Two-photon excitation microscopy

Two-photon excitation microscopy

Two-photon excitation microscopy setup

Schematic of two-photon excitation

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Two-photon excitation in biological tissues

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Diagram of a two-photon excitation microscope

Two-photon microscopy of in vivo brain function

Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a very high depth. It is a special variant of fluorescence microscopy that uses two photons of lower energy to excite a fluorophore, instead of one photon of higher energy. This technique was first developed in the 1990s and has since become a powerful tool in biological research.

Principle

Two-photon excitation relies on the simultaneous absorption of two photons by a fluorophore. Each photon has approximately half the energy (and thus twice the wavelength) required for excitation. This process is nonlinear and occurs only at the focal point of the laser, which allows for precise spatial localization of the excitation. The use of longer wavelengths reduces scattering in biological tissues, enabling deeper penetration and less photodamage compared to single-photon excitation.

Applications

Two-photon microscopy is widely used in neuroscience, cell biology, and developmental biology. It is particularly useful for imaging thick specimens, such as brain slices or whole embryos, where traditional fluorescence microscopy would be limited by scattering and absorption. The technique allows researchers to observe dynamic processes in living tissues, such as neuronal activity, blood flow, and cellular interactions.

Advantages

The main advantages of two-photon excitation microscopy include reduced photobleaching and phototoxicity, as well as improved imaging depth. The localized excitation reduces out-of-focus fluorescence, enhancing image contrast and resolution. Additionally, the use of infrared light minimizes damage to living tissues, making it ideal for long-term imaging studies.

Limitations

Despite its advantages, two-photon microscopy has some limitations. The requirement for high-intensity pulsed lasers can be costly and complex to operate. The technique also has lower temporal resolution compared to some other imaging methods, which can be a limitation for certain dynamic studies.

Related pages

• Fluorescence microscopy
• Confocal microscopy
• Neuroscience
• Cell biology

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  Diagram of a two-photon excitation microscope

• Two-photon microscopy of in vivo brain function