Comparing melatonin and 5-HT permeation using their fluorescence. (A) Schematic showing a monolayer of tsA201 cells on a coverslip locally perfused by a pipette containing control saline or saline supplemented by 10 mM melatonin (Mel) or 5-HT. The epi-illumination and fluorescence recordings use a 40× objective below the coverslip in a circular field of view of 39 µm that is several cell diameters wide. (B, top) Three idealized fluorescence recordings for a step pulse of perfusion with three conditions: no cells, with cells and an impermeant indoleamine, and with cells and a permeant indoleamine. (bottom) Idealized fluorescence difference traces ΔF of a record with cells minus a record without cells (see section “Influx and efflux detected by in-cell fluorescence”). (C) Five superimposed fluorescence traces from five chips without cells (gray) and from five chips with cells (purple) as melatonin and 5-HT are locally perfused for 20 s. (D) Mean difference fluorescence ΔF for the experiment in C; mean data with “No cells” are subtracted from mean data “With cells.”