Automatic DRG-neuron identification and quantification of phosphospecific fluorescence intensities. A) Example image of a microscope view field of the DRG neuron culture immunofluorescently labelled for the neuronal marker PGP9.5. Neurons show high degree of heterogeneity in respect to size, fluorescence intensity and extend of cell-cell contacts. B) Examples of objects selected as neurons (upper two rows) and rejected objects (lower two rows). Rejected objects include non-cellular debris, glia cells and clustered neurons. C) "Virtual wells" were created by randomly selecting 250, 1000, 5000, 10,000 or 20,000 cells per virtual well out of 49,503 measured cells. The normalized mean well intensity of 10,000 such virtual wells with the respective standard deviation is depicted indicating the sensitivity limit of studies based on the various average cell numbers per well (250 cells per well resulted in a well Mean Intensity of 1.0005 ± 0.1133, 1000 cells in 1.0003 ± 0.0564, 5000 cells in 0.9999 ± 0.0240, 10,000 cells in 0.9997 ± 0.0159, and 20,000 cells result 0.9998 ± 0.0098). D) IB4 lectin derived fluorescence intensities showed a continuous distribution of intensities. Coincubation with uncoupled lectin to block specific binding resulted in an intensity histogram (n = 5002 cells, black line) matching the first peak of the unblocked IB4-intensity histogram (n = 23266 cells, red line) identifying these cells as unspecificly binding and thus as IB4(-)-cells. E) Diameter histogram of 49,503 neurons (yellow bars). 40,706 neurons were labeled for IB4. IB4(+)-neurons (red bars) show a higher average diameter than IB4(-)-neurons (blue bars). F) Comparison of the normalized Mean Intensity of phosphorylated and total Erk1/2 in IB4(+)- and IB4(-)-neurons (n = 1421 IB4(+)-neurons and n = 963 IB4(-)-neurons). IB4(+)-neurons show a higher amount of pErk1/2 but also a higher expression rate of Erk1/2 in comparison to IB4(-)-neurons (p < 0.001).