Development of digital fluorescence light sheet microscopy for high-resolution optical imaging

In modern biology investigating the morphological development of an organism is an inevitable task. Optical microscopy has been extensively used to monitor the live tissue changes in a model organism and it is typically used in conjunction with white light or fluorescence microscopy. The fluorescence microscopy occupies a significant place in biological research due to its ability to address various biological question at subcellular level of a biological sample. One of the more recent developments in fluorescence imaging is selective plane illumination microscopy (SPIM), it is a wide-field fluorescence microscope that images the sample based on the optical sectioning by a light sheet. The main optical properties of SPIM depend on the thickness and the distribution of the light sheet. Controlling the characteristics of the light sheet is possible by using engineered beams such as the Bessel, Airy, tiling beams, etc. Ideally, a SPIM should be able to apply different engineered light sheets to provide the user with the direct chance to evaluate different light sheets on the same sample for optimum image quality. The first section of this thesis provides insights into SSPIM, which is developed to achieve better imaging system. SSPIM is a hologram producer for beam engineering that converts a Gaussian beam to all possible light sheet variants. We demonstrate that the SSPIM is readily integrated into the SPIM setup and can enhance the imaging capabilities of a large biological sample. The second part of this thesis focuses on the development of a novel SPIM setup, which addresses some of the existing limitations of SPIM in resolution caused by the non- transparency of a large biological sample. The core of this SPIM relies upon the Multiview SPIM (M-SPIM) setup, which enables the observer to image the sample from different views simultaneously. To accomplish this, M-SPIM is build up as a four lenses SPIM set up to illuminate the sample simultaneously on two sides and detect the emission signals in orthogonal directions. In order to engineer the light sheet within this setup, the M-SPIM was combined with a spatial light modulator (SLM). We take advantage of the SSPIM software to produce several holograms as virtual lenses for applying to tile thin light sheets over a large field of view. We demonstrate that the combination of the tiling technique and M-SPIM, called MT-SPIM, has several advantages compared to previous methods including sample rotation. The first advantage is that a fairly large sample can be imagedin sub-cellular resolution without any mechanical rotation, preventing the sample from unwanted drift over the imaging. The other advantage is that the spatial and temporal resolution is homogenous in different planes, resulting in a successful 3D image registration and fusion. The results show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two which improves imaging quality of nuclei and MyosinII from cellular to subcellular level during early embryogenesis of Drosophila melanogaster.