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Optical methods

Calcium Imaging
The firing of an action potential is accompanied by an influx of calcium ions into the cell. This increasing intracellular calcium ion concentration can be detected by fluorescent dyes (e.g. Fluo-4) or genetically encoded fluorescent sensor proteins (e.g. GCaMP), that have been introduced into the cell beforehand, allowing video recording of neural activity.

One way is to load the cell culture with a dye like Fluo-4-AM (Invitrogen). Upon cellular activity the Ca binds to Fluo-4 leading to a conformational change and the increase in fluorescence. The other method is to introduce a genetically encoded calcium sensor like GCaMP into the cell. GCaMP consists of calcium binding domains and a circularly permuted GFP. Similar to the Fluo-4 the protein changes its conformation upon Ca2+ binding leading to an increase in fluorescence. While using Fluo-4 is a fast and comparable easy method, GCaMP requires a well-planned experimental setup. However using a genetically encoded sensor provides more opportunities in selectively image special neuronal types.

To process the calcium imaging movies we established an analysis software written in MATLAB for automatic ROI detection and signal propagation. With this software we are able to extract the important data like propagation direction and network synchrony.


Sketch of the script used to analyze sequences from calcium imaging. (A) An additional binning is applied to obtain a resolution closer to cell dimensions. (B) Raw data are extracted from sequences and normalized prior to filter application. Increases in fluorescence intensity related to action potentials are identified by the first derivative of the filtered data and translated in an event trace. (C) A summation of the events over time results in a histogram enabling identification of substrate spanning excitation. (D) Color-coded delay plot of the substrate spanning excitation marked in red in the histogram in C. The insert in the lower left corner indicates the first event in the substrate spanning excitation. Scale bar: 50 μm.