Novel Lipophilic Fluorophores with Highly Acidity‐Dependent Two‐Photon Response

Abstract

Lipophilic fluorophores are widely implemented in nonlinear microscopy; however, few existing membrane‐specific probes combine the high brightness of two‐photon excited fluorescence (2PEF) with pH sensitivity. Herein we describe four novel two‐photon excited fluorophores, based on a coumarin 151 core structure, where lipophilicity is induced by a covalently attached phosphazene moiety. Changing the environmental acidity using trifluoromethanesulfonic (triflic) acid leads to profound changes in the linear fluorescence and 2PEF characteristics, due to chromophores’ switching between neutral‐ and protonated forms. We characterize this dependence by measuring the two‐photon absorption (2PA) spectra over the region λ2PA=550–1000 nm, observing 2PA cross sections of σ2PA=10–20 GM, with an associated 2PEF brightness of 10–13 GM, in neutral solutions of both acetonitrile and n‐octanol. Although quantum chemical modelling and NMR measurements show that, at high chromophore concentrations, protonation may be accompanied by a dimerization process, these dimers likely do not form at the lower concentrations used in optical spectroscopy.