This method can help answer key questions in the plant development field regarding the distribution of growth regulatory phytohormones. By using genetically encoded FRET biosensors, for example nlsGPS1 which detects gibberellins, it is possible to measure the dynamic distribution of important compounds in Arabidopsis tissues at the cellular solution. Generally, individuals new to this matter will struggle, because analyzing FRET biosensors involves ratiometric imaging which has increased noise and additional analytical steps compared to intention metric imaging.

Begin by sowing approximately 20 sterilized Arabidopsis seeds on 1/2 Murashige and Skoog or MS agar square plates. Seal the plates with pore surgical tape and wrap them with aluminum foil for one to three days of incubation at four degrees Celsius for stratification. For root imaging, transfer the plates to a growth chamber in a vertical orientation for three days under the indicated conditions.

For dark-grown Hypocotyl, transfer the plates to the growth chamber for a one to four hour light pulse to synchronize the germination. Then wrap the plates with aluminum foil, and place them into the growth chamber in a vertical orientation for three days. For steady state measurements, add 50 microliters of mock solution to a clean microscope slide and gently place three nuclear localized gibberellin perception sensor one or nlsGPS1 expressing seedlings onto the slide.

Then spot a drop a vacuum grease on each corner of a clean cover slip and gently place to cover slip over the seedlings. Carefully adding extra mock solution to remove any air bubbles as necessary. Before gibberellin four or GA4 treatment, use a 20 milliliter syringe filled with vacuum grease and equipped with a modified 200 microliter pipette tip with a one millimeter diameter opening to draw a uniformly layered three and a half centimeter long by two and a half centimeter wide rectangle on a clean glass slide.

Add 50 microliters of mock solution to the center of the rectangle and use clean forceps to lift nlsGPS1 expressing seedlings by the underside of their cotyledons for careful transfer into the mock solution. Place a cover slip spotted with vacuum grease as just demonstrated into the center of the vacuum grease rectangle and carefully fill the reservoir with extra mock solution without disturbing the seedlings. Then acquire images of the seedlings on a confocal microscope equipped with appropriate lasers for exciting CFP and YFP to perform Forster resonance energy transfer imaging.

For GA4 for treatment, remove the slide from the microscope stage and set a timer for 20 minutes. Immediately remove the mock solution from the right side of the cover slip while adding 17 microliters of one quarter MS liquid supplemented with one micro molar GA4 to the left side of the cover slip until all of the mock solution has been replaced. Then place the glass slide back onto the microscope stage and wait another 10 minutes before acquiring the after GA4 treatment image.

To set up a time course for a tissue of interest, add 200 microliters of mock solution to the center of the perfusion channel of an ibidi sticky-slide, and gently place nlsGPS1 seedlings into the mock solution as demonstrated. Use forceps to gently place a cover slip over the seedlings using the backside of the forceps to press gently on the outer edges of the cover slip until it forms a strong bond with the sticky material on the periphery of the sticky-slide. Next, use two 0.8 millimeter inner diameter elbow Luer connectors and 0.8 millimeter inner diameter piece of silicone tubing to connect the sticky-slide to a 20 millimeter syringe filled with mock solution and to connect the slide to an outlet container for collecting the outflow solution.

Gently depress the plunger to dispense enough solution to the chamber to ensure that there are no air bubbles and then load the syringe onto a programmable syringe pump. Then start the pump to initiate the time course. For GA4 treatments during the time course, pause the pump to stop the perfusion And replace the mock buffer containing syringe with a syringe filled with one quarter MS liquid supplemented with GA4.

For 3D image analysis, import the files into the IMARIS image analysis software program and open the surfaces wizard. Set the background subtraction to three microns and the thresholding to default. During the segmentation of the nuclei take care not to include the many nuclei with dim fluorescence as the signal-to-noise ration of the object with near background levels of signal will be too low for ratiometric image analysis.

Then mask the CFP and FRET emission channels based on the surfaces created using the YFP emission. Use the XT mean intensity ratio extension to compute a ratio of donor excitation acceptor emission divided by the donor excitation donor emission between the mean intensity values of the individual surfaces in the two channels. To color the individual surfaces with the nlsGPS1 emission ratio, select color coding with statistics and set the mean intensity ratio as the statistics type.

Under the statistics icon, export the ratios of individual values from the table and copy and paste the values into a spreadsheet for graphical representation of the data. In the Arabidopsis root, the nlsGPS1 emission ratio gradient is indicative of low GA4 levels in the meristematic and division zones and high GA levels in the late elongation zone. In contrast, an emission ratio gradient is not observed in nlsGPS1 nonresponsive roots, suggesting that the endogenous GA4 gradient is not an artifact.

An nlsGPS1 emission ratio gradient is also formed in dark-grown hypocotyls with low levels in the cotyledons and the typical hook and high levels in the rapidly elongating basal region of the hypocotyl. In contrast, an emission ratio gradient is not observed in the nlsGPS1 nonresponsive hypocotyls. Furthermore, exogenously supplied GA4 accumulates preferentially in the elongation zone compared to the division zone of the Arabidopsis root indicating that nlsGPS1 can be used to study endogenous and exogenous GA patterning.

In addition, time course analysis of GA4 treated nlsGPS1 seedlings, reveals a faster accumulation of exogenous GA4 in the root elongation so compared to the division zone. While attempting this procedure it is important to remember to avoid saturated pixels during the quantitative imaging to keep the imaging parameters constant and to minimize the drift and focal change issues during the sample perfusion. Following this procedure elaborated methods, like imaging roots growing in root chip type control profusion devices can be performed to answer additional questions about how GA gradients change in Arabidopsis root tips growing at different rates or in response to stress After its development, this technique paved the way for research in the field of plant growth to explore the dynamic distribution of the gibberellins in additional Arabidopsis thaliana tissues and organs.

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