Three-photon microscopy 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 1300nm or longer wavelength laser to excite the fluorescent dyes with three simultaneously absorbed photons, and then the fluorescent dyes emit one photon whose energy is three times the energy of each incident photon. Comparing to two-photon microscopy, three-photon microscopy reduces out of focus light by, which is two times of that of two-photon microscopy. In addition, three-photon microscopy employs near-infrared light with less tissue scattering effect, which 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 point scanning method; Both are able to image 3D sample by adjusting the position of the focus lens along axial and lateral directions; The structures of both systems do not require 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 excitation laser is about 1200nm or more in three photon microscopy with the emission wavelength less than one-third of the excitation wavelength. Three photon microscopy has deeper penetration since the lowest optical absorption band of the tissue is 1050 to 1100 nm which is within the range of 400 to 2400 nm. However, three-photon microscope needs the laser with higher power due to relatively smaller cross-section of the dyes for three photon excitation, which is on the order of, which is much smaller than the typical two-photon excitation cross-sections of. The Ultrashort pulses are usually around 100 fs.
Resolution
For three photon fluorescence scanning microscopy, the three dimensional intensity point-spread function can be denoted as, where denotes the 3-D convolution operation, denotes the intensity sensitivity of an incoherent detector, and, denotes the 3-D IPSF for the objective lens and collector lens in single-photon fluorescence, respectively. The 3-D IPSF can be expressed in where is a Bessel function of the first kind of order zero. The axial and radial coordinates and are defined by where is the numerical aperture of the objective lens, is the real defocus, and is the radial coordinates.
Coupling with other multiphoton techniques
Correlative images can be obtained using different multiphoton schemes such as 2PEF, 3PEF, and Third harmonic generation, in parallel. A multichannel image is then constructed. 3PEF is also compared to 2PEF : it generally give a smaller degradation of the signal-to-noise ratio with depth, even if the emitted signal is smaller than with 2PEF.
Development
After three-photon excited fluorescence was observed by Singh and Bradley and further validated by Hell, Chris Xu reported measurement of excitation cross sections of several native chromophores and biological indicators, which shows possibility of implementing three-photon excited fluorescence in Laser Scanning Microscopy. In November 1996, David Wokosin applied three photon excitation fluorescence for fixed in vivo biological specimen imaging. In 2010s, three photon microscopy is developed further. In January 2013, Horton, Wang and Kobat invented in vivo imaging of an intact mouse brain by employing point scanning method to three photon microscope. In May 2017, Rowlands applied wide-field three-photon excitation to three photon microscope for larger penetration depth. In Oct 2018, T Wang, D Ouzounov, and C Wu were able to image vasculature and GCaMP6 calcium using three photon microscope.
Applications
Three-photon microscopy has similar application fields with two-photon excitation microscopy including neuroscience, and oncology. However, comparing to standard single-photon or two-photon excitation, three-photon excitation has several benefits such as the use of longer wavelengths reduces the effects of light scattering and increasing the penetration depth of the illumination beam into the sample. The nonlinear nature of three photon microscopy confines the excitation target to a smaller volume, reducing out-of-focus light as well as minimizing photobleaching on the biological sample. These advantages of three-photon microscopy gives it an edge in visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue and Rapid volumetric imaging. In the recent study, Xu has demonstrated the potential of three-photon imaging for noninvasive studies of live biological systems. The paper used three-photon fluorescence microscopy at a spectral excitation window of 1,320 nm to imaging the mouse brain structure and function through the intact skull with high spatial and temporal resolution and large FOVs, and at substantial depth. This work demonstrates the advantage of higher-order nonlinear excitation for imaging through a highly scattering layer, which is in addition to the previously reported advantage of 3PM for deep imaging of densely labeled samples.
