C.elegans embryogenesis takes about 13 hours, and is usually monitored by filming one embryo at a time. Our protocol, however, allows the simultaneous 3D time lapse imaging of 80 to 100 embryos. The ability to collect data for large numbers of developing C.elegans embryos opens up a new range of experiments, including quantitative analysis of developmental events and large scale screens.
This protocol can be easily adapted to capture different developmental processes or to image embryos expressing any combination of markers in tissues of interest. It's challenging to properly disperse the embryos within the wells. They should be close together to enable the capture of multiple embryos per field without clumping in different Z planes.
We recommend using two researchers for side-by side dissection, on ice, and blocking off unused wells. We will also demonstrate some useful tricks for quickly setting up a well-staged 384-well plate. Begin by sealing a glass-bottomed 384-well plate with PCR adhesive foil to mask unused wells.
Use a razor or scalpel to cut away the foil to expose a subset of the wells for use. Add 70 microliters of freshly prepared TMHC into each well and keep the plate on ice. Exclude the outer two rows of the plate to prevent edge effects.
For rapid sample preparation, side-by-side dissection by two researchers is recommended. When all of the solution has been plated, use fine tweezers and a dissection microscope to transfer about 10 gravid adults into 150 microliters of ice-cold TMHC solution within one depression slide per condition. Using the tweezers and a scalpel, dissect the worms to release the embryos and load a pulled capillary pipette into a mouth aspirator.
Use the mouth pipette to transfer all of the two to eight-cell stage embryos into individual wells of the prepared plate and examine the plate to confirm that no aggregates are present within any wells. When all of the embryos have been collected, settle the specimens by centrifugation and use an ethanol soaked wipe to remove any residue from the bottom of the plate. Place the plate in the plate holder of a confocal microscope equipped with a temperature-controlled environment and use the 10X objective to perform a pre-scan of each well to identify fields with suitable embryos.
At the end of the scan, switch to the 60X objective, adjust the focal plane at each point to image one to four fields per well and acquire 18 Z-sections at two micrometer intervals every 20 minutes for 10 hours. When all of the images have been acquired, perform a low, whole well brightfield scan, approximately 20 to 24 hours following the start of the overnight imaging to assess the embryonic lethality. For automated cropping using embryoCropUI executable, first download the program from Zenodo and the testfiles.
zip to determine whether the program is functioning properly on the platform. Once downloaded, unzip and navigate to find the embryoCropUI executable file and double click to launch the program. Select open to load the first specific 4D field of view to crop.
When cropping a TIFF series with multiple dimensions load only the first image in the series within the folder. When all of the images have been loaded specify the imaging parameters, select background subtraction and attenuation correction, and set the image collection order and the microns per pixel. Then select run, a new subfolder labeled crop will be created in the same path as the uncropped folder and the cropped versions will be saved in this location.
For visualization, download the open and combined MSV2 ImageJ plugin and graphic user interface instructions Zenodorepov2. docx from Zenodo and open the plugin in IMageJ. Locate lines three and four and input the location at which the image folders will be stored after the cropping.
The image folder names of the conditions to be processed, and the unique alphanumeric identifier for each overnight experiment. When all of the information has been entered, click run. A window will appear that will launch a prompt to navigate to the outer folder containing the cropped image folders.
Once selected another window will appear which will allow specification of the imaging parameters. Click okay. The composite file will begin to assemble.
When the composite has been generated, review the files that are left open. When imaged in conjunction custom reporter strains provide informative readouts for most major developmental events. Including sulfate specification, dorsal intercalation, epidermal enclosure, elongation, and neurogenesis.
24 hours after injection is an optimal time point for imaging. As of this time maternal protein depletion and inhibition of zygotic gene expression are both effective yielding distinct, highly reproducible signature phenotypes across a broad spectrum of genes involved in sulfate specification and morphogenesis. It is important to be patient during the dissection and embryo selection steps.
Minimizing the clumping and selecting the appropriate focal planes are also essential to ensuring high quality data. Our protocol can be performed with a variety of fluorescent marker strains and in combination with RNAi or mutants. Our processing tools allow for streamlined, automated, or manual data analysis.
Using the strains, methods, and tools described here our group performed an RNAi-based high content screen targeting approximately 2, 000 genes required for embryonic development. Semi-high throughput methods were essential for this project.
