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Nematostella Vectensis belongs to the phylum cnidaria which is the closest relative of bilaterians and has become an important model organism for evolutionary development biology. However, because of its high light sensitivity, large size, and constant need to move freely in order to properly develop and survive, in toto live imaging of Nematostella is a major challenge in microscopy. Several important questions, e.g. how animal behavior during muscle hydraulics guides the animal morphology and how neuronal dynamics control muscle movements and body deformations that are necessary for animal development remain unanswered. In the field of microscopy, light-sheet fluorescence microscopy (LSFM) has emerged as a preferred tool to image light-sensitive large samples due to optical sectioning and high-speed functional imaging capabilities. In a conventional light microscope, the specimen needs to be embedded in agarose and placed within a narrow space between orthogonally lying illumination and detection objectives. These constraints however make it impossible to use on Nematostella or other freely moving animals. On the contrary, recently developed, so-called single objective light-sheet microscopes are capable of imaging freely moving animals, but the lateral field of view (FOV) in such existing techniques is limited to 1*1mm2 . To overcome these challenges, in this thesis I developed an oblique plane microscope for imaging freely moving specimens at mesoscopic scales (MesoOPM) that provides light-sheet scanning based rapid volumetric imaging capability with cellular resolution and over 2mm FOV. By utilizing a long working distance illumination objective and placing it at 65 degrees angle to the optic axis of the detection objective, we can maintain the open-top configuration that allows easy sample mounting. Furthermore, the rapid tilt-invariant light-sheet scanning along the image plane of the detection objective is achieved by precisely synchronizing an electrically tunable lens with the galvo scanner in the illumination arm. I performed meticulous optical design optimization of the microscope to maximize the spatial resolution under these conditions. The experimental PSF of the MesoOPM was then measured with diffraction limited beads and found to be 1.62*2.81µm2 in lateral dimensions and the 5.27µm in axial dimension. Finally, the imaging capabilities of the microscope are showcased by imaging the muscle structure and the nervous system of a freely moving live Nematostella at 300 frames per second, covering the entirety of the animal over a FOV of 1.56mm*0.78mm*240µm in two seconds. This is the first-ever report of imaging 3d volume of a live freely moving Nematostella in its entirety using a fluorescence microscope. This technology opens up a whole new direction of imaging an entire freely moving specimen which will allow us to study the interactions between animal behavior and the environment by visualizing the underlying cellular structures, which so far has been a challenge. |
