This video introduces the concepts behind calcium imaging and includes an example protocol for time-lapse imaging of worms contained in molded agarose wells.
This protocol is an excerpt from Turek et al, Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans, J. Vis. Exp. (2015).
1. Calcium Imaging
- Use transgenic strains expressing genetically encoded calcium sensors such as HBR16 (goeIs5[pnmr-1::SL1-GCaMP3.35-SL2::unc-54-3'UTR, unc-119(+)]).
- Use a compound microscope equipped for wide-field epifluorescence. Connect the TTL output of the EMCCD camera to the TTL input of the LED, so that each time the camera records a frame the sample will be illuminated. Use an exposure time of about 5 msec. Use EM gain in the range of 50 - 300.
- Specify a burst movie running for 24 hr with each worm being imaged every 15 - 30 min first for 20 sec with DIC, then for 20 sec with a GFP fluorescence to record GCaMP, and then take a final image of the mKate2 signal is taken to control for expression levels. Use a frame rate of 2/sec during each burst.
- For visual data inspection, use a false-color map to enhance the visibility of small changes in fluorescence intensity. Plot fluorescent data as ΔF/F, with F being the average baseline value of fluorescence. A detailed description of calcium data analysis can be found in the literature.
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|EMCCD camera||Andor||iXon 897||now sold as iXon Ultra 897|
|Z-stage for the microscope||Prior||Nano Scan Z|
|X-Y stage for the microscope||Prior||Proscan III|
|Red light filter||Chroma||ET660/50m|
|35 mm Petri dish||Falcon||Falcon Disposable Petri Dishes, Sterile, Corning|