The overall goal of this microscopy protocol is to measure in vivo plant calcium dynamics as insects feed from a leaf. This method can help answer key questions about plant aphid interactions such as how plants detect and respond to aphid pests. The main advantage of this technique is that it allows us to detect plant calcium signals in vivo whilst a live insect is feeding from the leaf.
Although this method can provide an insight into the role of calcium signaling during aphid feeding, it can also be applied to other systems, such as new insects or plants or even to detect signals in response to abiotic stresses, such as salt. Begin by growing 35S:GCaMP3 Arabidopsis plants and on Day 11 rearing aphids as described in the text protocol. On Day 19 of the experiment, remove the seedlings from the controlled environment room.
Detach the largest leaf from each plant using a pair of sharp scissors. Next, use a pair of tweezers to place each leaf, abaxial surface facing up, into separate wells of a 96-well plate containing 300 microliters of distilled water per well. To reduce detachment stress in the leaves, cover the plate with clear plastic wrap and aluminum foil and leave it at room temperature overnight.
On Day 20, collect the aged aphids from the colony established on Day 11 by moving the infested plant from the soil into a transparent box with a lid. Make sure to keep the insects contained in the box during the experiment. Remove the aluminum foil from the 96-well plate and transfer it to a fluorescent stereo microscope.
Adjust the light exposure until the GCaMP3 fluorescence can be visualized in the veins of the leaves. Adjust the magnification in zoom for the observation of four wells in one frame. Using a moist paintbrush, transfer one aphid to a detached leaf under the microscope.
Lightly touch the adjacent leaf with the empty paintbrush as a control. Place the plastic wrap back on top of the plate to prevent the aphid from escaping. Record the fluorescence of the leaves in pairs of one aphid treated and one untreated.
Begin by clicking Start Experiment in the built-in microscope software. After recording measurements for 50 minutes, click Stop Experiment. Remove the aphid from the leaf.
Repeat the measurements with further pairs of leaves. To begin data collection, open the Image software. Import the image files into Fiji or ImageJ.
Convert the recorded images to 32-bits by clicking on Image, Type, and then 32-bit. Start by clicking on the Image tab and selecting Properties. Set the measurement scale to pixels and the time frame to the same time interval as used during the microscopy.
Follow the insect movement under the microscope and discard the samples in which the aphids do not settle in one location for more than five minutes. Use the cursor to place a region of interest, or ROI, around the area of tissue that will be analyzed for GFP signal. Create the ROI by drawing an oval with the Oval Tool and edit the size using Edit, Selection, and then Specify.
Select ROIs on the untreated controls in regions of the leaf comparable to those selected on the treated leaf. To analyze the raw fluorescence values, or F in the ROI over time, use the Time Series Analyzer plugin by clicking Plugins and Time Series Analyzer at the ROI of interest, by selecting Add button. After selecting the ROI, select Get Average to display a table of F-values for each frame in that ROI and copy this data into a spreadsheet.
Next, select the region of the feeding site GFP signal using the Freehand Selection Tool. Outline the maximal signal in the feeding site and then calculate the area of this shape by clicking Analyze and then Measure. To calculate the speed of the feeding site, use the MTrackJ plugin by clicking Plugins, Tracking and then MTrackJ.
Next, click on the Add button and then on the cursor at the center of the signal when it is first visible. Click again on the edge of the signal at its point of furthest spread. Finally, to calculate the speed of the signal, click Measure.
To convert the 32-bit image files into heat maps, use the NucMed_Image LUTs plugin. Select NucMed from the Plugins menu and then click Lookup Tables. Select Blue Green Red to convert the images to heat maps.
If it is difficult to visualize the aphid associated GFP signals, enhance the contrast of the heat map by clicking Process, Enhance Contrast and then adjust Saturated pixel percent. Finally, use the Time Stamper to add time information by selecting Plugins and then Time Stamper. Set the starting time at zero and the interval based on the time interval used for microscopy.
Within a few minutes of aphid settling, there was a highly localized increase in GFP fluorescence around the feeding site, showing the elevation of the cytosolic calcium in the treated leaf. In the control leaf, on the other hand, the calcium dynamics stay relatively stable. Some treated leaves show secondary increase in GFP fluorescence after the initial peak.
The time course video of the aphid treated leaf shows the dynamics of the GCaMP3 fluorescence over time. In systemic midrib and the systemic lateral tissue regions, areas that are not around the aphid feeding site, no calcium elevation is detected in either treated or control samples. While attempting this procedure, it's important to remember not to touch or disturb the leaves or the insects unnecessarily, as this may result in touch-induced calcium signals or prevent the aphids from settling.
Generally, individuals new to this method might struggle to ensure the aphids settle on the leaf to feed, but this can be minimized by handling the insects as little as possible. After watching this video, you should have a good understanding of how to measure plant calcium dynamics in vivo using a fluorescent biosensor when insects feed from a leaf. Extending from this procedure, leaves from mutant plants can be used in order to answer additional questions like the genetic mechanisms involved in detecting the aphid and enlisting the calcium signal.
