THESIS
2021
1 online resource (xviii, 182 pages) : illustrations (some color)
Abstract
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.
Post a Comment