Home › Botany › Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor
Botany JoVE (Open Access) Citable · DOI

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

DOI: 10.3791/56142-v
What you'll learn
  • ✓ Set up live-cell calcium imaging of plant immune responses using GCaMP3 biosensor
  • ✓ Detect and quantify real-time calcium dynamics during aphid feeding on Arabidopsis
  • ✓ Analyze spatial and temporal fluorescence changes to characterize plant stress signaling
Protocol

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Difficulty
intermediate
Total time
~2–3 hours per experiment (including microscopy setup, live imaging, and data processing)
Model organism
Arabidopsis thaliana (GCaMP3 transgenic)

Steps

1
Detach and prepare Arabidopsis leaves for imaging

Remove leaves from GCaMP3-transgenic Arabidopsis plants and prepare them for microscopy. This preserves the leaf tissue while enabling optical access to cellular compartments.

▶ 00:44
2
Configure fluorescence microscopy system for live imaging

Set up the fluorescence microscope, adjust focus, and optimize laser/detection parameters to capture GCaMP3 signal before introducing insects. Ensures proper baseline imaging conditions.

▶ 01:38
3
Record calcium fluorescence during aphid feeding

Place aphids on the leaf and acquire continuous real-time video frames to capture calcium dynamics at the feeding site. High temporal resolution resolves dynamic cellular responses.

▶ 03:06
4
Generate time-course video and extract data

Compile recorded image sequences into time-lapse video and quantify fluorescence intensity changes over time. Enables both visual and quantitative analysis of calcium signals.

▶ 05:39
5
Analyze GCaMP3 fluorescence signal localization and kinetics

Quantify spatial distribution and temporal dynamics of calcium responses at and around aphid feeding sites. Determines specificity and magnitude of plant immune calcium signaling.

▶ 06:39
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