Methods

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Multi-photon microscopy

Multi-photon microscopy

– Coherent Mira Model 900-F Laser

In traditional fluorescence spectroscopy, one photon is used to excite a molecule from its ground state to a higher energy state. Afterwards, the molecule decays to an intermediate energy state, emitting a photon of light (fluorescence) with energy equal to the difference of those two levels. In multi-photon excitation, however, two or more photons with lower energy (longer wavelength) excite the molecule together.

One advantage of multi-photon excitation is that the induced fluorescence only occurs at, or near, the focal point of the beam. This improves the signal-to-noise ratio by eliminating fluorescence from outside the focal plane. The pinhole is therefore unnecessary, because all of the fluorescent light originates from the laser focus spot. Furthermore, longer wavelengths penetrate deeper in biological materials and are not scattered as much as shorter wavelengths.

Multi-photon microscopy