Figure 2.

Schematic drawing of FP-based sensors for application in acidic organelles. (A) mKeima emits single emission at 620 nm, with dual excitation peaks at 438 and 550 nm, corresponding to protonated and deprotonated state of the chromophore. Under starvation-induced autophagy LC3-tagged mKeima is incorporated into autophagosomes, then recruited into lysosomes. This acidification induces color change of mKeima. LC3: microtubule-associated protein light chain 3; (B) D1-SG is a FRET-based and low-affinity Ca²⁺ sensor applicable at pH levels between 5.5 and 7.4 (K_d ~ 120–190 μM). Citrine can serve as an intentiometric pH sensor with direct excitation of ~500 nm, due to its inherent pH sensitivity (pK_a = 5.8). tPA: tissue plasminogen activator (secretory granule localization); (C) eZinCh is FRET-based, with low-affinity Zn²⁺ sensors composed of Cerulean and Citrine with Cys and His substitutions directed outside the β-barrel. Simultaneous use with pH sensor is necessary for pH calibration of the sensor. VAMP2: vesicle-associated membrane protein 2 (secretory granule localization); (D) ClopHensor records both Cl⁻ and pH in a ratiometric manner by applying three lasers for excitation (458, 488 and 543 nm). E²GFP changes the ratio of two excitation peaks and fluorescence intensity (protonated and deprotonated forms) depending on Cl⁻ concentration and pH. A pH isosbestic point exists at ~458 nm so that E²GFP emission under 458 nm excitation is pH-insensitive.