Fp 8600 spectrophotometer

The UV-vis absorption and photoluminescence (PL) spectra were measured with a V-670 spectrometer (Jasco) and a FP-8600 spectrophotometer (Jasco), respectively, using degassed spectral grade solvents. The absolute PL quantum yields (Φ_PL) were determined using an ILF-835 integrating sphere system (Jasco). The transient PL decay measurements were carried out using a C11367 Quantaurus-tau fluorescence lifetime spectrometer (Hamamatsu Photonics; λ = 340 nm, pulse width = 100 ps, and repetition rate = 20 Hz) under N₂, and a C9300 streak camera (Hamamatsu Photonics) with an N₂ gas laser (λ = 337 nm, pulse width = 500 ps, and repetition rate = 20 Hz) under vacuum (<4 × 10^–1 Pa). The HOMO energies of materials in neat films were determined using an AC-2 ultraviolet photoelectron spectrometer (Riken-Keiki). The LUMO energies were estimated by subtracting the optical energy gaps (E_g) from the measured HOMO energies; the E_g values were determined from the onset positions of the PL spectra of the thin films.

The fluorescence of ATP indicators in vitro was tested in buffer C, (50 mM HEPES-KOH [pH 7.7], 200 mM KCl, 1 mM MgCl₂, and 0.05% Triton X-100), at 25°C unless otherwise stated. Buffer C is designed to be closer to the reported values of pH, K⁺, and free Mg²⁺ found in E. coli cytosol20 (link)22 (link)23 (link) compared to buffer B (50 mM MOPS-KOH [pH 7.3], 50 mM KCl, 0.5 mM MgCl₂, and 0.05% Triton X-100) used in a previous report13 (link). A 200 mM stock of ATP-Mg²⁺ or ADP-Mg²⁺ (equimolar mixture of MgCl₂ and ATP or ADP) was added to the ATP indicator solution to change the ATP-Mg²⁺ concentration, and the fluorescence was measured with a FP8600 spectrophotometer (JASCO), unless otherwise specified. For the spectra measurements, the measurements were started once fluorescence stabilized. When the excitation spectra of single FP-type indicators were measured, the solution was excited at 380–505 nm, and emission was recorded at 513 nm. When the emission spectra of FRET-type indicators were measured, the solution was excited at 435 nm, and emission was recorded at 460–600 nm. For the k_on and k_off measurements, single FP-type indicators were excited at 494 nm, and emission at 513 nm was recorded. ATP-Mg²⁺ was added to the solutions during measurement.