Both dyes were internalized during constant illumination.

<p>A. Di-4-ANEPPS was bath-applied for 24 hours and then washed out. Photos were taken every 10 minutes for 1 hour. The plot shows the frequency density of pixel brightness of the somata area. While in general the frequency of smaller grey values increased over time (due to bleaching), there were fewer dark pixels in the 20 minutes picture (blue) than in the initial picture (orange; bottom inset) as indicated by the shift of the frequency density to the right. No fluorescence: grey value of 0; highest fluorescence: grey value of 255. Binwidth: 1. For display purposes only bins 1 to 250 are shown. Top inset: Photos showing neuron staining immediately after dye wash-out and after 20 minutes of illumination. Immediately after washout cell bodies are dark while their surrounding is brightly stained. After 20 minutes of illumination the cell cytoplasm shows fluorescence and the cell nucleus is visible (orange arrow). B. Photos were analyzed and internalization was noted if at least the nucleus of one cell was visible. Group 1: Preparations that had been used for 180 minutes staining experiments (including illumination) (RH795: N = 6, Di-4-ANEPPS: N = 4). Photos were inspected for signs of internalization after the 180 minutes of staining experiments, after 24 hours staining and after bleaching. Group 2: Preparations were stained for 24 hours. Internalization was measured after the staining process and then again after 1 hour of bleaching (RH795: N = 4, Di-4-ANEPPS: N = 7). Group 1 Di-4-ANEPPS values after bleaching and group 2 RH795 values after bleaching were included for the sake of completeness (all preparations had shown internalization in the previous experimental step already).