Spectroscopic characterization of oral epithelial dysplasia and squamous cell carcinoma using multiphoton autofluorescence micro-spectroscopy.

OBJECTIVE: Multiphoton autofluorescence microscopy (MPAM) has shown potential in identifying features that are directly related to tissue microstructural and biochemical changes throughout epithelial neoplasia. In this study, we evaluate the autofluorescence spectral characteristics of neoplastic epithelium in dysplasia and oral squamous cell carcinoma (OSCC) using multiphoton autofluorescence spectroscopy (MPAS) in an in vivo hamster model of oral neoplasia in order to identify unique signatures that could be used to delineate normal oral mucosa from neoplasia. MATERIALS/METHODS: A 9,10-dimethyl-1,2-benzanthracene (DMBA) hamster model of oral precancer and OSCC was used for in vivo MPAM and MPAS. Multiphoton Imaging and spectroscopy were performed with 780 nm excitation while a bandpass emission 450-650 nm was used for MPAM. Autofluorescence spectra was collected in the spectral window of 400-650 nm. RESULTS: MPAS with fluorescence excitation at 780 nm revealed an overall red shift of a primary blue-green peak (480-520 nm) that is attributed to NADH and FAD. In the case of oral squamous cell carcinoma (OSCC) and some high-grade dysplasia an additional prominent peak at 635 nm, attributed to PpIX was observed. The fluorescence intensity at 635 nm and an intensity ratio of the primary blue-green peak versus 635 nm peak, showed statistically significant difference between control and neoplastic tissue. DISCUSSION: Neoplastic transformation in the epithelium is known to alter the intracellular homeostasis of important tissue metabolites such as NADH, FAD, and PpIX, which was observed by MPAS in their native environment. A combination of deep tissue microscopy owing to higher penetration depth of multiphoton excitation and depth resolved spectroscopy could prove to be invaluable in identification of cytologic as well as biomolecular spectral characteristic of oral epithelial neoplasia. Lasers Surg. Med. 49:866-873, 2017. © 2017 Wiley Periodicals, Inc.

Production and assessment of decellularized pig and human lung scaffolds.

The authors have previously shown that acellular (AC) trachea-lung scaffolds can (1) be produced from natural rat lungs, (2) retain critical components of the extracellular matrix (ECM) such as collagen-1 and elastin, and (3) be used to produce lung tissue after recellularization with murine embryonic stem cells. The aim of this study was to produce large (porcine or human) AC lung scaffolds to determine the feasibility of producing scaffolds with potential clinical applicability. We report here the first attempt to produce AC pig or human trachea-lung scaffold. Using a combination of freezing and sodium dodecyl sulfate washes, pig trachea-lungs and human trachea-lungs were decellularized. Once decellularization was complete we evaluated the structural integrity of the AC lung scaffolds using bronchoscopy, multiphoton microscopy (MPM), assessment of the ECM utilizing immunocytochemistry and evaluation of mechanics through the use of pulmonary function tests (PFTs). Immunocytochemistry indicated that there was loss of collagen type IV and laminin in the AC lung scaffold, but retention of collagen-1, elastin, and fibronectin in some regions. MPM scoring was also used to examine the AC lung scaffold ECM structure and to evaluate the amount of collagen I in normal and AC lung. MPM was used to examine the physical arrangement of collagen-1 and elastin in the pleura, distal lung, lung borders, and trachea or bronchi. MPM and bronchoscopy of trachea and lung tissues showed that no cells or cell debris remained in the AC scaffolds. PFT measurements of the trachea-lungs showed no relevant differences in peak pressure, dynamic or static compliance, and a nonrestricted flow pattern in AC compared to normal lungs. Although there were changes in content of collagen I and elastin this did not affect the mechanics of lung function as evidenced by normal PFT values. When repopulated with a variety of stem or adult cells including human adult primary alveolar epithelial type II cells both pig and human AC scaffolds supported cell attachment and cell viability. Examination of scaffolds produced using a variety of detergents indicated that detergent choice influenced human immune response in terms of T cell activation and chemokine production.

In vivo layer-resolved characterization of oral dysplasia via nonlinear optical micro-spectroscopy.

Optical spectroscopy has proven to be a powerful technique for studying neoplastic transformation in epithelial tissue. Since specific intra-layer precancerous changes originate in the stratified layers of the oral mucosa, layer-resolved analysis will likely improve both our understanding of the mechanism of premalignant transformation, and clinical diagnostic outcomes. However, the native fluorescence signal in linear spectroscopy typically originates from a multi-layered focal volume. In this study, nonlinear spectroscopy was exploited for in vivo layer-resolved discrimination between normal and dysplastic tissue for the first time. Our results revealed numerous intra-layer specific differences.

Gold nanorods for intravital vascular imaging of preneoplastic oral mucosa.

We explore the feasibility of using gold nanorods with efficient two-photon luminescence properties as contrast agents for intravital imaging of neoplasia. This investigation spanned ex vivo characterization in cells/tissue to in vivo implementation in an oral carcinogenesis model. GNRs were >40 times brighter than surrounding tissue. Intravital imaging revealed 3D microvasculature, and in dysplasia, abnormal vessels (dense and tortuous) compared to normal. GNRs were diffusely distributed in lesions after 24 hours. No known previous study has revealed abnormal vessel structure in dysplasia by imaging. Results suggest GNRs can function as high-contrast agents for in vivo visualization of carcinogenesis features.

Hybrid shear force feedback/scanning quantitative phase microscopy applied to subsurface imaging.

Quantitative phase microscopy allows for the study of the surface morphology and dynamics of transparent biological specimens. Although phase data often contains coupled subsurface information, decoupling the surface and subsurface components is often very difficult or impossible. We hereby present a simple procedure which exploits simultaneous obtained quantitative phase and shear-force feedback topography data to extract subsurface sample information. Our results reveal subsurface features in fabricated samples and fish erythrocytes.

Trimodal imaging system capable of quantitative phase imaging without 2 pi ambiguities.

A neoteric approach to interferometric phase imaging unencumbered by 2 pi phase ambiguities is presented. This technique utilizes an actively controlled angular displacement glass plate positioned in the reference arm of an environmentally stabilized pseudoheterodyne Mach-Zehnder interferometer. The plate is continually adjusted to maintain a constant interferometric output phase, as a phase object in the sample arm is raster scanned. Using a 632.8 nm source, unwrapped phase images of translucent samples ranging from approximately 150 nm to 1.5 microm thick were obtained. This system is incorporated into a conventional near-field scanning optical microscope, which permits simultaneous phase, intensity, and surface morphology studies.