RESUMEN
In a synovial joint, the articular cartilage is directly affected during the progression of Osteoarthritis (OA). The characterization of early stage modification in extra-cellular matrix of cartilage is essential for detection as well as understanding the progression of disease. The objective of this study is to demonstrate the potential and capability of nonlinear optical microscopy for the morphological investigation of early stage osteoarthritic cartilage. ICRS Grade-I cartilage sections were obtained from the femoral condyle of the human knee. The surface of articular cartilage was imaged by second harmonic generation and two-photon excited fluorescence microscopy. Novel morphological features like microsplits and wrinkles were observed, which would otherwise not be visible in other clinical imaging modalities (e.g., CT, MRI, ultrasound and arthroscope. The presence of superficial layer with distinct collagen fibrils parallel to the articular surface in 4 specimens out of 14 specimens, indicates that different phases of OA within ICRS Grade-I can be detected by SHG microscopy. All together, the observed novel morphologies in early stage osteoarthritic cartilage indicates that SHG microscopy might be a significant tool for the assessment of cartilage disorder.
RESUMEN
Due to specific structural organization at the molecular level, several biomolecules (e.g., collagen, myosin etc.) which are strong generators of second harmonic generation (SHG) signals, exhibit unique responses depending on the polarization of the excitation light. By using the polarization second harmonic generation (p-SHG) technique, the values of the second order susceptibility components can be used to differentiate the types of molecule, which cannot be done by the use of a standard SHG intensity image. In this report we discuss how to implement p-SHG on a commercial multiphoton microscope and overcome potential artifacts in susceptibility (χ) image. Furthermore we explore the potential of p-SHG microscopy by applying the technique to different types of tissue in order to determine corresponding reference values of the ratio of second-order χ tensor elements. These values may be used as a bio-marker to detect any structural alterations in pathological tissue for diagnostic purposes. The SHG intensity image (red) in (a) shows the distribution of collagen fibers in ovary tissue but cannot determine the type of collagen fiber. However, the histogram distribution (b) for the values of the χ tensor element ratio can be used to quantitatively identify the types of collagen fibers.