Second-harmonic generation (SHG) microscopy has intrinsic contrast for imaging fibrillar collagen and has shown great promise for disease characterization and diagnostics. In addition to morphology, additional information is achievable as the initially emitted SHG radiation directionality is related to subresolution fibril size and distribution. We show that by two parameter fittings, both the emission pattern (F SHG /B SHG ) creation and the reduced scattering coefficient μ ′ s , can be obtained from the best fits between three-dimensional experimental data and Monte Carlo simulations. The improved simulation framework accounts for collection apertures for the detected forward and backward components. We apply the new simulation framework to mouse tail tendon for validation and show that the spectral slope of μ ′ s obtained is similar to that from bulk optical measurements and that the (F SHG /B SHG ) creation values are also similar to previous results. Additionally, we find that the SHG emission becomes increasingly forward directed at longer wavelengths, which is consistent with decreased dispersion in refractive index between the laser and SHG wavelengths. As both the spectral slope of μ ′ s and (F SHG /B SHG ) creation have been linked to the underlying tissue structure, simultaneously obtaining these parameters on a microscope platform from the same tissue provides a powerful method for tissue characterization.
The cervix softens and shortens as its collagen microstructure rearranges in preparation for birth, but premature change may lead to premature birth. The global preterm birth rate has not decreased despite decades of research, likely because cervical microstructure is poorly understood. Our group has developed a multilevel approach to evaluating the human cervix. We are developing quantitative ultrasound (QUS) techniques for noninvasive interrogation of cervical microstructure and corroborating those results with high-resolution images of microstructure from second harmonic generation imaging (SHG) microscopy. We obtain ultrasound measurements from hysterectomy specimens, prepare the tissue for SHG, and stitch together several hundred images to create a comprehensive view of large areas of cervix. The images are analyzed for collagen orientation and alignment with curvelet transform, and registered with QUS data, facilitating multiscale analysis in which the micron-scale SHG images and millimeter-scale ultrasound data interpretation inform each other. This novel combination of modalities allows comprehensive characterization of cervical microstructure in high resolution. Through a detailed comparative study, we demonstrate that SHG imaging both corroborates the quantitative ultrasound measurements and provides further insight. Ultimately, a comprehensive understanding of specific microstructural cervical change in pregnancy should lead to novel approaches to the prevention of preterm birth.
A system for making wavefront corrections for use in multiphoton microscopy has been constructed. Corrections are
made using a high-resolution nematic liquid crystal device which has a phase stroke of 2π. The device has a design
wavelength of 1064 nm. A simple way for setting the device up for lower wavelengths (here 800 nm) is presented. It
was found that the device has an undesired zero-order diffraction component of 30%. A scheme for filtering this portion
out is presented and it was demonstrated that this can eliminate the component completely. The device was used to
optically simulate a thin lens with a specified focal length, which was found to match within error bounds. Finally the
modulator was used to compensate for a mechanical defocus that was applied intentionally.