The host-guest GMG2 chemodosimeter for visualizing and quantifying of EP in vitro and in vivo.

a The working principle of the developed and optimized CMG2 sensor for specific and rapid determination of EP. b Fluorescence imaging and real-time quantification of EP in cell membrane, brain tissue, and zebrafish. c Schematic diagram of fiber optic construction of multiple brain regions in mice.

Design and synthesis of host–guest supramolecular fluorescent chemodosimeters for selective and rapid response toward EP.

a Schematics of guest recognition molecules G1–G3 with varies alkyl chain lengths, host molecules CB[n]s with different cavity sizes, and neurotransmitters to be tested. b The self-assembly of the developed and optimized CMG2 supramolecular fluorescent sensor for EP. c Fluorescence titration spectra of MB (10 μm) toward addition of CB¹⁰-G2 (concentration ratio, 1:1) with different concentration (0−15 μm) in PBS buffer (10 mm, pH = 7.4) containing 0.05% DMSO; Illustration: CB¹⁰-G2 with MB assembled molar ratio. Data are presented as mean ± S.D. Error bars: S.D., n  =  5 independent experiments. d Interference of G1, G2, G3, CM8G2 and CMG2 on NE and DA (Set the response value of CMG2 for EP to 1.0). Data are presented as mean ± S.D. Error bars: S.D., n  =  5 independent experiments. e Kinetic fluorescence responses of the G1–G3 (each 10 μm) and the assembled host-guest supramolecular fluorescent sensor toward addition of 5 μm EP in PBS (10 mm, pH 7.4). f Reaction rate constants (k_obs) were measured for the G1–G3 and the assembled host-guest supramolecular fluorescent sensor in response to EP. The pseudo-first-order rate constants (k_obs) obtained from the slopes of plot of ln[(F_max–F_t)/F_max] vs time. F_max is the maximum fluorescence intensity during the measurement time, F_t is the fluorescence intensity at the corresponding time points (F_max, F_t: The fluorescent emission wavelength is 570 nm). g The molecular structure of CMG2 after DFT optimization and the quantification of the distance between two recognition sites of G2. The above-mentioned source data are provided as a Source Data file.

Fluorescence titration of CMG2 toward EP and mechanism evaluation.

a The working principle of the developed and optimized CMG2 for specific and rapid determination of EP. b Fluorescence spectra of 10.0 μm CMG2 with the addition of EP at different concentrations (0– 15 μm) in cell lysis buffer (10 mm, pH = 7.4) containing 0.05% DMSO excited at 480 nm. c Relative fluorescence intensity of the CMG2 versus EP concentration (0–15 μm). Data are presented as mean ± S.D. Error bars: S.D., n  =  5 independent experiments. d Maldi-TOF mass spectrometry of CMG2 + EP. e¹HNMR spectra of G2, MB and CMG2 ([G2] = [MB] = [CB¹⁰] = [CMG2] = 5 mm). f¹HNMR spectra of CMG2 and CMG2 assembled with EP ([CMG2] = [EP] = 5 mm, in DMSO-d₆: D₂O = 1: 1 at 298 K). g Transient absorption spectroscopy for CMG2 (left), and CMG2 + EP (right). h Principal spectral components from the global analysis of time-resolved absorption spectra from 480 nm excitation for CMG2 (left), and CMG2 + EP (right). The above-mentioned source data are provided as a Source Data file.

Fluorescence imaging and real-time quantification of EP in neurons.

a Fluorescence imaging and real-time quantification of EP in cell membrane. b Confocal fluorescence images of neurons costained with CMG2 and a commercial membrane probe (Dio). Three independent experiments were repeated and similar results were obtained. Scale bar: 15 μm. c Representative images showing the fluorescence images of CMG2 at cell membrane and their response to 100 μm EP. Scale bar: 15 μm. d Representative traces of CMG2 in response to 100 μm EP. e Dynamic response summary of CMG2 in response to 100 μm EP (n = 15 cells). f Schematic diagram of electrically stimulated neurons. g Confocal fluorescence images of electrically stimulated neurons. Scale bar: 10 μm. h Time-lapse confocal fluorescence images of CMG2-incubated neurons after electrical stimulation. Scale bar: 15 μm. i Representative traces of CMG2 after electrical stimulation (n = 15 cells). j Dynamic response summary of CMG2 after electrical stimulation. The above-mentioned data are all presented as mean ± S.D. Error bars: S.D., gray dots represent individual data points. Statistical significance is calculated with a two-tailed unpaired t-test and P values are indicated (***p <  0.001). Source data are provided as a Source Data file.

Fluorescence imaging and real-time quantification of EP in brain tissues and zebrafish.

a Fluorescence imaging and real-time quantification of EP in brain tissue and zebrafish. b Fluorescence images of brain tissue slices from S1BF, CA1, LD and Cpu regions incubated with CMG2 after electrical stimulation. Scale bar: 75 μm. c Representative traces of CMG2 in S1BF, CA1, LD and Cpu after electrical stimulation. d Dynamic response summary of CMG2 after electrical stimulation in S1BF, CA1, LD and Cpu (n  =  15 independent experiments). e Fluorescence images of zebrafish incubated with CMG2 after electrical stimulation. Scale bar: 130 μm and 10 μm. f Representative traces of CMG2 in zebrafish after electrical stimulation. g Dynamic response summary of CMG2 after electrical stimulation in zebrafish (n  =  15 independent experiments) The above-mentioned data are all presented as mean ± S.D. Error bars: S.D., gray dots represent individual data points. Statistical significance is calculated with a two-tailed unpaired t-test and P values are indicated (^nsp > 0.05, *p  <  0.05, **p  <  0.01, and ***p  <  0.001). Source data are provided as a Source Data file.

Real-time monitoring and quantifying of EP in 26 brain regions.

a Schematic illustration depicting probe injection and fluorescence imaging in vivo. Example image showing CMG2 in FrA in a coronal brain slice; Scale bar: 500 μm. b Schematic cartoon illustrating tail suspension experiments. In vivo imaging of normal and afraid-model mouse brains stained with CMG2. c An overhead and lateral view of the brain surface implanted with A multi-fiber microarray, including anterior (A) middle (M) and posterior (P). d Distribution of brain slices in different areas and three-dimensional remapping of Allen brain Atlas. e Fluorescence signal F570/F695 ratio changes in seven representative brain regions with time after tail suspension stimulation. f Quantized maps of fluorescence signals in 26 different brain regions (n  =  15 independent experiments). The above-mentioned data are all presented as mean ± S.D. Error bars: S.D., white dots represent individual data points. Statistical significance is calculated with a two-tailed unpaired t-test and P values are indicated (^nsp > 0.05, *p  <  0.05, **p  <  0.01, and ***p <  0.001). Source data are provided as a Source Data file.