Three-photon microscopy (3PEF) is a high-resolution fluorescence microscopy based on nonlinear excitation effect. Different from two-photon excitation microscopy, it uses three exciting photons. It typically uses 1300 nm or longer wavelength lasers to excite the fluorescent dyes with three simultaneously absorbed photons. The fluorescent dyes then emit one photon whose energy is (slightly smaller than) three times the energy of each incident photon. Compared to two-photon microscopy, three-photon microscopy reduces the fluorescence away from the focal plane by 1 / z 4 {\displaystyle 1/z^{4}} , which is much faster than that of two-photon microscopy by 1 / z 2 {\displaystyle 1/z^{2}} . In addition, three-photon microscopy employs near-infrared light with less tissue scattering effect. This causes three-photon microscopy to have higher resolution than conventional microscopy.
Concept Three-photon excited fluorescence was first observed by Singh and Bradley in 1964 when they estimated the three-photon absorption cross section of naphthalene crystals. In 1996, Stefan W. Hell designed experiments to validate the feasibility of applying three-photon excitation to scanning fluorescence microscopy, which further proved the concept of three-photon excited fluorescence. Three-photon microscopy shares a few similarities with Two-photon excitation microscopy. Both of them employ the point scanning method. Both are able to image 3D samples by adjusting the position of the focus lens along the axial and lateral directions. The structures of both systems do not require a pinhole to block out-focus light. However, three-photon microscopy differs from Two-photon excitation microscopy in their Point spread function, resolution, penetration depth, resistance to out-of-focus light and strength of photobleaching. In three-photon excitation, the fluorophore absorbs three photons almost simultaneously. The wavelength of the excitation laser is about 1200 nm or more in three photon microscopy with the emission wavelength slightly longer than one-third of the excitation wavelength. Three photon microscopy has deeper tissue penetration because of the longer excitation wavelengths and the higher order nonlinear excitation. However, a three-photon microscope needs a laser with higher power due to relatively smaller cross-section of the dyes for three-photon excitation, which is on the order of 10 − 82 cm 6 ( s / photon ) 2 {\displaystyle 10^{-82}{\text{cm}}^{6}(s/{\text{photon}})^{2}} . This is much smaller than the typical two-photon excitation cross-sections of 10 − 49 cm 4 s / photon {\displaystyle 10^{-49}{\text{cm}}^{4}s/{\text{photon}}} . The Ultrashort pulses are usually around 100 fs.
Resolution For three photon fluorescence scanning microscopy, the three dimensional intensity point-spread function (IPSF) can be denoted as,
h i ( ν , u ) = | I 1 ( ν / 3 , u / 3 ) | 3 I 2 ( ν , u ) ⊗ 3 D {\displaystyle h_{i}(\nu ,u)=\left|I_{1}(\nu /3,u/3)\right|^{3}I_{2}(\nu ,u)\otimes _{3}D} , where ⊗ 3 {\displaystyle \otimes _{3}} denotes the 3-D convolution operation, D {\displaystyle D} denotes the intensity sensitivity of an incoherent detector, and I 1 ( ν , u ) {\displaystyle I_{1}(\nu ,u)} , I 2 ( ν , u ) {\displaystyle I_{2}(\nu ,u)} denotes the 3-D IPSF for the objective lens and collector lens in single-photon fluorescence, respectively. The 3-D IPSF I 1 ( ν , u ) {\displaystyle I_{1}(\nu ,u)} can be expressed in
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