Encyclopedia of Experiments: Biology
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Transcript
- Place one transgenic worm expressing GCaMP-- a calcium sensing protein-- in an imaging chamber along with bacterial food for the worm. Then seal the dish with the lid and laboratory film to prevent evaporation. View it under a compound microscope equipped for wide field epifluorescence. Record time lapse images of the worms to track their behavior and capture fluorescence using imaging software.
Transgenic worms express a GCaMP sensor protein under the control of a promoter that drives GCaMP expression in a specific neuron. When that neuron is activated, it fires an action potential, which depolarizes the plasma membrane. Upon de-polarization, voltage-gated calcium channels open in the plasma membrane. This causes an influx of calcium ions into the cell, resulting in neuronal excitation.
GCaMP in the excited neuron binds the calcium ions. This causes GCaMP to fluoresce when the worms are imaged with a low intensity fluorescence excitation light. In the example protocol, we will use calcium imaging to visualize AVA interneuron activity in transgenic C. elegans in agarose microchambers.
- Calcium imaging is performed on a compound microscope equipped for wide field epifluorescence. To limit light exposure, use a transistor-transistor logic signal that triggers an LED to illuminate the sample at the same time the camera records a frame.
Run a burst movie for 24 hours that images each worm every 15 to 30 minutes. First, record 20 seconds with DIC, then 20 seconds with GFP fluorescence, and finally, an image of the mKate2 signal for control expression levels.
For visual data inspection, use a false color map to enhance the visibility of small changes in fluorescence intensity. Lastly, perform calcium data analysis using standard procedures.
