Multi-photon Fluorescence Imaging
Conventional fluorescence microscopy techniques are based on single-photon excitation fluorescence, in which a fluorescent molecule absorbs a photon, transiting from the ground state to an excited state, followed with emission of a long wavelength photon by transiting to the ground state after the energy relaxation. The photon energy of excitation light used here must be higher than that of the emitted fluorescence wavelength for energy conservation. If long-wavelength photons to excite the fluorescent molecule, it will not produce fluorescence. In 1931, Maria Goppert-Mayer predicted that if one photon does not have enough energy to excite the fluorescent molecule, but in a short time to encounter a second or more photons, the same molecule can simultaneously absorb two or more photons, each of the absorption produces the molecule excitement with an equivalent energy of the absorbed photon. Conse?quently, the fluorescent molecule, after gaining sufficient photon energy, will also fluoresce after relaxation. This multiphoton excitation process, was not observed until the 1960s when two-photon excited fluorescence was first discovered in CaF2: Eu3+ by Kaiser and Garret , there-photon excited fluorescence was observed and the three-photon absorption cross section for naphthalene crystals was estimated by Singh and Bradley .
As shown in Fig. 2.2, multiphoton fluorescence involves the absorption of multiple photons to an excited electronic state followed by the relaxation of the molecule to the ground electronic state through the emission of a single photon. The wavelength of the emitted photon is approximately equal to the excitation wavelength divided by the number of photons absorbed.
Two-photon excitation wavelength is twice single-photon excitation wavelength. For example, to excite the fluorescent probe Indo-l with Ar+ laser, we use 351 nm laser, while two-photon excitation is necessary to use 700 nm laser. Despite there is a difference between single-photon and multi-photon excitation process, the fluorescence emission spectra are identical. That is, the multi-photon technology can detect ultraviolet fluorescent probe without the use of an ultraviolet light source. But to achieve multi-photon excitation, it usually requires ultra-fast femtosecond laser pulses to produce a high density of photons focused on suitable fluorescent medium, and induce multi-photon transition with a sufficiently high probability, due to the fact that the probability that more than one photon can be absorbed simultaneously scales with intensity raised to the nth power where n is equal to the number of photons absorbed. Thus the multi-photon excitation has high spatial local characteristics, only samples in the center area of focus can absorb enough photons to give off fluorescence, which naturally reduces size of emission spots on sample upon laser illumination. Based on this principle, multi-photon excitation can obtain clearer three-dimensional fluorescence image than the single-photon confocal (Figs. 2.3 and 2.4).
In addition, multi-photon fluorescence excitation uses red or infrared light, which minimizes scattering in the tissue. Further the background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes, typically 5-20 times deeper than other types of fluorescent microscopes. Two-photon
Fig. 2.2 Single photon versus multi-photon excitation
Fig. 2.3 (a shark choroid plexus stained with fluorescein) provide a comparison of confocal and two-photon microscopy imaging quality 
excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and that not only greatly reduces the phototoxicity of cells, but also extends the observation time of living organisms [8, 15, 20, 45, 55].
Single-molecule biophysical approaches to live-cell studies based on fluorescence imaging have greatly enriched our knowledge on the behavior of single biomolecules in their native environments and their roles in cellular processes . And it can detect a variety of small molecules in vitro and allow imaging of the dynamic changes and cell-to-cell variation in the intracellular levels  (Fig.2.5).