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

introduction and overview
I am interested in tissue regeneration in the zebrafish, especially brain regeneration after traumatic injury. In my PhD, I focus on immune cells that are main players in the inflammation because we characterized acute inflammation as a positive regulator of the brain regeneration. Considering dynamic property of those cells and days-long process of inflammation, obtaining spatio-temporal information microscopy in which specimen gets minimum photo-toxicity and photo-bleaching. Therefore, we decided to study dynamics of immune cells by SPIM. In this course I would like to observe neuron dynamics in the zebrafish brain after traumatic injury induced by two-photon laser. I visited Gene Myers lab as they have excellent equipment on their SPIM, two-photon light sheet laser which is used to make injury and single-photon light sheet microscope which allows immediate observation. In addition, I used an Open-SPIM only for observation after the ablation with Myers' SPIM because it has more similar equipment to ours which we are going to assemble in a month.

Laser-induced ablation and SPIM observation @ Myers' lab
'''Sample Preparation and Laser Ablation 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 fixed to a metal-based sample holder with adhesive tape as shown in the picture on right. 3. To make the nearest access of the laser to the brain, the tube was rotated dorsal-side upright (see the scheme). 4. 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 Ras-GFP: labeling cellular membrane
 * Laser power: <600mW
 * Wave length: 920 nm
 * Duration: <2 sec

'''Imaging and result The movie shows the brain shrank soon after the ablation and tissue components running out of the injured tissue were captured. After shrinkage the brain bunched up due to either development or tissue elasticity of the brain as often referred as sponge-like structure. Observation of the other brain hemisphere with lower magnification will define the cause of this morphological
 * 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 12hr
 * Special thanks to Nicola

SPIM observation with Open-SPIM
'''Sample Preparation and Laser Ablation
 * 3 days post fertilization
 * PTU treated: to inhibit pigmentation
 * Expressing HuC-GFP: labeling neuron

'''Imaging and Result Unfortunately we could not find an injured spot with this sample. When we were trying to make an injury, we could not define ROI well because, somehow, this transgenic line emitted very blur fluorescence. It might be because of the highly-compacted population of the neurons in the brain. In this time-lapse movie, neurons are coloured according to the depth of them within the sample. The movie shows dynamics of neurons over developmental stages at single-cell level. During imaging the fish moved and the focusing Z positions were shifted. This movement is most likely due to the development not voluntary movement. This is inevitable when working with zebrafish larvae, therefore, It would be solved by better manipulation of data processing or development of a new function of 4D-SPIM stage coupled with the camera, for instance, auto-chasing of a certain remarkable structure in the specimen.
 * Objective lens: 20x 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 18.5hr
 * Special thanks to Pete

'''Data Processing Procedure Since we could not register the images in a way the majority of the people does, probably because of high level of neuron compaction in the brain, we took another way of processing. This might be a unique case, thus, I would like to report here what steps we followed. '''Concatenation of the images '''Hyperstacking into a 4D image '''Temporal Colour Code To show the depth of each Z stack, temporal colour code will be useful '''Time Stamp To show each time points on                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          9time-lapse data, time stamp is required.
 * Open all the tif images that you would like to make into time-lapse.
 * Image>Stacks>Tools>Concatenate>All Open Windows
 * The resulting file contains the Z-stacks and the time points, however, it does not have two channels that are for Z-projection and time lapse.
 * Image>Hyperstacks>Stack to Hyperstack
 * Order:default, Channels: # of colours, Slices: # of Z stacks, Frames: # of time points
 * Image>Hyperstacks>Re-order Hyperstack
 * Change boxes to: size(z)->Frames(t), Frames(t)->slides(z)
 * Image>Hyperstacks>Temporal-Color code
 * LUT = Spectrum
 * Image>Stacks>Time Stamper
 * Special thanks to Olivieer

Initial question and approach
Can we image the full periphery of an organism without generating a huge amount of data that require extensive post processing time to be reconstruct? One idea is to perform the imaging around the main axis of the body, like camembert slices (figure 1). To keep the time resolution high and the time to set up the experiment reasonable the acquisition of the stacks would need to require 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 and to center the embryo in the agarose tube my idea is to us hydrodynamic forces. 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 due to the hydrodynamic drag, 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. 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.

Initial setup idea

first attempt
making of the canal

after the removal of the plunger

Because of the double surrounding wall around the pdms the extraction of the glass capillary was impossible and it broke. Once the entrance is full of glasses pieces the canal become unusable

Image processing
[[Media:Movie.ogv]]