This protocol enables the efficient tracing of cell lineages and identities in live embryos while simultaneously analyzing detailed cell morphologies such as axons and dendrites in developing C.elegans neurons. Compared to confocal microscopy, dual-view inverted selective plane illumination microscopy or diSPIM offers higher signal to noise, more isotropic spatial resolution, and is better suited for longterm in vivo imaging. Begin by adding 500 microliters of 1%methylcellulose solution into the depression of a concave microscope slide.
Using a platinum wire pick, transfer adults from a nematode growth medium plate into the M9 methylcellulose solution. Use the sharpened tips of 18 gauge hypodermic needles to slice each animal transversely at the mid body to at least one to four cell embryos. With an aspirator mouthpiece held gently between the teeth, prefill a microcapillary pipette with 10 to 15 microliters of M9 buffer and gently aspirate several embryos from the slide into the capillary.
Dispense the embryos into the central rectangle of the coverslip of a steel imaging chamber filled with fresh M9 buffer. Gently orient the embryos vertically so that the long axis of each embryo is perpendicular to the long axis of the coverslip. Then place the steel imaging chamber into the sample holder on the stage of a diSPIM.
To prepare the embryos for imaging, open micro manager and set the laser intensities to 179 microwatts 0.5%equivalence for 488 nanometers and 79 microwatts 0.25%equivalence for 561 nanometers. The calibration between software adjustment and true laser output must be done ahead of time in order to set the scope for specific microwatts. In the plugins menu, select device control and applied scientific imaging diSPIM to open the applied scientific imaging dual-view inverted selective plane illumination microscopy window.
Under the acquisition tab, confirm that the parameters are set as indicated. Under the autofocus tab, set the parameters as indicated. Note that the autofocus channel should specify the nuclear fluorescence channel for the planned lineaging experiments.
Check the beam and sheet boxes for path A or B and click live to begin the image acquisition. A live view window will open. Then draw a box around the embryo of interest to select the autofocus analysis region of the embryo and click start acquisition to initiate the longterm multidimensional image capture.
For image viewing, processing and cell lineage analysis, after downloading and extracting the software bundle, double-click on the CytoSHOW_app. jnlp file to begin running CytoSHOW. Under the file menu, select new and diSPIM monitor micro manager to locate the root data set folder where the raw micro manager images were saved.
Select any time point folder and click open. Multidimensional navigation windows will be opened automatically for both SPIM A and SPIM B.Using the polygon selection tool, click just outside the anterior, posterior, dorsal and ventral edges of the embryo of interest to generate a bow tie pattern over the embryo in both the SPIM A and SPIM B views and click diSPIM. Align the green and red channels for each SPIM arm and click diSPIM again.
The shifts will be triggered in all of the other position windows. Next, click diSPIM and fuse to open the deconvolved fuse diSPIM raw data volumes dialog box and set the parameters as indicated. When all of the parameters have been set, click yes and specify the output directory in which to save the processed files before clicking OK.In the preview window, move the t-scroll bar to time point two of the window and move the z-slider until the view directly along the long axis of the embryo is shown.
Move the t-scroll bar back to time point one in the preview window and use the line selection tool to draw a line from the ventral most round nucleus through the plane of the AB cell metaphase plates. Click the diSPIM preview button to save the fine adjustments to the orientation of the previewed imaged volume. Set the t-scroll bars of the diSPIM monitor windows to the starting and ending time points of the full span of images to process.
Then click OK.For an efficient and accurate cell lineage analysis, it is critical to achieve a precise ADL orientation of the data volumes prior to Starry Night processing. To open the Starry Night lineage trace series in AceTree, open the provided customized AceTree file and select open configure file to locate the previously indicated output directory. Open the fuse subfolder for the embryo of interest and select the edited file.
Then click open and proceed with the lineage visualization and editing as previously described. Optimized protocols do not induce any detectable phototoxicity to the embryos as assessed by the time of hatching and the timing related to developmental milestones. Using this protocol as demonstrated, the unidentified cells in this transgenic green fluorescent protein expressing nematode strain were determined to correspond to motor neurons, the excretory canal cell, and two muscle cells.
The identified neurons were obliquely shaped as early as 360 minutes post fertilization with the longer cellular axis representing the subsequent axis for neurite outgrowth. From 385 to 410 minutes post fertilization, the RMDD neurites extended approximately six micrometers anterior of the cell bodies. From 415 to 445 minutes post fertilization, both neurites extended dorsally into and around the presumptive nerve ring.
On average, each RMDD neurite extended approximately 11 micrometers from the cell body before synchronously meeting its contralateral counterpart at the apex of the ring. Taken together, these data demonstrate that neuronal developmental features for single identifiable cells are able to be examined, compared and quantified using this integrated protocol. It is important to remember to orient the embryos vertically so that the long axis of each embryo is perpendicular to the long axis of the coverslip.
Using this protocol, we have begun to explore the nascent embryonic nervous system from all angles. For example, during cell birth, migration and differentiation, neurite formation, outgrowth and fasciculation.
