An investigation of squaraines as a new class of fluorophores with long-wavelength excitation and emission.

We present an overview of our investigation of the spectral properties and photostabilities of squaraine derivatives. The objective was to identify long-wavelength probes with reasonable quantum yields, reasonably long lifetimes, and good photostabilities for use in fluorescence-based assays and/or imaging. Both symmetrical and unsymmetrical squaraines were studied. Based on this investigation the most suitable probes for use in a biological application were found to be the symmetrical indolenine derivatives of the squaraines, which display the highest photostability. Importantly, their quantum yields and lifetimes increase significantly upon covalent and noncovalent binding to proteins (bovine serum albumin, antibodies), demonstrating the usefulness of the squaraines as long wavelength probes. The squaraine absorbance maxima between 630 and 650 nm allows the use of the new commercially available 635- and 650-nm diode lasers.