Second Harmonic Generation (SHG) imaging is a non-invasive microscopy technique that creates detailed images of biological tissues. It achieves high-resolution visualization without needing external dyes or stains. This method provides insights into tissue architecture by interacting with specific molecular structures.
The Fundamental Principle
Second Harmonic Generation is a non-linear optical process. In this process, two photons of the same frequency simultaneously interact with a material. These two photons are converted into a single new photon with double the original frequency, or half the original wavelength. This transformation occurs without any energy absorption by the material.
This optical phenomenon occurs only in materials with a non-centrosymmetric molecular structure, meaning they lack a center of symmetry at the molecular level. High-intensity, pulsed lasers, such as femtosecond lasers, are employed to generate a detectable signal. These lasers deliver light in extremely short bursts, providing the necessary photon density.
Unlike linear optical processes like fluorescence, SHG does not involve energy absorption by the sample followed by delayed emission. Instead, the SHG signal is generated instantaneously upon interaction with the laser light. The emitted SHG light is coherent and highly directional, propagating in the forward direction. This directionality helps distinguish the SHG signal from scattered light.
Key Structures Visualized
SHG imaging visualizes biological components with a non-centrosymmetric molecular structure. Collagen is the most prominent biological molecule generating SHG signals. Its triple-helical, fibrillar structure is inherently non-centrosymmetric and highly organized, making it readily detectable. Collagen is abundant in various connective tissues, including the skin, tendons, bone, and cartilage, where its fibrillar networks provide structural support and integrity.
Myosin, a protein found in muscle fibers, also exhibits an organized structure that produces an SHG signal. This allows for detailed visualization of muscle tissue architecture, including the sarcomeres within muscle cells. The ordered arrangement of myosin filaments contributes to its SHG activity. Other structures, such as microtubules, can also generate SHG signals due to their inherent organization.
Applications Across Disciplines
SHG imaging offers broad utility across various scientific and medical disciplines. In medicine and pathology, it plays a role in disease diagnosis and monitoring. For instance, in cancer detection, SHG can reveal subtle changes in collagen organization within the tumor microenvironment, which often becomes stiffer and more aligned compared to healthy tissue. This altered collagen architecture can serve as an indicator for tumor progression or malignancy in tissues like breast or ovarian cancer.
The technique is also valuable for assessing fibrosis in organs such as the liver, lung, and kidney. Fibrosis involves the excessive accumulation of collagen, and SHG imaging can quantify the density and structural arrangement of these collagen fibers, providing insights into disease severity and progression. In tissue engineering, SHG helps monitor the integration of scaffolds and the regeneration of new tissues by visualizing newly formed collagen networks. This allows researchers to assess the success of regenerative therapies and the structural integrity of engineered tissues.
In developmental biology, SHG imaging is used to observe dynamic processes like tissue development and morphogenesis. Researchers can track the formation and remodeling of collagen matrices during embryonic development or wound healing in real-time. This provides insights into how tissues form and repair themselves.
Neuroscience benefits from SHG by imaging collagen within the extracellular matrix of the brain or peripheral nerves. It can visualize the myelin sheath around nerve fibers, as well as collagen in blood vessel walls within neural tissue, without interfering with neuronal activity. In pharmacology and drug discovery, SHG imaging can assess the impact of new drugs on tissue structure and integrity. For example, it can evaluate whether a therapeutic agent promotes healthy collagen remodeling or reduces fibrotic scarring in response to injury or disease.
Distinct Advantages
A primary advantage is its label-free imaging capability. This means external dyes or fluorescent tags are not required to visualize specific structures. This preserves the integrity of the biological sample, avoids potential phototoxicity, and simplifies sample preparation. Structures are visualized intrinsically, preserving the native state of the biological sample.
The technique also allows deep tissue penetration due to its use of near-infrared (NIR) light. NIR light scatters less in biological tissues than visible light, enabling deeper imaging than conventional microscopy, even in living organisms.
SHG imaging provides high spatial resolution, allowing detailed visualization of microstructures. The non-linear process confines signal generation to the highly focused laser spot, enabling resolution of fine architectural details like collagen fibers or muscle sarcomeres down to sub-micrometer scales.
The method is non-destructive, making it suitable for live cell and tissue imaging. This allows researchers to observe dynamic changes in tissue organization over extended periods, such as during wound healing or developmental processes. The contrast in SHG imaging is derived directly from the inherent molecular organization of the tissue itself, providing specific structural information without background interference.