Team SiedyZsig

Imaging a fixed, large spheroid (&oslash; &asymp; 500 µm) on Zeiss Lightsheet Z.1
We used a cell line expressing GFP-tagged histone 2B. The spheroid was embedded together with beads in 1.5 % agarose in a glass capillary (black label).
 * Sample preparation


 * Imaging
 * 1 timepoint, 6 angles, dual-side illumination
 * excitation: 488 nm
 * detection: 20x ...

After approximately 2 h 6 min (2 h to set up the acquisition, 6 min to run it) we had a nice 36 GB data set. Post-processing in progress, images will follow...
 * Results

Outlook: Overnight time-lapse imaging of living, 4-days-old spheroids (&oslash; &asymp; 50 µm) on Zeiss Lightsheet Z.1
H2B-GFP cells were additionally labelled with the live-dye SiR-tubulin (Lukinavičius et al. 2014). They were embedded as single cells in Matrigel and injected into chambers made of either 1 % Gelrite or 1.5 % agarose. We incubated the chambers, and for the past four days the cells have been proliferating and forming small spheroids.
 * Sample preparation


 * Imaging

TBD


 * Results

TBD

Sample Preparation and Laser Ablation
'''Sample used 1. The zebrafish are anesthetized with tricane (200µg/mL) and embedded into FEP tubes (inner and outer diameters: 0.8mm and 1.6mm, respectively)filled with 0.1% agarose containing tricane. 2. A tube was mechanically attached to a metal-based sample holder. 3. By confining area of light sheet illumination with high-power two-photon laser, we succeeded to make well-targeted injury as shown on the right. '''Parameters for laser ablation
 * 3 days post fertilization
 * PTU treated: to inhibit pigmentation
 * Expressing HuC-GFP or Ras-GFP: labeling neuron or cellular membrane, respectively
 * Laser power: <600mW
 * Wave length: 920 nm
 * Duration: <2 sec

Imaging & Results
'''Myers' SPIM
 * Objective lens: 40x 0.8 N.A. Water dipping, NIKON
 * Area: 331x331µm sq, 1µm x 100 Z stacks/time point, Single angle
 * Excitation: 488nm, <2mW, exposure for 16 msec in average, single side illumination
 * Time-lapse: Every 10min for 24hr

'''Open SPIM
 * Objective lens: 40x 0.8N.A. Water dipping,
 * Area: 26 x 22mm sq, µm x 134 Z stacks/time point, Single angle
 * Excitation: 488nm, <, single side illumination
 * Time-lapse: Every 10min for 24hr

Initial question and approach
How can we image a entire organism without generating a huge amount of data that require extensive post processing time to reconstruct? One idea is to perform the imaging around the main axis of the body, like camembert slices (illustration to come). To keep the time resolution high and the time to set up the acquisition low those stacks would have to involve only the rotation motor and not the translation ones but that means that the main axis of the embryo must be align with the one of the rotation motor.

To align the capillary and the motor axis 3D printers would give us a precision lower than 10 µm. To center the embryo in the agarose tube my idea is to us hydrodynamic forces during the mounting. Indeed the mounting of the embryo in the agarose happens at low Reynolds number, therefore a Poiseuille flow is established in the capillary during the aspiration of the embryo, i.e. the speed of the fluid is maximal at the center of the canal and null on the side. In theory, for an embryo with high aspect ratio like drosophila one of the equilibrium position in this parabolic flow is at the center of the capillary with its long axis parallel to the flow, due to the hydrodynamic drag. The idea is to temper the capillary containing the agarose and the sample while the flow is still running to freeze the sample in position.