, and can thus be easily observed with conventional photomultiplier tubes. Similar to SHG, SFG probes the second-order nonlinear susceptibility χ(2), but because SFG can be tuned into selected vibrational resonances, the second-order response can be assigned to specific chemical groups of the molecule. It is clear that SFG spectroscopic contrast would complement the existing contrast in a nonlinear, multi-modal optical microscope. Several attempts have been made to design an excitation and detection configuration for efficient SFG microscopic imaging. Inspired by the configuration used in SFG spectroscopy experiments, the first SFG microscopes were based on wide-field illumination (illumination area is hundreds of μm2) of the sample, where the SFG radiation is captured by a high numerical aperture (NA) lens to form an image on a camera [3]. However, modern nonlinear optical (NLO) imaging techniques, such as SHG, two-photon excited fluorescence (TPEF) and coherent Raman scattering (CRS) microscopy, are based on laser scanning technologies to rapidly acquire images. In addition, NLO microscopes are driven by high-repetition rate ultrafast lasers, which are user-friendly and exhibit an optimized balance between peak power and average power for rapid imaging. On the other hand, most SFG microscopes to date have used amplified laser systems, whose low repetition rates are not optimized for laser scanning microscopy. [4–6] Unfortunately, the combination of non-ideal light sources and incompatible excitation schemes has contributed to the perception that SFG microscopy is not suitable for high-resolution, fast laser-scanning imaging applications. NM4C.2.pdf Optics in the Life Sciences 2017 (BODA, NTM, OMP, OTA, Brain) © 2017 OSA