Polarization-Sensitive Sum-Frequency Generation Microscopy of Collagen Fibers

Abstract

Point-scanning sum-frequency generation (SFG) microscopy enables the generation of images of collagen I fibers in tissues by tuning into specific vibrational resonances of the polypeptide. It is shown that when collagen-rich tissues are visualized near the 2954 cm -1 stretching vibration of methylene groups, the SFG image contrast is higher compared to the contrast seen in nonresonant second-harmonic generation (SHG) imaging.Polarization and spectrally resolved analysis of the SFG signal as a function of fiber orientation in the CH-stretching range of the vibrational spectrum enabled a comparative characterization of the achiral tensor elements of collagen's second-order susceptibility. This analysis reveals that selected on-resonance tensor elements are enhanced over other elements, giving rise to a much stronger anisotropy ρ of the signal for SFG ( ρ≈15 ) compared to SHG ( ρ≈3 ). The improved anisotropy of the vibrationally resonant signal contributes to the higher contrast seen in the SFG tissue images.

Imaging the Nonlinear Susceptibility Tensor of Collagen by Nonlinear Optical Stokes Ellipsometry

“…Most commonly, the individual collagen fibers are considered to be lying within the image plane, resulting in polar tilt angles, q, of 90 , defined relative to the optical axis. This assumption holds reasonably well for highly aligned collagen tissues such as tendon samples sectioned along the major axis, and is used in numerous previous studies (15,29,30). However, for the meshlike collagen structures such as those found in skin tissues and basement membrane, the collagen fibers can no longer be considered to be lying within the imaging plane at each pixel.…”

“…The recovery of the orientation information of collagen has been one of the overall goals of polarization-dependent SHG measurements (28). The influence of q on the polarization-dependent nonlinear optical measurements has been explored and discussed qualitatively (29). However, quantitative recovery of q at each individual pixel of the image has remained an elusive goal.…”

Metrology of Multiphoton Microscopes Using Second Harmonic Generation Nanoprobes

“…The use of wavelengths beyond 1 µm raises implementation and metrology issues because microscope optics are not necessarily corrected nor characterized in this regime. Suitable microscope metrology procedures are also much needed for nonlinear techniques relying on multicolor excitation, such as coherent anti‐Stokes Raman Scattering (CARS), stimulated Raman scattering (SRS), sum‐frequency generation, or multicolor multiphoton imaging using wavelength mixing . Indeed, these methods typically use two laser beams with at least two different wavelengths, and require to maintain foci coalignment at the sub‐micrometer scale over a large field of view during beam scanning.…”

Advances in Vibrationally Resonant Sum-frequency Generation Microscopy

“…[8] Hence, the SFG and CRS techniques complement each other, offering the microscopist a rich palette of unique contrast mechanisms. Recent advances include the implementation of phase-sensitive detection [9], which enables a direct view of chemical group orientation of molecular compounds in tissue, and the use of polarization sensitive SFG [10], which allows a detailed examination of the tensorial properties of χ (2) -active materials in the tissue. Although these applications demonstrate the utility of SFG microscopy, several technical challenges remain.…”