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What is Multi-Photon Microscopy?
Multi-photon microscopy is an advanced optical imaging technique that uses ultrashort laser pulses to excite fluorescent molecules within biological samples. It enables high-resolution imaging of living tissue while limiting unwanted excitation outside the focal plane.
Unlike conventional fluorescence microscopy, multi-photon excitation uses longer-wavelength infrared light that can penetrate deeper into biological tissue with reduced scattering and photodamage.
HOW Multi-Photon Microscopy Works
The principle can be understood through three main steps:
Ultrafast laser excitation
A femtosecond pulsed laser delivers short pulses at a wavelength suited to the fluorescent molecules being studied.
Two-photon absorption
Two lower-energy photons are absorbed almost simultaneously by a fluorescent molecule, providing the energy required to reach an excited state. This nonlinear process occurs mainly at the tightly focused laser spot.
Fluorescence detection
The excited molecules emit fluorescence, which is collected to construct a high-resolution image of the biological sample.
