SHG Fiberscopy Assessment of Collagen Morphology and Its Potential for Breast Cancer Optical Histology.

OBJECTIVE: This study is to investigate the feasibility of our recently developed nonlinear fiberscope for label-free in situ breast tumor detection and lymph node status assessment based on second harmonic generation (SHG) imaging of fibrillar collagen matrix with histological details. The long-term goal is to improve the current biopsy-based cancer paradigm with reduced sampling errors. METHODS: In this pilot study we undertook retrospective SHG imaging study of ex vivo invasive ductal carcinoma human biopsy tissue samples, and carried out quantitative image analysis to search for collagen structural signatures that are associated with the malignance of breast cancer. RESULTS: SHG fiberscopy image-based quantitative assessment of collagen fiber morphology reveals that: 1) cancerous tissues contain generally less extracellular collagen fibers compared with tumor-adjacent normal tissues, and 2) collagen fibers in lymph node positive biopsies are more aligned than lymph node negative counterparts. CONCLUSION/SIGNIFICANCE: The results demonstrate the promising potential of our SHG fiberscope for in situ breast tumor detection and lymph node involvement assessment and for offering real-time guidance during ongoing tissue biopsy.

A biopsy-needle compatible varifocal multiphoton rigid probe for depth-resolved optical biopsy.

In this work, we report a biopsy-needle compatible rigid probe, capable of performing three-dimensional (3D) two-photon optical biopsy. The probe has a small outer diameter of 1.75 mm and fits inside a gauge-14 biopsy needle to reach internal organs. A carefully designed focus scanning mechanism has been implemented in the rigid probe, which, along with a rapid two-dimensional MEMS scanner, enables 3D imaging. Fast image acquisition up to 10 frames per second is possible, dramatically reducing motion artifacts during in vivo imaging. Equipped with a high-numerical aperture micro-objective, the miniature rigid probe offers a high two-photon resolution (0.833 × 6.11 µm, lateral × axial), a lateral field of view of 120 µm, and an axial focus tuning range of 200 µm. In addition to imaging of mouse internal organs and subcutaneous tumor in vivo, first-of-its-kind depth-resolved two-photon optical biopsy of an internal organ has been successfully demonstrated on mouse kidney in vivo and in situ.

Spectro-temporal dispersion management of femtosecond pulses for fiber-optic two-photon endomicroscopy.

The emerging fiber-optic two-photon endomicroscopy technology holds a strong promise for enabling translational applications of nonlinear optical imaging. Effective femtosecond pulse dispersion management is critical for achieving high-quality imaging. Here we report systematic analyses and performance characterization of a dual-fiber spectro-temporal dispersion management scheme involving a grating pair as the pulse stretcher. Compared with conventional linear-only compensation, the grating-based spectro-temporal compensation also takes into account nonlinear effects and enhances the two-photon signal by ~3-fold as experimentally demonstrated. Numerical simulations were carried out to systematically investigate the influence of several key design parameters on the overall compensation efficacy. Furthermore, comprehensive performance comparison with an ideal grism-pair counterpart reveals that a grating-pair stretcher affords much higher power throughput and thus is preferable for portable endomicroscopy systems with limited laser source power.

Fitting-free algorithm for efficient quantification of collagen fiber alignment in SHG imaging applications.

Collagen fiber alignment derived from second harmonic generation (SHG) microscopy images can be important for disease diagnostics. Image processing algorithms are needed to robustly quantify the alignment in images with high sensitivity and reliability. Fourier transform (FT) magnitude, 2D power spectrum, and image autocorrelation have previously been used to extract fiber information from images by assuming a certain mathematical model (e.g. Gaussian distribution of the fiber-related parameters) and fitting. The fitting process is slow and fails to converge when the data is not Gaussian. Herein we present an efficient constant-time deterministic algorithm which characterizes the symmetricity of the FT magnitude image in terms of a single parameter, named the fiber alignment anisotropy R ranging from 0 (randomized fibers) to 1 (perfect alignment). This represents an important improvement of the technology and may bring us one step closer to utilizing the technology for various applications in real time. In addition, we present a digital image phantom-based framework for characterizing and validating the algorithm, as well as assessing the robustness of the algorithm against different perturbations.

Nonlinear optical endomicroscopy for label-free functional histology in vivo.

