Second Harmonic Generation (SHG) is a nonlinear optical phenomenon produced by materials with a non-centrosymmetric structure. The SHG signal originates when two excitation photons, with the same frequency, interact with the material, and are subsequently combined into a single emitted photon, with exactly double the original energy and frequency (half the wavelength). It is described as a nonlinear coherent scattering process, in which no energy is lost.
Several biological materials can produce SHG signal, including collagen, microtubule bundles, muscle myosin, and starch granules, meaning that these structures can be readily imaged with a specially designed microscope without staining. SHG microscopy offers several advantages for in situ imaging of biological samples, such as very low phototoxicity and photodamage, high axial and lateral resolution, and deep tissue penetration (several hundred microns).
In MC², the Zeiss LSM880 confocal microscope is equipped with the necessary components to image SHG signals. The Spectra-Physics Insight DS+ multiphoton laser system is a near-IR Ti:Sapphire femtosecond pulsed laser, that can be freely tuned between 690-1300nm, offering both the excitation power and wavelengths required for SHG signal generation. In addition, the microscope is equipped with four external, non-descanned, detectors that allow the effective detection of the generated SHG signals in both the transmitted and reflective pathway, with high efficiency and minimal loss of signal.
To showcase the robustness of the technique, we present in the image above some initial results from a trial to resolve the structure of collagen in swine menisci. Imaging was done at the thickest part of the meniscus. As we imaged deeper into the tissue, we observe that the orientation of the collagen fibrils change. Each image (A-C) is a maximum projection of 27-29 serial optical sections, covering a thickness of about 120μm. A, in the lower 120μm collagen shows a uniform orientation, with fibrils arranged in parallel to each other; B, a transitional zone, with fibrils in mixed orientation; C, collagen shows again uniform orientation, but the direction shows a 90° turn in comparison to A. As this is a work in progress, no definite conclusions can be drawn, but it is highly possible, that this change in the orientation of the collagen fibrils, act to strengthen the meniscus, which is known to attract and dampen significant forces within the knee.