To monitor the development of three dimensional structure of large biological specimens as a function of time (4D fluorescent imaging) has always been difficult for fluorescent microscope. Even though the sectioning ability of confocal microscope has been proved in imaging of large biological specimens, its temporal resolution is a limitation when imaging living samples. In this thesis, I will review our recent work of building up the light-sheet microscope to solve the above problem. The light-sheet microscope in our lab has 300 nanometer (nm) isotropically spatial resolution with two views using Gaussian beam light-sheet illumination. To solve the problem of limited field of view (FOV) for thin light-sheet created by Gaussian beam, we introduced Bessel beam light-sheet microscope. We...[ Read more ]

To monitor the development of three dimensional structure of large biological specimens as a function of time (4D fluorescent imaging) has always been difficult for fluorescent microscope. Even though the sectioning ability of confocal microscope has been proved in imaging of large biological specimens, its temporal resolution is a limitation when imaging living samples. In this thesis, I will review our recent work of building up the light-sheet microscope to solve the above problem. The light-sheet microscope in our lab has 300 nanometer (nm) isotropically spatial resolution with two views using Gaussian beam light-sheet illumination. To solve the problem of limited field of view (FOV) for thin light-sheet created by Gaussian beam, we introduced Bessel beam light-sheet microscope. We proved that due to its unique illumination geometry, light-sheet microscope has high spatial and temporal resolution, high signal-to-noise ratio (SNR), low photon damage, and low photon toxicity, which make it the optimal choice in 4D fluorescent imaging of live specimens with large size.