3D second harmonic generation imaging tomography by multi-view excitation.

Biological tissues have complex 3D collagen fiber architecture that cannot be fully visualized by conventional second harmonic generation (SHG) microscopy due to electric dipole considerations. We have developed a multi-view SHG imaging platform that successfully visualizes all orientations of collagen fibers. This is achieved by rotating tissues relative to the excitation laser plane of incidence, where the complete fibrillar structure is then visualized following registration and reconstruction.

3D texture analysis for classification of second harmonic generation images of human ovarian cancer.

Remodeling of the collagen architecture in the extracellular matrix (ECM) has been implicated in ovarian cancer. To quantify these alterations we implemented a form of 3D texture analysis to delineate the fibrillar morphology observed in 3D Second Harmonic Generation (SHG) microscopy image data of normal (1) and high risk (2) ovarian stroma, benign ovarian tumors (3), low grade (4) and high grade (5) serous tumors, and endometrioid tumors (6). We developed a tailored set of 3D filters which extract textural features in the 3D image sets to build (or learn) statistical models of each tissue class.

Texture analysis applied to second harmonic generation image data for ovarian cancer classification.

Remodeling of the extracellular matrix has been implicated in ovarian cancer. To quantitate the remodeling, we implement a form of texture analysis to delineate the collagen fibrillar morphology observed in second harmonic generation microscopy images of human normal and high grade malignant ovarian tissues. In the learning stage, a dictionary of “textons”—frequently occurring texture features that are identified by measuring the image response to a filter bank of various shapes, sizes, and orientations—is created.

Multi-view second-harmonic generation imaging of mouse tail tendon via reflective micro-prisms.

Here we experimentally show that second-harmonic generation (SHG) imaging is not sensitive to collagen fibers oriented parallel to the direction of laser propagation and, as a consequence, can potentially miss important structural information. As an alternative approach, we demonstrate the use of reflective micro-prisms to enable multi-view SHG imaging of mouse tail tendon by redirecting the focused excitation and collection of subsequent emission. Our approach data corroborates the theoretical treatment on vanishing and nonvanishing orientations, where fibers along the laser direction are largely transparent by SHG.

Second harmonic generation microscopy analysis of extracellular matrix changes in human idiopathic pulmonary fibrosis.

Patients with idiopathic fibrosis (IPF) have poor long-term survival as there are limited diagnostic/prognostic tools or successful therapies. Remodeling of the extracellular matrix (ECM) has been implicated in IPF progression; however, the structural consequences on the collagen architecture have not received considerable attention. Here, we demonstrate that second harmonic generation (SHG) and multiphoton fluorescence microscopy can quantitatively differentiate normal and IPF human tissues.