First-generation ABS fluorescence probes for FA based on the aza-Cope rearrangement reaction. (a) Reaction scheme for FA detection with the silicon rhodamine-based dye FAP-1. (b) Fluorescence response of 10 μM FAP-1 to 100 μM FA over 120 min (red trace). (c) Confocal microscopy images of MCF7 cells stained with 10 μM FAP-1 at 37 °C for 60 min: (left) vehicle (Ctrl) with no additive; (right) 20 μM tranylcypromine (TCP), an inhibitor of the demethylase enzyme lysine-specific demethylase 1 (LSD1). The bar graph presents fluorescence intensities (mean ± SEM, n = 6; *, P ≤ 0.005). (d) FA-dependent fluorescence responses of folate derivatives analyzed by FAP-1, showing that THF, DHF, and 5,10-me-THF derivatives are high folate producers. The bar graph presents fluorescence intensities (mean ± SEM, n = 3). Adapted from refs 14 and 36. Copyright 2015 American Chemical Society.