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.

Abstract. 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.

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.

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 β 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 β 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 β far from resonance and the optimal excitation is within the 800–900 nm range. The impact is larger for backward collected SHG.

Abstract. 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 ðF SHG ∕B SHG Þ creation and the reduced scattering coefficient μ 0 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 0 s obtained is similar to that from bulk optical measurements and that the ðF SHG ∕B SHG Þ 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 0 s and ðF SHG ∕B SHG Þ 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.

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.

We demonstrate three-dimensional ͑3D͒ 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. © 2011 American Institute of Physics. ͓doi:10.1063/1.3583379͔Liquid-based variable-focus microlenses are emerging as important components in optical imaging due to their compact structure, high transmission and simple fabrication. 1,2Variable-focus liquid microlenses have been demonstrated by different mechanisms, including mechanical-wetting using liquid pressure, 3 electrowetting of a liquid droplet 2,4 and actuation of stimuli-responsive materials. 5 The optical properties of a liquid lens depend heavily on its geometric profile which is determined by many fabrication factors, such as surface smoothness and control of surface wetting. Accurate three-dimensional ͑3D͒ profiling, therefore, is critical to the optimization of the fabrication process and the optical properties of liquid microlenses. Conventional goniometers are incapable of 3D measurements, and they are not applicable where the surrounding structure has poor optical transparency. Limited by the characteristics of liquids, the liquid-toliquid interface can hardly be directly measured by laser range finder or mechanical profiler which is normally used for solid microlenses. 6,7 Here, we report on the use of a Shack-Hartmann wave front sensor for performing accurate 3D surface profiling of the liquid-liquid interfaces in liquid microlenses. To demonstrate our method, the example we used was a variable-focus liquid microlens actuated by thermoresponsive hydrogels. 5,8,9 A physical model is presented to calculate the surface profile from the obtained wave front profile through the liquid lens. The hydrogel-actuated liquid microlens was measured at focal lengths of 15.2 mm, 23.0 mm, and 34.7 mm, respectively. The liquid-liquid interface was found to have good spherical shape at the center, but become highly linear near the margin of the aperture. The measured water-oil interface was studied by simulation software and the simulated focal length and aberration coefficients are well matched with experiment results. The spherical aberration of the liquid lens was found to increase with decreasing focal lengths. The 3D surface profiling using Shack-Hartmann wave front sensor accomplishes a comprehensive analysis of the surface and determination of optical properties of liquid microlenses. Figure 1͑a͒ shows the cross section schematic and an image of the tunable liquid microlens actuated by a thermoresponsive hydrogel. The fabrication process was based on liquid phase photopolymerization. 8 The inner s...

We present a 3-D surface profiling method for microlenses that utilizes a Shack-Hartmann wavefront sensor. This method applies to both solid microlenses and liquid-liquid interfaces in liquid microlenses. The wavefront at the aperture stop of a microlens is measured by a Shack-Hartmann wavefront sensor and is then used to calculate the 3-D surface profile of the microlens. Three types of microlenses-a photoresist microlens, a hydrogel-driven tunable liquid lens, and an electrowettingdriven tunable liquid lens-were fabricated and measured. The variable-focus liquid lenses were tested within a wide focal length range. The obtained surface profiles were fitted to spherical and conical surface models to study their geometrical properties. The photoresist microlens was found to be approximately spherical. For the hydrogel-driven microlens, the profile was smooth and nearly spherical at the center but became steep and linear at the aperture edges. The electrowetting-driven liquid lens was also fitted better with the conical model, and its conic constant was determined. The obtained surface profiles were used to estimate the optical properties of microlenses in an optical analysis software package. The comparison between the simulation and experiment results indicated that the accuracy of the estimation is rough and the error could be due to the wavefront measurement and surface fitting approximation.[2011-0321.R1]