Overview of image analysis by NA³.

<p><b>a</b>, Motoneuron loaded with a fluorescent calcium indicator. Average intensity projection of 1000 images to show the neuronal morphology. The neuron shifts spontaneously from a low activity state to a high activity state. Rainbow pseudocolor shows low intensity values in blue and high intensity values in red. <b>b</b>, Workflow for the analysis of calcium imaging raw data. After threshold determination according to a rule, the user defines the window size for x,y-grid, and chooses a signal-to-noise ratio value to tune the stringency of the tool. Two computations are started: (1) the signal intensity calculation, (2) the wavelet transform. A result pdf is automatically created. <b>c</b>, The signal-to-noise ratio (SNR) defines the stringency of the computation. The signal average threshold (SAT) can be used to set a signal threshold. The SAT can be close to the black level without having a strong impact on the computation result. <b>d</b>, The documentation pdf defines activity events, marks them, and counts them. All traces showing one or more activity events are given and used to count the total activity. An overview image is created that shows the grid over the first image of the movie, red circles to visualize the activity state of a grid window, and the total activity value, which is a computed value to describe the overall activity state of the neurons. The number of counted activity events per grid window events is shown in a text fil in the results folder. <b>e</b>, Overview of the NA³ workflow. The tool combines functions in ImageJ with “R”. Video processing and signal extraction occurs in ImageJ, before the signals are automatically transferred to “R”. In “R”, the event computing takes place. The result is created in “R” and exported as a pdf-file. The results are also transferred back to ImageJ to allow an interactive access to the data for image processing, ROI selection, or data evaluation.