What is Multiphoton Excitation?
Multiphoton excitation is a fluorescence imaging technique that uses the simultaneous absorption of two or more photons to excite a fluorophore. Unlike traditional single-photon excitation, which uses a single high-energy photon, multiphoton excitation uses lower-energy photons in the near-infrared range. This technique is especially valuable in histology for its ability to image deeper into biological tissues with reduced photodamage and photobleaching.
How Does Multiphoton Excitation Work?
Multiphoton excitation relies on the principle that two or more photons can be absorbed simultaneously if their combined energy matches the energy required to excite a fluorophore. This process requires a high photon density, typically achieved using a pulsed laser. Because the probability of simultaneous photon absorption is low, excitation is confined to the focal point of the laser, providing intrinsic
optical sectioning and reducing out-of-focus light.
Deeper tissue penetration: Near-infrared light used in multiphoton excitation penetrates deeper into tissues than visible light, allowing for imaging of thicker specimens.
Reduced photodamage: Lower energy photons cause less damage to biological samples, preserving the integrity of delicate tissues.
Reduced photobleaching: Because excitation is confined to the focal point, regions outside the focal plane experience less light exposure, minimizing photobleaching of fluorophores.
Intrinsic optical sectioning: The nonlinear nature of multiphoton excitation provides inherent optical sectioning capability, eliminating the need for physical sectioning of tissues and improving three-dimensional imaging.
Live tissue imaging: The reduced photodamage and deeper penetration make it suitable for imaging live tissues and studying dynamic processes in real-time.
Thick tissue imaging: It is ideal for imaging thick tissue sections, such as brain slices, without the need for extensive sample preparation.
Label-free imaging: Multiphoton excitation can be used for
label-free imaging techniques like second harmonic generation (SHG) and third harmonic generation (THG), providing structural information without the need for fluorescent dyes.
High-resolution imaging: It enables high-resolution imaging of subcellular structures, aiding in the detailed study of cellular morphology and function.
Cost: The equipment required for multiphoton excitation, including pulsed lasers and advanced microscopes, is expensive, limiting accessibility for some laboratories.
Complexity: The technique requires specialized knowledge and expertise to operate and interpret results, making it less accessible to non-experts.
Fluorophore limitations: Not all fluorophores are suitable for multiphoton excitation, and the choice of fluorophores may be limited by their absorption spectra.
Conclusion
Multiphoton excitation is a powerful tool in histology, offering deep tissue imaging, reduced photodamage, and intrinsic optical sectioning. While it has some limitations, its advantages make it invaluable for studying complex biological tissues and dynamic processes. As technology advances and becomes more accessible, its applications in histology will likely continue to expand, providing new insights into the structure and function of biological tissues.
