*2.1. Probe Design and Synthesis*

The synthetic scheme of the probe 3HF18 (**4**) is illustrated in Figure 1.

**Figure 1.** Synthetic scheme of the 3HF18 probe (**4**). *Reagents and Conditions*: (i) K2CO<sup>3</sup> , DMF dry, 80 ◦C, 4 h; (ii) KI, K2CO<sup>3</sup> , CH3CN dry, reflux, 48 h; (iii) (a) NaOMe, DMF dry, RT, 24 h; (b) EtOH, NaOMe, H2O<sup>2</sup> , reflux, 30 min; (iv) K2CO<sup>3</sup> , CH3CN dry, reflux 48 h.

Compound **1** was prepared according to a reported method [16]. The introduction of the C<sup>18</sup> hydrocarbon chain was obtained by reaction with N-methyloctadecylamine in the presence of a base (step ii). Compound **2** was condensed with N,N-diethylaminobenzaldehyde under basic conditions, and then oxidized in the presence of hydrogen peroxide (step iii), so affording the fluorescent 3-hydroxyflavone core by oxidative cyclization (Algar–Flynn–Oyamada modified reaction) [22]. The quaternization of the amine by reaction with 1,3-propanesultone gave the fluorescent probe product 3HF18 (**4**) with a zwitterionic group. The products of all steps were purified by column chromatography or preparative TLC and fully characterized. NMR and mass spectrometry have confirmed the structure of the final product.
