Portera-Cailliau Laboratory - In Vivo 2-Photon Calcium Imaging

In Vivo 2-Photon Calcium Imaging

OGB Injection
Cartoon of bolus loading of calcium indicators for in vivo calcium imaging

*News Flash* Peyman's calcium imaging paper has been accepted by the Journal of Neuroscience and is now in press!

To study how neuronal activity emerges within networks of immature cortical neurons, we use 2-photon imaging of fluorescent calcium indicator dyes (like Fluo4-AM or Oregon Green BAPTA1-AM) and electrophysiology.

This technique allows us to study the spatio-temporal dynamics of activity in large ensembles of Layer 2/3 neurons. Below are two in vivo calcium imaging 'movies' acquired at 4Hz.

in vivo calcium imaging of OGB-loaded Layer 2/3 neurons in barrel cortex at postnatal day 5 (left) and 26 (right)

Correlation matrix of the activity of a subset of 172 (left; P5) and 114 (right; P26) Layer 2/3 neurons recorded simultaneously with 2-photon calcium imaging.
We use custom-written programs in MatLab to analyze the calcium imaging data. For example, we can create correlation matrix diagrams that reflect how synchronous activity is across large numbers of neurons. We also use temporal deconvolution algorithms to extrapolate firing rates of individual neurons.

After each experiment, the exact location of the imaging within barrel cortex is identified using careful reconstructions in fixed tissue. To do so, we match the blood vessel imprint on the surface of the brain to a photograph of the same blood vessels taken through the glass covered cranial window at the time of the experiment. We then use cytochrome oxidase histochemistry to reveal the barrel structure in Layer 4, and overlay the 2-photon image of the Layer 2/3 neurons over the barrels.