Calcium imaging · Lobeworks/17

Calcium imaging watches neurons fire by filming a fluorescent protein that lights up when calcium enters the cell, and it is the standard way to record thousands of identified neurons at once in a living animal.

Calcium imaging. Calcium imaging watches neurons fire by filming a fluorescent protein that lights up when calcium enters the cell, and it is the standard way to record thousands of identified neurons at once in a living animal.

Every action potential lets calcium into the cell through voltage-gated channels. Neurons engineered to express an indicator such as GCaMP, a fluorescent protein fused to a calcium-binding domain, glow more brightly for a short time after each burst of spikes. A microscope focused through a window in the skull then films a field of hundreds to thousands of cells, and software turns each cell's brightness over time into an estimate of its activity. Two-photon microscopes, which excite fluorescence only at the focal point with infrared light, see to about a millimetre deep in mouse cortex; three-photon microscopes have reached below the cortex into the hippocampus.

It sees who, and less exactly when. The calcium signal rises and decays over tens to hundreds of milliseconds, so single spikes and precise timing are blurred, while the identity and position of every cell are known.

It depends on gene delivery and optical access, which is why it is almost entirely an animal method: a human use would need a gene introduced into brain cells and a clear path for light.

Integrators keep a record. Variants such as CaMPARI change colour permanently from green to red when calcium is high while a violet light is on, freezing a snapshot of which cells were active during a chosen moment.

Light limits depth. Scattering in tissue sets the millimetre ceiling, so deep structures require lenses or fibres pushed into the brain.

Questions: Can light-sheet microscopy record a whole living brain at work? Only in animals small and transparent enough to need no clearing. In 2013 a light-sheet microscope recorded the brain of a larval zebrafish carrying a calcium indicator, capturing more than 80 % of its neurons at single-cell resolution, the whole volume about 0.8 times a second. At that rate it follows the slow calcium signal, too coarse to separate single spikes. A mouse or human brain scatters light too much, so in those the method works on cleared, fixed tissue, which keeps the cells and loses their activity. What did MICrONS add to a wiring diagram? It recorded what the neurons did before mapping how they were wired. While a mouse watched films, calcium imaging followed the responses of about 75,000 neurons in a cubic millimetre of visual cortex; the same cube was then cut and imaged by electron microscopy, and 523 million synapses were traced among its 200,000 cells. Because each reconstructed neuron could be matched to its recorded responses, the map could be asked who connects to whom by function. One answer was a like-to-like rule: excitatory neurons that respond to similar features are more likely to be connected, within and across layers and areas. Why is calcium imaging done almost only in animals? It needs two things a person would have to consent to as a medical intervention: a gene for the indicator introduced into brain cells, usually by a viral vector, and an optical path to the cortex, a window in the skull or a lens pushed into the tissue. Gene delivery into the brain is permanent in practice and is offered in people only as a treatment for disease, so the risk is hard to justify for a recording that brings the participant no benefit. The method's insights reach people through animal studies, while human recordings rely on electrodes placed for clinical reasons. How do optogenetics and calcium imaging divide the work of writing to neurons and reading them? Both rely on genes delivered into chosen neurons and on light: calcium imaging makes active cells glow so a microscope can read which ones fired, and optogenetics makes cells fire or fall silent when light reaches them. Combined in one experiment, with an indicator and an opsin excited by different colours, they close the loop, so a researcher can watch an ensemble form during a task and later switch the same cells on to test what they do. Both are limited to the depth that light can reach and to animals into which genes can be introduced.