Quantifying collagen structure in breast biopsies using second-harmonic generation imaging.

Quantitative second-harmonic generation imaging is employed to assess stromal collagen in normal, hyperplastic, dysplastic, and malignant breast tissues. The cellular scale organization is quantified using Fourier transform-second harmonic generation imaging (FT-SHG), while the molecular scale organization is quantified using polarization-resolved second-harmonic generation measurements (P-SHG). In the case of FT-SHG, we apply a parameter that quantifies the regularity in collagen fiber orientation and find that malignant tissue contains locally aligned fibers compared to other tissue conditions. Alternatively, using P-SHG we calculate the ratio of tensor elements (d(15)/d(31), d(22)/d(31), and d(33)/d(31)) of the second-order susceptibility χ(2) for collagen fibers in breast biopsies. In particular, d(15)/d(31) shows potential differences across the tissue pathology. We also find that trigonal symmetry (3m) is a more appropriate model to describe collagen fibers in malignant tissues as opposed to the conventionally used hexagonal symmetry (C6). This novel method of targeting collagen fibers using a combination of two quantitative SHG techniques, FT-SHG and P-SHG, holds promise for breast tissue analysis and applications to characterizing cancer in a manner that is compatible with clinical practice.

Quantification of collagen fiber organization using three-dimensional Fourier transform-second-harmonic generation imaging.

We present three-dimensional Fourier transform-second-harmonic generation (3D FT-SHG) imaging, a generalization of the previously reported two-dimensional FT-SHG, to quantify collagen fiber organization from 3D image stacks of biological tissues. The current implementation calculates 3D preferred orientation of a region of interest, and classifies regions of interest based on orientation anisotropy and average voxel intensity. Presented are some example applications of the technique which reveal the layered structure of collagen fibers in porcine sclera, and estimates the cut angle of porcine tendon tissues. This technique shows promising potential for studying biological tissues that contain fibrillar structures in 3D.

Quantitative second-harmonic generation microscopy for imaging porcine cortical bone: comparison to SEM and its potential to investigate age-related changes.

We propose the use of second-harmonic generation (SHG) microscopy for imaging collagen fibers in porcine femoral cortical bone. The technique is compared with scanning electron microscopy (SEM). SHG microscopy is shown to have excellent potential for bone imaging primarily due its intrinsic specificity to collagen fibers, which results in high contrast images without the need for specimen staining. Furthermore, this technique's ability to quantitatively assess collagen fiber organization is evaluated through an exploratory examination of bone structure as a function of age, from very young to mature bone. In particular, four different age groups: 1 month, 3.5 months, 6 months, and 30 months, were studied. Specifically, we employ the recently developed Fourier transform-second harmonic generation (FT-SHG) imaging technique for the quantification of the structural changes, and observe that as the bone develops, there is an overall reduction in porosity, the number of osteons increases, and the collagen fibers become comparatively more organized. It is also observed that the variations in structure across the whole cross-section of the bone increase with age. The results of this work show that quantitative SHG microscopy can serve as a valuable tool for evaluating the structural organization of collagen fibers in ex vivo bone studies.

The effect of keratoconus on the structural, mechanical, and optical properties of the cornea.

Keratoconus is an eye disorder wherein the cornea weakens due to structural and/or compositional anomalies. This weakened cornea is no longer able to preserve its normal shape against the intraocular pressure in the eye and therefore bulges outward, leading to a conical shape and subsequent distorted vision. Changes in structure and composition often manifest as a change in shape (or geometry) as well as in mechanical and optical properties. Thus, understanding the properties and structure of keratoconic corneas could help elucidate etiology and pathogenesis, to develop treatments, and to understand other diseases of the eye. In this review, we discuss the changes in structure, composition, and mechanical and optical properties of the cornea with keratoconus. Current treatments for keratoconus and a novel proposed treatment using two-photon excitation therapy are also discussed. The intended audiences are mechanical engineers, materials engineers, optical engineers, and bioengineers.

Nonlinear optical response from arrays of Au bowtie nanoantennas.

We demonstrate that the optical response of a single Au bowtie nanoantenna can be favorably modified to increase the local intensity by a factor of 10(3) in the feed gap region when a periodic array of antennas are used. We find that the array periodicity can be used to modulate and shape the spectral emission. An analysis of the emission confirms the presence of second-harmonic generation and two-photon photoluminescence, typical of gold nanostructures, but also reveals a portion of the emitted spectrum that cannot be attributed to a single multiphoton process. Our investigations have important implications for understanding the role of resonant nanostructures in designing optical antennas for next-generation photonic technologies.

Quantitative analysis of collagen fiber organization in injured tendons using Fourier transform-second harmonic generation imaging.

Fourier transform-second harmonic generation (FT-SHG) imaging is used as a technique for evaluating collagenase-induced injury in horse tendons. The differences in collagen fiber organization between normal and injured tendon are quantified. Results indicate that the organization of collagen fibers is regularly oriented in normal tendons and randomly organized in injured tendons. This is further supported through the use of additional metrics, in particular, the number of dark (no/minimal signal) and isotropic (no preferred fiber orientation) regions in the images, and the ratio of forward-to-backward second-harmonic intensity. FT-SHG microscopy is also compared with the conventional polarized light microscopy and is shown to be more sensitive to assessing injured tendons than the latter. Moreover, sample preparation artifacts that affect the quantitative evaluation of collagen fiber organization can be circumvented by using FT-SHG microscopy. The technique has potential as an assessment tool for evaluating the impact of various injuries that affect collagen fiber organization.

Photopatternable Polymeric Membranes for Optical Oxygen Sensors.

A new class of optical oxygen sensor that can be photopatternable by traditional UV lithography is presented. They are fabricated using photopatternable spin-on silicone (polydimethyl-siloxane, PDMS) with oxygen sensitive luminescent dyes. It has a good adhesion property and can be applied on glass or on photopolymer (SU-8) without any additional surface treatments. The optimum mixture composition for patternable oxygen sensitive membranes is investigated and its optical properties are characterized. Proof-of-concepts for two applications, intensity-based oxygen sensing with SU-8 based structure and self-calibration fluidic oxygen sensor, are described. These photopatternable optical membranes will find many applications wherever small patterns of oxygen sensitive membranes are required.