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

Kirby R Campbell,Bruce Wen,Benjamin L Cox,Emily M Shelton,Kevin Eliceiri,Robert Swader,Paul J Campagnola

doi:10.1364/optica.4.001171

Kirby R Campbell, Bruce Wen + Show 5 more

Open Access

https://doi.org/10.1364/optica.4.001171

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Abstract

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. We evaluated high frequency and Gaussian weighted fusion reconstruction algorithms, and found the former approach performs better in terms of the resulting resolution. The new approach is a first step toward SHG tomography.

Highlights

Second harmonic generation (SHG) microscopy has been highly successful in visualizing and quantifying complex collagen assembly as it targets fibrillar collagen with high sensitivity and specificity
Using these types of metrics, SHG imaging has been used for revealing extracellular matrix (ECM) structural changes in a wide range of diseases such as cancers, connective tissue disorders, and fibroses [2,3,4,5,6,7]
Fibers at high tilt angles will appear at lower intensities. This is a significant limitation as the native ECM in most tissues has intrinsic 3D architecture, where not all the collagen fibers lie in the plane of incidence or at small tilt angles relative to the plane

Summary

Introduction

Second harmonic generation (SHG) microscopy has been highly successful in visualizing and quantifying complex collagen assembly as it targets fibrillar collagen with high sensitivity and specificity. The collagen fibers are the structures viewed in the microscope and can be analyzed through image analysis approaches. Using these types of metrics, SHG imaging has been used for revealing extracellular matrix (ECM) structural changes in a wide range of diseases such as cancers, connective tissue disorders, and fibroses [2,3,4,5,6,7]. Imaging of collagen and other SHG active scaffolds (e.g., cellulose and silk) [12,13] in tissue engineering applications would be enhanced through this capability To achieve this goal, a new SHG imaging approach is necessary that can acquire data from different views to reveal fibers of all orientations

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