Virtual laboratory / Cells and circuits
Two-photon calcium imaging
Imaging of a population of neurons in visual cortex and of the response of each neuron to oriented gratings.
Guided experiment: Two-photon calcium imaging of visual cortex.
Two-photon calcium imaging
Calcium imaging records the activity of many neurons at once. When a neuron fires, calcium enters it, and genetically encoded indicators such as GCaMP become brighter when they bind calcium. A two-photon microscope can image hundreds of neurons in the living brain. Indicators of the GCaMP6 family detect single action potentials in the mouse visual cortex (Chen et al., 2013).
Design the experiment
Questions to investigate
- Compare the fast and the slow indicator at a low frame rate. Which one detects more single spikes, and why?
- Reduce the number of trials per orientation. How reliable are the tuning curves and the orientation selectivity values?
- Lower the laser power. What happens to the signal-to-noise ratio and to the number of responsive cells?
- Increase the frame rate. What does it gain, and what would it cost in a real microscope?
In a real laboratory
The indicator is delivered by viral injection or a transgenic line, and a cranial window is implanted over the cortex. The head-fixed animal is accustomed to the setup before imaging. Images are corrected for brain motion, cells are detected by software and a fluorescence trace is extracted for each cell. Laser power is limited to avoid damaging the tissue. All procedures require approval by an Institutional Animal Ethics Committee (in India, under the CCSEA and the Prevention of Cruelty to Animals Act 1960; elsewhere, the equivalent national body).
How the simulation works
Each neuron fires as a Poisson process whose rate depends on the orientation of the grating through a tuning curve. Fluorescence is the spike train convolved with the rise and decay of the indicator and averaged over each frame. Noise grows as laser power falls and as frame rate rises. The kinetics of the two indicators are approximate. Indicator saturation, neuropil contamination and brain motion are not simulated.
- Chen, T.-W., Wardill, T. J., Sun, Y., et al. (2013). Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature, 499(7458), 295–300. https://doi.org/10.1038/nature12354
About the simulations
Each simulator is a simplified version of a published model of cells, signals, instruments or behaviour, and runs in the web browser. The simulations show how the techniques work and how design choices change the results; they do not replace training in a laboratory.
All data are simulated and generated afresh on each run, so results vary between runs, as in real experiments. Nothing is stored or sent: designs and results are lost when the page is closed, unless the protocol is copied or the data file is downloaded.
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