4D microscopy is a valuable tool for reconstructing the complete cell lineage of C.elegans embryo or any other specimen at the level of single cell. Nomarski optics can be used to highlight cell shape changes during development with a standard microscope and transmitted light without the need for transgenic animals or fluorescence microscopy. 4D microscope allows to study the cell cycling, proliferation, differentiation, and apoptosis in C.elegans and in other systems such as mammalians or tissue cell culture.
Cell lineage tracing on DIC images is performed manually. It's time consuming and requires some practice to perform a full embryo analysis. Demonstrating the procedure will be Begona Ezcurra, a technician in our lab.
Before preparing the embryos, adjust the recording temperature of the 4D microscope to 15, 20, or 25 degrees Celsius and set the microscope to record 1, 500, 30 focal plane z-stacks one micron apart with 30 second intervals between the beginning of each stack. Next, aliquot 500 to 1000 microliters of liquid 4.5%agar into three millimeter glass tubes and carefully seal the tubes with wax film to avoid desiccation. To prepare a thin homogenous agar pad for the recording, carefully heat and aliquot of agar over an alcohol burner until the agar is melted and place the tube in an 80 degree Celsius heat block.
Place a glass microscope slide onto a piece of plastic and use a pasture pipette in one hand to place a small drop of melted agar into the center of the slide, then immediately use the other hand to place a second slide onto the agar drop to create a very thin agar pad. To mount the embryos, first, use a picker to transfer 5 to 10 gravid hermaphrodites into a watchmaker glass filled with water under a stereo microscope. Use a scalpel to cut open the hermaphrodite nematodes and extract one to four early eggs from the uterus.
Gently slip the top slide off of the agar pad and use a capillary tube to place an early egg into the center of the pad. Use an eyelash glued to the end of a toothpick to adjust the egg position as necessary, and use the capillary pipette to remove any excess water. Use a scalpel to slowly and obliquely drape the cover slip over the preparation before using a micropipet to fill 3/4 the space surrounding the agar pad with water.
Then seal the cover slip with petroleum jelly to avoid desiccation during long recording periods. After sealing, place the slide onto the 4D microscope stage and use a low magnification objective to bring the embryo into focus. Select the 100X immersion objective and focus the condenser.
Completely open the aperture of the condenser and close the field diaphragm to provide a higher numerical aperture and greater resolution. Turn the Wollaston prism to obtain a three dimensional image of the embryo illuminated on one side, then turn the prism in the other direction to get the effect of having the embryo illuminated on the other side and begin the 4D recording. For cell lineage reconstruction of a 4D movie recorded embryo, open the lineage tracing software and select File and New project.
Select the appropriate cell lineage template depending on the recording temperature and set the scan count to 1500. The time between scans to 30 seconds, the level count to 30, and the distance between levels to one micron. Select the image file, format, and directory where the images were saved.
Select whether the images were saved as single images or as multi-image z-stacks and set the file naming and image formats. Then click Channel processing enabled to detect and set the appropriate light channels of the 4D movie. For cell lineage tracing, initiate the cell lineage tracing software.
A video window and cell lineage window will appear. In the lineage window, select the lineage branch and click the cell nucleus corresponding to this cell in the video window. Play the 4D movie forward, backward, or up or down a level to follow the cell of interest spatially over time.
Periodically click on the cell nucleus to generate a point in the lineage branch and to register the spatial coordinates of the cell at a specific time. As a result, cell lineage progressions and 3D reconstructions of the embryo are possible. Then press the return key to mark mitosis and select one of the daughter cells to follow the cell progression as just demonstrated.
Repeat the analysis for the rest of the embryonic cells to trace the complete cell lineage or to follow specific cells of interest, such as those undergoing apoptosis. To allow the comparison of mutant lineages of interest with the stereotyped wild-type C.elegans cell lineage. Worm plates are incubated within cardboard boxes to avoid condensation.
Once the recording is complete, the cell lineage can be reconstructed using lineage tracing software as demonstrated. For example, in this representative analysis, the traced gsr-1 mutant embryo cell lineage was compared with the C.elegans wild-type lineage. And the detection of a progressive delay in the cell cycle during embryonic development was revealed.
As a consequence, mutant embryos arrested at intermediate stages whereas wild-type embryos progressed and finally hatched as larvae. In addition, other important features of embryonic development, such as apoptosis can be dynamically visualized using 4D microscopy. Lineage tracing of the DIC images must be manually processed.
A software processing of unmarked cells on visible light in early stages of development are not accurate for embryo analysis. The application of image recognition system to field of visible light microscopy will bring about a great advance in developmental biology field.
