Fig. 1. Mechanism and system of multiphoton fluorescence imaging. (a) Schematic of Jablonski energy level in which the fluorophore is excited to produce fluorescence after absorbing single photon, two photons, and three photons; (b) spatial distributions of fluorescence intensity under linear excitation and nonlinear excitation^[4]; (c) schematic of one-photon wide-field microscopy system and multiphoton laser scanning microscopy system^[27]

Fig. 2. Properties of NIR light in biological tissues. (a) Interactions of excitation light with tissues^[28]; (b) absorption spectra of oxyhaemoglobin and deoxyhaemoglobin in visible and near-infrared bands^[28]; (c) scattering coefficient of NIR light in mouse brain tissue^[28]; (d) attenuation length of light in mouse brain cortex^[29]; (e) schematic of in vivo multiphoton brain imaging excited under NIR-Ⅰ light and NIR-Ⅱ light^[48]

Fig. 3. Deep brain vascular multiphoton imaging. (a) Reconstructed 3D in vivo two-photon fluorescence image of brain vasculatures under 1280 nm excitation (middle) and 2D cross section images at different depth (on each side)^[51]; (b) reconstructed 3D in vivo three-photon fluorescence image of brain vasculatures under 1675 nm excitation^[52]; (c) 2D cross section images at different depth^[52]; (d) three-photon fluorescence signal distribution of the straight lines across the blood vessels in Fig.3(c)^[52]

Fig. 4. Dyes-assisted deep brain vascular multiphoton imaging. (a) Reconstructed 3D in vivo three-photon fluorescence image of brain vasculatures labeled by Qtracker 655 under 1700 nm excitation^[55]; (b) three-photon action cross sections of several fluorescent probes^[55]; (c) cross sections of 2D three-photon angiography at different depths and corresponding signal-to-noise ratio^[55]; (d) polymer dots assisted in vivo multiphoton fluorescence image of brain vasculatures under 1225 nm excitation^[56]; (e) multicolor three-photon fluorescence brain imaging under 1340 nm excitation^[63]

Fig. 5. Multiphoton brain neuronal imaging. (a) 3D reconstruction of three-photon fluorescence images of GCaMP6s-labeled neurons in the mouse cortex and the hippocampus under 1300 nm excitation^[65]; (b) two-color two-photon fluorescence imaging of neurons labeled with Cal-590 and OGB-1 under 1000 nm excitation^[68]; (c) hybrid multiplexed sculpted light microscopy (HyMS) for simultaneous volumetric two-photon and three-photon imaging of mouse neurons in cortex and hippocampus region^[69]; (d) schematic of motion-corrected AO three-photon microscope and three-photon microscopy of mouse visual cortex and hippocampus under 1300 nm excitation^[72]

Fig. 6. Multiphoton brain imaging through skull. (a) Comparison of 1320 nm excited three-photon and 920 nm excited two-photon for in vivo brain vascular imaging through intact skull^[76]; (b) 1320-nm-excited three-photon fluorescence neurons imaging through intact skull^[76]; (c) 1660-nm-excited third-harmonic generation (THG) and three-photon fluorescence imaging of osteocytes in skull bone^[77]; (d) 1550-nm-excited three-photon fluorescence imaging of brain blood vessels labeled by DCDPP-2TPA nanoparticles through skull^[78]; (e) two-photon fluorescence images of brain blood vessels labeled by ultrasmall conjugated polymer dots under different laser excitations^[48]; (f) in vivo two-photon fluorescence imaging of bone marrow^[48]; (g) 3D reconstructed two-photon images of brain blood vessels upon 1200 nm excitation through intact skull^[48]

Fig. 7. Multiphoton fluorescence imaging of tumor and cardiovascular diseases. (a) In vivo two-photon fluorescence images of blood vessels in normal and tumor tissues under NIR-Ⅱ excitation^[87]; (b) long time circulation of nanoparticle probes in blood^[87]; (c) 3D reconstructed two-photon fluorescence images of tumor blood vessel network under NIR-Ⅱ and NIR-Ⅰ excitations^[87]; (d) 3D reconstructed two-photon fluorescence images of nanoparticle distributions in tumor microenvironment under NIR-Ⅱ and NIR-Ⅰ excitations^[87]; (e) in vivo three-photon fluorescence imaging of lipids in fatty liver, carotid artery, and brain artery under NIR-Ⅱ excitation^[99]