Two-photon excitation microscopy

Two-photon excitation microscopy is a powerful fluorescence microscopy technique that allows imaging of living tissue up to a very high depth. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation microscopy utilizes the simultaneous absorption of two photons of longer wavelength to excite a fluorophore. This technique was first described by Maria Goeppert-Mayer in her doctoral dissertation in 1931, but it was not until the development of pulsed lasers in the 1990s that it became practically feasible for biological imaging.

Principles

The fundamental principle behind two-photon excitation microscopy is the simultaneous absorption of two photons by a fluorophore. This process requires the fluorophores to be in an intense light field, typically provided by a laser emitting short pulses at high peak power. The energy of the two absorbed photons adds up to the energy required to excite the fluorophore, after which it emits a photon at a shorter wavelength, producing an image.

One of the key advantages of this technique is its inherent three-dimensional resolution. Since two-photon absorption is highly dependent on the intensity of the light, it occurs significantly only at the focal point of the laser beam. This eliminates the need for a physical pinhole to achieve optical sectioning, as is necessary in confocal microscopy. Additionally, the use of longer wavelength light allows deeper penetration into biological tissues with reduced photobleaching and photodamage outside the focal volume.

Applications

Two-photon excitation microscopy is widely used in the study of neuroscience, cell biology, and tissue engineering. It is particularly useful for imaging deep within living organisms, such as the brain, where it can be used to study neuron function, dendritic activity, and blood flow. It has also been applied in the development of biophotonics techniques, including fluorescence lifetime imaging microscopy (FLIM) and photon counting.

Advantages and Limitations

The main advantages of two-photon excitation microscopy include:

• Deep tissue penetration due to the use of near-infrared light.
• Reduced photobleaching and photodamage outside the focal volume.
• Inherent optical sectioning capability without the need for a pinhole.

However, there are also limitations to consider:

• The requirement for expensive, high-power pulsed lasers.
• Lower signal-to-noise ratio compared to one-photon excitation in certain conditions.
• Potential thermal damage to the sample due to the high intensity of the laser beam.

Future Directions

The future of two-photon excitation microscopy lies in the development of new fluorophores optimized for two-photon absorption, improvements in laser technology, and the integration with other imaging modalities. Advances in these areas will likely expand the applications of two-photon excitation microscopy in both basic and clinical research.

See Also

• Fluorescence microscopy
• Confocal microscopy
• Optical microscopy
• Laser scanning confocal microscopy