This manuscript reports on the first two-photon, label-free, metabolic imaging of biological tissues in vivo at histological resolution on an extremely compact, fiber-optic endomicroscopy platform. This system provides new opportunities for performing non-invasive and functional histological imaging of internal organs in vivo, in situ and in real time. As a routine clinical procedure, traditional histology has made significant impacts on medicine. However, the procedure is invasive and time consuming, suffers random sampling errors, and cannot provide in vivo functional information. The technology reported here features an extremely compact and flexible fiber-optic probe ~2 mm in diameter, enabling direct access to internal organs. Unprecedented two-photon imaging quality comparable to a large bench-top laser scanning microscope was achieved through technological innovations in double-clad fiber optics and miniature objective lenses (among many others). In addition to real-time label-free visualization of biological tissues in situ with subcellular histological detail, we demonstrated for the first time in vivo two-photon endomicroscopic metabolic imaging on a functioning mouse kidney model. Such breakthroughs in nonlinear endoscopic imaging capability present numerous promising opportunities for paradigm-shifting applications in both clinical diagnosis and basic research.

Experimental and simulation study of the wavelength dependent second harmonic generation of collagen in scattering tissues.

We report on the wavelength dependence of second harmonic generation (SHG) of collagen in scattering tissues over the wavelength range of 800-1200 nm. The study incorporates inclusion of the molecular hyperpolarizability ß of collagen and optical scattering, both of which are wavelength dependent. Using 3D SHG imaging and Monte Carlo simulations, we find the wavelength dependence of ß is not well described by a two-state model based on known absorption bands. We further find that longer wavelength excitation is inefficient as the reduction in scattering is overcome by the decreased ß far from resonance and the optimal excitation is within the 800-900 nm range. The impact is larger for backward collected SHG.

Simultaneous determination of the second-harmonic generation emission directionality and reduced scattering coefficient from three-dimensional imaging of thick tissues.

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 (FSHG/BSHG)creation and the reduced scattering coefficient µs', 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 µs' obtained is similar to that from bulk optical measurements and that the (FSHG/BSHG)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 µs' and (FSHG/BSHG)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.

Nonlinear optical microscopy and ultrasound imaging of human cervical structure.

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.

Goniometric measurements of thick tissue using Monte Carlo simulations to obtain the single scattering anisotropy coefficient.

The scattering anisotropy, g, of tissue can be a powerful metric of tissue structure, and is most directly measured via goniometry and fitting to the Henyey-Greenstein phase function. We present a method based on an independent attenuation measurement of the scattering coefficient along with Monte Carlo simulations to account for multiple scattering, allowing the accurate determination of measurement of g for tissues of thickness within the quasi-ballistic regime. Simulations incorporating the experimental geometry and bulk optical properties show that significant errors occur in extraction of g values, even for tissues of thickness less than one scattering length without modeling corrections. Experimental validation is provided by determination of g in mouse muscle tissues and it is shown that the obtained values are independent of thickness. In addition we present a simple deconvolution-based method and show that it provides excellent estimates for high anisotropy values (above 0.95) when coupled with an independent attenuation measurement.

Three-dimensional surface profiling and optical characterization of liquid microlens using a Shack-Hartmann wave front sensor.

We demonstrate three-dimensional (3D) surface profiling of the water-oil interface in a tunable liquid microlens using a Shack-Hartmann wave front sensor. The principles and the optical setup for achieving 3D surface measurements are presented and a hydrogel-actuated liquid lens was measured at different focal lengths. The 3D surface profiles are then used to study the optical properties of the liquid lens. Our method of 3D surface profiling could foster the improvement of liquid lens design and fabrication, including surface treatment and aberration reduction.

Fast localized wavefront correction using area-mapped phase-shift interferometry.

We propose an innovative method for localized wavefront correction based on area-mapped phase-shift (AMPS) interferometry. In this Letter, we present the theory and then experimentally compare it with a previously demonstrated method based on spot-optimized phase-stepping (SOPS) interferometry. We found that AMPS outperforms SOPS interferometry in terms of speed by threefold, although in noisy environments the improvements may be larger. AMPS yielded similar point-spread functions (PSF) as SOPS for moderate system-induced aberrations, but yielded a slightly less ideal PSF for larger aberrations. The method described in this Letter may prove crucial for applications where the phase-stepping solution does not have sufficient speed.