Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. This simple protocol for 3D time-lapse imaging can directly study cell wall dynamics over a long period. This protocol uses calcofluor dye to mark the dynamics of cell wall thickness, and therefore does not require a transformation of a fluorescent protein.
The calcofluor signals are stable and can last for more than a week. This method may be applied to many other systems with simple structures containing cell walls and can be stained by calcofluor such as in flowering plants. In this protocol, the calcofluor signals are stable when this dye is mixed within the medium during the whole process of time-lapse imaging.
To begin, disperse the seven day old moss tissue into a 1.5 milliliter micro tube containing 20 microliters of sterilized water. For the wild type use forceps to separate the protonemata. For the ggb mutant, separate the protonemata using AP-1000 pipette.
Add 10 microliters of the sterilized calcofluor-white into the microtube and leave the tube upright in the hood for two minutes for staining. Immediately after staining, mix the plants with 200 microliters of cooled BCDATG containing BCDAT medium by pipetting five times with a P1000 pipette and transfer the mixture into a 27 millimeter glass base dish. Ensure that the 200 microliters of BCDATG with samples is evenly distributed on the surface of the 27 millimeter glass bottom dish to produce a thin layer of film so that the samples are within the objective working distance during imaging.
After solidifying for two minutes at room temperature, cover the thin layer with three milliliters of cooled BCDATG and let it set for 10 minutes to solidify again. On the bottom of the dish draw nine lines each in parallel and perpendicular directions and mark the rows with characters from A to H, and the columns with numbers from one to eight. Use upper, lower, right, middle, and left to mark the positions within each square.
For example, if a plant is in a square in the second row, in the sixth column and is positioned in the upper left part of the square, then this plant is given the name of b6 upper left. Place the glass bottom dish under white light for one to two days before being subjected to time-lapse imaging every day, for up to three days. To reconstruct the 3D images open the ND2 file and click volume.
Reconstruct the cell wall at day zero, day one, day three, and day four. After taking each image, put the glass based dish back into the grow chamber. Dynamic changes in cross walls were shown by calcofluor-white staining in wild type, and ggb mutants.
For ggb mutants, white dashed lines below each image indicate the broken surface cell wall in the expanding cells and orange lines indicate the cells beneath the cell wall surface in ggb. The differences in calcofluor-white signal can be detected on the surface positions of the cells and less densely stained regions indicated cell expansion in ggb mutants or newly branching sites in the wild type. For wild type pre culture, the plants in weak red light from the above for five days is required to induce the phototropic responses of protonemata so that the plants can be attached to the bottom of dishes and the plants can be emerged within the objective upworking distance during imaging because calcofluor-white can be used with other dye such as microsphere.
Additional information about the cell expansion can also be studied. Dynamics of plant cell wall is critical for efficient doing growth and development as well as for response to environmental signal. This protocol allows for direct of the division of changes in cell wall signals and integrating.
