Pre-Clinical Translation of Second Harmonic Microscopy of Meniscal and Articular Cartilage Using a Prototype Nonlinear Microendoscope

Abstract

Previous studies using nonlinear microscopy have demonstrated that osteoarthritis (OA) is characterized by the gradual replacement of Type II collagen with Type I collagen. The objective of this study was to develop a prototype nonlinear laser scanning microendoscope capable of resolving the structural differences of collagen in various orthopaedically relevant cartilaginous surfaces. The current prototype developed a miniaturized femtosecond laser scanning instrument, mounted on an articulated positioning system, capable of both conventional arthroscopy and second-harmonic laser-scanning microscopy. Its optical system includes a multi-resolution optical system using a gradient index objective lens and a customized multi-purpose fiber optic sheath to maximize the collection of backscattered photons or provide joint capsule illumination. The stability and suitability of the prototype arthroscope to approach and image cartilage were evaluated through preliminary testing on fresh, minimally processed, and partially intact porcine knee joints. Image quality was sufficient to distinguish between hyaline cartilage and fibrocartilage through unique Type I and Type II collagen-specific characteristics. Imaging the meniscus revealed that the system was able to visualize differences in the collagen arrangement between the superficial and lamellar layers. Such detailed in vivo imaging of the cartilage surfaces could obviate the need to perform biopsies for ex vivo histological analysis in the future, and provide an alternative to conventional external imaging to characterize and diagnose progressive and degenerative cartilage diseases such as OA. Moreover, this system is readily customizable and may provide a suitable and modular platform for developing additional tools utilizing femtosecond lasers for tissue cutting within the familiar confines of two or three portal arthroscopy techniques.

Collagen Organization Within the Cartilage of Trpv4−/− Mice Studied with Two‐Photon Microscopy and Polarized Second Harmonic Generation

“…Understanding the different stages of the disease progression requires finding new techniques for longitudinal intravital imaging of the articular cartilage. These regions are not easily accessible with microscope objectives, but recent developments of GRIN lenses for the exploration of deep organs and cavities in live animals , as well as the first report on preclinical translation of SHG microscopy to the meniscal and articular cartilage , could provide solutions for this problem.…”

Assessment of Articular Cartilage by Second Harmonic Microscopy: Challenges and Opportunities

“…As a recent breakthrough in technological advancement, SHM images acquired using a flexible fiber-optic-based probe head possess a quality level comparable to images captured by large bench-top laser-scanning microscopes was demonstrated by Liang et al, that shows a promising potential of SHM for in-vivo arthroscopic applications [17]. In another study Stephen et al, recently demonstrated the pre-clinical translation of SHM using a microendoscope and a prototype of a SHM-based arthroscope for the in-vivo assessment of articular cartilage [16]. A summary of major recent advancements in articular cartilage assessments by SHM are included in Table 1.…”

“…Recently, SHM has been demonstrated for real time in-vivo endomicroscopy images with resolution 2.6 µm [16]. The acquired SHM micrographs are capable of detecting microsplits and ultra-structure fibril arrangements of collagen fiber in articular cartilage.…”

“…The acquired SHM micrographs are capable of detecting microsplits and ultra-structure fibril arrangements of collagen fiber in articular cartilage. Such technical advancements in SHM imaging at the endomicroscopic level are highly valuable for both translational and basic research [16,17].…”

“…Baskey et al [16] 2019 Detection of collagen reorganization at different layers due to applied mechanical loading (strain) on articular cartilage. p-SHM Animal (bovine)/human Mansfield et al [31] 2018…”