Two-photon fluorescence imaging and specifically biosensing of norepinephrine on a 100-ms timescale

Abstract

Norepinephrine (NE) is a key neurotransmitter in the central nervous system of organisms; however, specifically tracking the transient NE dynamics with high spatiotemporal resolution in living systems remains a great challenge. Herein, we develop a small molecular fluorescent probe that can precisely anchor on neuronal cytomembranes and specifically respond to NE on a 100-ms timescale. A unique dual acceleration mechanism of molecular-folding and water-bridging is disclosed, which boosts the reaction kinetics by ˃10⁵ and ˃10³ times, respectively. Benefiting from its excellent spatiotemporal resolution, the probe is applied to monitor NE dynamics at the single-neuron level, thereby, successfully snapshotting the fast fluctuation of NE levels at neuronal cytomembranes within 2 s. Moreover, two-photon fluorescence imaging of acute brain tissue slices reveals a close correlation between downregulated NE levels and Alzheimer's disease pathology as well as antioxidant therapy.

Fig. 1. Schematic of the sensing mechanism and the chemical structures of relevant compounds.

a Schematic illustration of the ultrafast sensing mechanism toward norepinephrine by the two-photon fluorescence probe BPS3, boosted by the dual acceleration of conformational-folding and water-bridging. b Molecular structures of the compounds referred in this work.

Fig. 2. Sensing performances and mechanism.

a Fluorescence spectrum of BPS3 (5 μM) upon addition of NE (0–300 nM), two-photon excited at 720 nm. b Plot and linear fitting of the fluorescence variation rates (ΔF/F₀) at 480 nm versus the concentration of NE (0–300 nM). Data are presented as mean ± S.D. Error bars: S.D., n = 3 independent experiments. c Selectivity and (d) competition tests of the probe BPS3 toward NE against other neuron transmitters and amino acids (norepinephrine (NE), epinephrine (EP), dopamine (DA), serotonin (5-HT), γ-aminobutyric acid (GABA), acetylcholine (Ach), cysteine (Cys), homocysteine (Hcy), glutathione (GSH), lysine (Lys), threonine (Thr), serine (Ser)), measured after 10 min of mixing. The concentrations of NE, EP, and DA were 300 nM, and other species were all 1 mM. Data are presented as mean ± S.D. Error bars: S.D., n = 3 independent experiments. e HPLC spectra of (i) NE, (ii) BPS3, (iii) BPS3 reacted with NE, and (iv) BPS3-OH. f HR-MS spectrum of the product of BPS3 reacted with NE in aqueous solution. g The sequential nucleophilic substitution-cyclization mechanism of the reaction between probe BPS3 and NE.

Fig. 3. Conformation-dependent reaction kinetics for NE sensing.

a Normalized fluorescence response dynamics (recorded at 480 nm, excited at 720 nm) of 5 μM aqueous solution of BPS2, BPS3, BPS4, and BPS3 + CB[7] (1 equiv.) with addition of 100 nM NE. For the curve BPS3 + CB[7], an additional 1 equiv. of AdOH was added at the time of 1200 s after the addition of NE. Inset: Enlarged view of the partial curve for the response of BPS3 and BPS4 toward NE. b Partial 2D NOESY NMR spectrum of BPS3 (1 mM in D₂O), at 298 K. c IGMH isosurface of BPS3 in folded conformation. d The calculated Hirshfeld Charge of the carbonothioate carbon of BPS3 in folded and stretched conformations, respectively. e Schematic of molecular conformational regulations by covalent or supramolecular approaches.

Fig. 4. Mechanism studies of the water-bridging-boosted reaction kinetics for NE detection.

a Normalized fluorescence response dynamics (recorded at 480 nm, excited at 720 nm) of 5 μM BPS3 solution in different solvents with addition of 100 nM NE. b The pathway and possible transition states of the reactions between the stretched probe BPS3 and NE, with or without the involvement of H₂O. c Calculated relative energies of the compounds and possible transition states during the reactions between the probe BPS3 and NE in different pathways.

Fig. 5. Bioimaging performances.

a Confocal fluorescence images of neurons co-stained with BPS3 and a commercial membrane probe (DiI). Three independent experiments were repeated and similar results were obtained. b Time-lapse confocal fluorescence images of BPS3-incubated neurons stimulated by PBS buffer or high concentration of potassium, respectively. c Time-course of the fluorescence variation rate (ΔF/F₀) of neurons stimulated by PBS (Phosphate-Buffered Saline) or high concentration of potassium, respectively (interval of 1 s). Data are presented as mean ± S.D. Error bars: S.D., n = 5 cells. d Illustration of the possible procedure of NE release upon stimulation with high concentration of potassium. e Three-dimensional two-photon confocal fluorescence images of the hippocampus region in mouse brain labeled with the BPS3 probe, two-photon excited at 720 nm. f Two-photon fluorescence images of the BPS3-incubated tissue slices from cornu ammonis of hippocampus (CA1), primary somatosensory cortex (S1BF), laterodorsal thalamic nucleus (LD), and caudate putamen (CPu) regions of normal and Alzheimer’s disease (AD) mouse brains, as well as the N-acetylcysteine (NAC)-treated AD mouse brain. Orange arrows point to the representative single cells. g Histogram of the relative fluorescence variation rates corresponding to panel (f). Statistical differences were analyzed by Student’s one-sided t-test. Data are presented as mean ± S.D. Error bars: S.D., n = 25 cells. p = 0.0006, 0.0083, 0.0007, 0.0074, 0.0077, 0.0156, 0.0068, 0.0233 from left to right, respectively, *p < 0.05, **p < 0.01, and ***p < 0.001.