Electronic Nature of Neutral and Charged Two-Photon Absorbing Squaraines for Fluorescence Bioimaging Application

The electronic properties of neutral 2,4-bis­(4-bis­(2-hydroxyethyl) amino-2-hydroxy-6-(2-(2-(2-methoxyethoxy)­ethoxy)­ethoxy)­phenyl)­squaraine (1) and charged 2-((3-octadecylbenzothiazol-2­(3H)-ylidene)­methyl)-3-oxo-4-((3-(4-(pyridinium-1-yl)­butyl)­benzo-thiazol-3-ium-2-yl)­methylene)­cyclobut-1-enolate iodide (2) squaraine derivatives were analyzed based on comprehensive linear photophysical, photochemical, nonlinear optical studies (including two-photon absorption (2PA) and femtosecond transient absorption spectroscopy measurements), and quantum chemical calculations. The steady-state absorption, fluorescence, and excitation anisotropy spectra of these new squaraines revealed the values and mutual orientations of the main transition dipoles of 1 and 2 in solvents of different polarity, while their role in specific nonlinear optical properties was shown. The degenerate 2PA spectra of 1 and 2 exhibited similar shapes, with maximum cross sections of ∼300–400 GM, which were determined by the open aperture Z-scan method over a broad spectral range. The nature of the time-resolved excited-state absorption spectra of 1 and 2 was analyzed using a femtosecond transient absorption pump–probe technique and the characteristic relaxation times of 4–5 ps were revealed. Quantum chemical analyses of the electronic properties of 1 and 2 were performed using the ZINDO/S//DFT theory level, affording good agreement with experimental data. To demonstrate the potential of squaraines 1 and 2 as fluorescent probes for bioimaging, laser scanning fluorescence microscopy images of HeLa cells incubated with new squaraines were obtained.