TY  - GEN
A1  - Singh, Rajwinder
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/31603/
N2  - 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.
AV  - public
CY  - Heidelberg
TI  - Development of a mesoscale oblique plane microscope for freely moving animals
Y1  - 2022///
ID  - heidok31603
ER  -