The aim of the following experiment is to visualize the early events of epidermal development after seed germination at the cellular level. This is achieved by first removing the seed coat to allow a clear view of the kosin as a second step. The kosin are mounted under a layer of agar media that immobilizes them and supports their development next time-Lapse imaging is performed over several days in order to observe the growth and division of epidermal cells in vivo.
Results were obtained that show dynamic gene expression and protein localization based on visible fluorescence. The main advantage of this technique over existing methods, such as still images of Gus staining or GFP fusions, is that it shows dynamic movement of proteins during cell division and differentiation. The Arabidopsis seeds for this experiment are sterilized in a seed sterilization solution made of 33%household bleach and 0.1%Triton X 100 place seeds carrying the desired fluorescent reporter constructs and genotypes in a 1.7 milliliter tube and apply one milliliter of sterilization solution, incubate on a mutator for 15 minutes.
In a sterile hood, use a pipette to remove the sterilization solution from the tube leaving the seeds behind. Rinse with one milliliter of sterile water. Repeat four times after the fourth rinse.
Incubate at four degrees Celsius for two or more days. To prepare the chamber slide media mix 20 milliliters of 0.5%solution of bact to agar in water in a 200 milliliter flask and microwave until dissolved. Be cautious when boiling the solution.
Call the solution to around 60 degrees Celsius. A solution that is too hot or applied too quickly may melt the glue or crack the glass of the chamber slide. When the agar solution is cool enough, collect one milliliter with a pipette and slowly apply it to the cover glass inside the chamber slide immediately.
Add a second milliliter of agar solution. Agar Media should now completely cover the bottom of the chamber slide. This minimal media is sufficient to support development in a plant olein, which contains stored nutrients for four to five days by maintaining moisture and allowing gas diffusion.
Call the slide with the chamber covered on the benchtop. To begin this procedure, remove the tube of sterile seeds from four degrees Celsius storage. Place a paper tissue on the stage of a dissecting microscope, moist them with water and adjust to create a smooth surface.
The tissue is used to stabilize the seeds during dissection. Pipette 20 to 30 seeds from the tube onto the tissue, being careful to place them in the field of view of the dissecting scope. Dissecting the co leadin is the most challenging aspect of this procedure.
Using low magnification and sharp forceps and being very careful will help to ensure a successful dissection Using sharp forceps. Carefully remove the seed coat from the seedling inside, both outer and inner integuments must be removed. Next, use a scalpel to slice the cot leadin free of the hyper cottle and radical.
It is beneficial to remove the hyper cot and radical for imaging because if intact, the hyper cott will straighten and lengthen dramatically. Moving the Koss Leadin out of the time-lapse field of view, keep the dissected cos leadin immersed in water. Repeat the seed dissection until the desired number of Olean is reached.
15 to 20 successful dissections are recommended before mounting olean in the chamber slide. To prepare the chamber slide for mounting cotan, use a small cover slip to cut through the agar media and lift the entire layer up from the glass base of the chamber. Slide pipette dissected cotan with a minimal amount of water from the holding tube into the chamber.
Slide under the lifted agar layer. Gently lower the agar onto the sedins. If excess water is present, soak it away with a tissue.
Being careful not to disturb the sedins when mounting is complete. Specimens should no longer move easily. Place the cover on the chamber slide and transfer the slide to the microscope stage.
Prior to imaging, set up the inverted confocal microscope to image the desired Fluor. The LSM 700 parameters are for GFP excitation of 488 nanometers and collection. With a band pass filter at 440 to 530 nanometers and for RFP excitation at 555 nanometers and collection.
With a band pass filter at 570 to 610 nanometers with a 20 times objective. Focus on ACO lead-in and move the objective to the center of the chamber. Slide under acquisition mode change zoom to 0.5 and frame size to 48 by 48 pixels.
Select the positions checkbox and scan overview image and then increase horizontal and vertical tile numbers to 30 to 35. Click the scan button to begin scanning. When the scan is complete, click the positions button under the dimensions tab and place Cross hairs at each cot leadin to observe.
Inspect the mounted specimens for damage. Delete positions corresponding to damaged coss leadin to leave only suitable specimens for imaging. Next, set up a Z series encompassing the coss leadin epidermis of all samples.
Click the Zack checkbox. Select each position under position list and click the move to button. Focus on the uppermost plane of the cos lead in epidermis and click the set first button under Zack.
Focus to the lowest position at which the epidermis is usefully visible. And click the set last button. Click the button next to optimal, which shows the best Z slice thickness to set.
Check each kos lead-in to confirm that these settings encompass all samples. The Z parameters cannot be set for individual positions. In the Zen 2009 software used here set up time series with the desired resolution and length intervals of 15 to 30 minutes for three days are effective for protein dynamics in epidermal cell division, but intervals can be changed as necessary.
Click the time series check box under time series set cycles for the number of images desired by typing in the text field set interval to 30 and use the pull down menu to select MIN to begin XY, ZT multi-position scan change frame size to the desired resolution and collect start experiment. Note that the number of positions must be limited by the scan specifications. If total scan time is greater than the desired interval, the time points will not be correct.
Using this system, a maximum of six simultaneous positions are possible when imaging GFP and RFP at 59 Z slices in a 30 minute interval. An example of a set of informative time points collected with this method is shown here. Cell membranes are labeled with RFP and GFP fused a polar protein under its native promoter.
The images in series A show that polar GFP initially appears evenly distributed then approximately two hours before asymmetric divisions indicated by arrowheads. Polar GFP segregates away from the site of the incipient meister OID in series B following the initial asymmetric division indicated by the arrowhead at 47 hours. Polar GFP disappears in both cells.
Implying rapid transition to guard mother cell state by the meris oid, the guard mother cell indicated by the asterisk divide symmetrically and differentiates to form stomatal guard cells while the cysto cell regains polar GFP expression presumably preceding a later asymmetric division. Shown here is a streamlined registered video of polar GFP localization during amplification and spacing of one stomatal precursor cell. Initially polar GFP appears uniformly in the cell by 39 hours after germination.
It polarizes strongly just before a division at 40 hours placing a smaller maid at the opposite end of the cell. The larger cell on the right also regains stomatal lineage identity and by 45 hours Polar GFP localization moves adjacent to the stomatal precursor. The division at 47 hours produces a new steroid on the right oriented away from the existing me stamoid on the left from the first me stamoid.
The end result is two stoma are separated by one cell. After watching this video, you should have a good understanding of how to use long-term time lapse of a germinating co leadin to assess gene expression and protein localization in the epidermis during development.
