Norcyanine dyes with benzo[c,d]indolium moiety: Spectral sensitivity with pH change for fluorescence pH imaging in living cells

Abstract

Fluorescence pH imaging in living cells is a rapidly expanding research direction, however, it relies on the development of pH-sensitive fluorescent imaging agents. Here four norcyanine dyes with benzo[c,d]indolium moiety, exhibiting high spectral sensitivity with pH changes, were synthesized for fluorescence pH imaging in living cells, and characterized by 1H NMR, 13C NMR, IR, UV–Vis and HRMS. The investigation of their spectral properties in methanol and water showed that the absorption and emission maxima were in the region 488–618nm and 583–651nm, respectively, and four dyes exhibited high photostability. The pH spectral titrations showed that selective dye D1 had pH-dependent absorption spectral changes within the pH range of 2.4 to 9.4, and high fluorescent spectral sensitivity at pH5.0–8.0, with a pKa of 5.0. A cell association study indicated that dye D1 exhibited no or mild cytotoxicity at the application dose and duration, and could be accumulated in cells and mainly distributed in the cytoplasm, giving red fluorescence imaging. In particular, dye D1 could achieve pH-dependent fluorescence imaging in living cells with the increase of pH from 3.0 to 8.0, at excitation wavelength of 543nm and receiving wavelength of 655–755nm, which was valuable for studying the weak acidic, neutral and weak alkaline biological tissue compartments.