Multiphoton microscopy (MPM) has greatly advanced biological research owing to its unique advantages over conventional optical technologies. First, the multiple modalities of MPM, such as multiphoton fluorescence and harmonic generation, can provide different information of biological structures and functionalities. Second, by utilizing nonlinear effect and longer excitation wavelength, MPM can achieve intrinsic three-dimensional imaging capability and larger penetration depth. Third, the ultrafast laser in MPM can also be used for laser microsurgery to specifically ablate target cells or tissues. These unique properties make MPM the method of choice for in vivo imaging of small animals. However, high-resolution, large field-of-view and deep tissue imaging is still challenging due to th...[ Read more ]

Multiphoton microscopy (MPM) has greatly advanced biological research owing to its unique advantages over conventional optical technologies. First, the multiple modalities of MPM, such as multiphoton fluorescence and harmonic generation, can provide different information of biological structures and functionalities. Second, by utilizing nonlinear effect and longer excitation wavelength, MPM can achieve intrinsic three-dimensional imaging capability and larger penetration depth. Third, the ultrafast laser in MPM can also be used for laser microsurgery to specifically ablate target cells or tissues. These unique properties make MPM the method of choice for in vivo imaging of small animals. However, high-resolution, large field-of-view and deep tissue imaging is still challenging due to the aberration and scattering of light.

Since MPM has such advantages and challenges, my thesis work mainly focused on advancing the microscopy technologies and applying them for morphological and functional imaging of biological tissues. Specifically, we first build up a combined single- and two-photon microscopy with time- and spectral-resolved detection capability to study new fluorescent signal produced by laser ablation and explore its biological applications. Next, we advance MPM by using adaptive optics (AO) to overcome the aberrations and recover optimal imaging performance in vivo. Firstly, we develop an AO two-photon microscopy to correct ocular aberrations based on direct wavefront sensing and achieve submicron resolution for structural and functional imaging of mouse retina. Secondly, we advance the two-photon endomicroscopy by adding adaptive optics, which restores diffraction-limited resolution for deep-brain imaging. Here, a new precompensation strategy plays a critical role to correct aberrations over large volumes. Thirdly, we optimize the AO two-photon microscopy for minimally-invasive brain imaging through the thinned-skull window and improve the wavefront sensing algorithm for reliable aberration determination below the scattering skull and brain tissue. Finally, we integrate AO with three-photon microscopy to further push the depth limit of in vivo imaging, by combating both the aberration and scattering. Here, we develop two major innovations: direct focus sensing with a phase-sensitive detection and conjugate AO with remote focusing. We achieve high-resolution imaging of mouse cortex up to 750 μm below the intact skull and subcortical structures as deep as 1.1 mm within the intact brain. Our results demonstrate the great potential of AO multiphoton microscopy to advance in vivo imaging techniques and facilitate biological research in living animals.