FIGURE 3.

Principle of different optical imaging modalities and a conventional three-photon microscopy (3PM) setup. And the characteristics and performance of two-photon microscope (2PM) and 3PM. (A) Left: Jablonski diagrams of one- (top), two- (middle), and three-photon (bottom) fluorescence. Right: A schematic depicting the distribution of laser excitation and fluorescence emission in 1P (top), 2P (middle), and 3P (bottom) excitation scenarios. Gray dots show photons. The repetition rate of the laser source used in 3PM is lower than that of 2PM. In general, the interval of pulses used in 2PM is tens of nanoseconds; The interval of pulses used in 3PM is several microseconds. Circles indicate cross sections of excitation/emission in the x, y plane. Color represents wavelengths and photon intensity. For example, to excite GFP, blue light is used in 1P excitation, long-wavelength light is used in 2P excitation, and longer-wavelength light is used in 3P excitation. (B) Schematic of a basic 3PM. Light red shows optical path of excitation light. Green shows optical path. Adapted with permission from Denk et al. (1990). (C) Schematic of a focused Gaussian beam in a scattering tissue, with the corresponding axial fluorescence distribution as a function of depth shown in panel (D). (D) Wavelength-dependent attenuation length affected by scattering and absorption. Reproduced with permission from Hontani et al. (2022). (E) Example images of GCaMP6s with 2P and 3P excitation, focused 200∼600 μm below the pial surface of visual cortex. Adapted with permission from Takasaki et al. (2020).