Photophysics of a Water−Soluble Rylene Dye:  Comparison with Other Fluorescent Molecules for Biological Applications

Abstract

The photophysical properties of a new water-soluble fluorescent dye based on the perylene diimide chromophore are investigated at the ensemble and single molecule level. Water solubility was obtained by attaching hydrophilic substituents in the bay region. The fluorophore has an absorption maximum at 567 nm and a high quantum yield of fluorescence (0.6). In addition, the two-photon absorption cross section has higher or comparable values with the ones reported in the literature for other water-soluble dyes used in biological studies. The most important finding is its improved photostability in single molecule experiments when compared to other water-soluble dyes. This is demonstrated by the increased observation time of immobilized single molecules, which is almost 10 times higher than for commonly studied fluorophores. A possible reason is the lower intersystem crossing rate constant of perylene diimide in comparison, for example, to rhodamine derivatives. A similar conclusion can be drawn for this chromophore in an aqueous solution, because single molecule measurements using fluorescence correlation spectroscopy demonstrate a very low contribution of the triplet state in the autocorrelation function. The possibility of imaging the new molecule in living cells is demonstrated by one-photon confocal microscopy and by fluorescence lifetime microscopy with two-photon excitation.

Single-Molecule Spectroscopy and Imaging of Biomolecules in Living Cells

“…Commonly used classes of organic fluorophores include the following: Cy3 and other carbocyanine dyes, rhodamines,44 fluoresceins,43 DCDHFs,34 terrylene and rylenes,45 etc.…”

Computational study of the one- and two-photon absorption properties of macrocyclic thiophene derivatives

“…Then, a program (FTRNLO) compiled by our group is used to calculate the second hyperpolarizability g and the TPA cross-section according to Eq. (1)e (3). Before that, the ground state OPA spectra, transition dipole moment and the corresponding transition energy are predicted for the TPA properties of the studied molecules.…”

Voltage sensitive dyes used for FLIM studied by two-photon-excitation

“…Annine6-plus [6] was a kind gift from P. Fromherz (Max-Planck Institute of Biochemistry, Munich, Germany); di-4-anepps=4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium, hydroxide, inner salt (Invitrogen, Karlsruhe, Germany); di-8-anepps=4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-, inner salt (Invitrogen, Karlsruhe, Germany); *t.gensch@fz-juelich.de; phone +49-2461-618068; fax +49-2461-618068; http://www.fz-juelich.de/inb/inb-1/, http://www.fzjuelich.de/inb/inb-1/gensch_t PMI-COOH=n-(2,6-diisopropylphenyl)-9-(4-carboxyphenyl]perylene-3,4-dicarboxyimide, inner salt [7] and PDI=tetrachloroperylenetetracarboxdiimide, inner salt (see for a similar compound Margineanu et al [8] Fluorescence spectra were measured with a fluorescence spectrophotometer (QM-4, PTI, Birmingham, NJ, USA) operating in right-angle configuration and equippedwith a Xenon-arc lamp as excitation source, two monochromators in the excitation and emission pathway with continuously adjustable slits, and a photomultiplier with sensitivity enhancement in the red spectral region for detection. For measurements of spectra on HEK293 and Hela cells, a cover slip with the adjacent cells was standing vertically (under 45 degree relative to the excitation light) in a 1x1x4 cm cuvette filled with extracellular solution.…”