The Effect of Branching on the One‐ and Two‐Photon Absorption, Cell Viability, and Localization of Cationic Triarylborane Chromophores with Dipolar versus Octupolar Charge Distributions for Cellular Imaging†

Abstract Two different chromophores, namely a dipolar and an octupolar system, were prepared and their linear and nonlinear optical properties as well as their bioimaging capabilities were compared. Both contain triphenylamine as the donor and a triarylborane as the acceptor, the latter modified with cationic trimethylammonio groups to provide solubility in aqueous media. The octupolar system exhibits a much higher two‐photon brightness, and also better cell viability and enhanced selectivity for lysosomes compared with the dipolar chromophore. Furthermore, both dyes were applied in two‐photon excited fluorescence (TPEF) live‐cell imaging.

General photophysical measurements. All measurements were performed in standard quartz cuvettes (1 cm x 1 cm cross-section). UV-visible absorption spectra were recorded using an Agilent 8453 diode array UV-visible spectrophotometer. The molar extinction coefficients were calculated from three independently prepared samples in MeCN (1M, 2M) and hexane (1,2) solution.
The emission spectra were recorded using an Edinburgh Instruments FLSP920 spectrometer equipped with a double monochromator for both excitation and emission, operating in rightangle geometry mode, and all spectra were fully corrected for the spectral response of the instrument. All solutions used in photophysical measurements had a concentration lower than 5 × 10 -6 M to minimize inner filter effects during fluorescence measurements.
Fluorescence quantum yield measurements. The fluorescence quantum yields were measured using a calibrated integrating sphere (inner diameter: 150 mm) from Edinburgh Instruments combined with the FLSP920 spectrometer described above. For solution-state measurements, the longest-wavelength absorption maximum of the compound in the respective solvent was chosen as the excitation wavelength, unless stated otherwise.
Lifetime measurements. Fluorescence lifetimes were recorded using the time-correlated single-photon counting (TCSPC) method using an Edinburgh Instruments FLS980 spectrometer equipped with a high speed photomultiplier tube positioned after a single emission monochromator. Measurements were made in right-angle geometry mode, and the emission was collected through a polarizer set to the magic angle. Solutions were excited with a pulsed diode laser at a wavelength of 378 nm (for 1 and 2) and 419 nm (for 1M and 2M) at repetition rates of 10 or 20 MHz, as appropriate. The full-width-at-half-maximum (FWHM) of the pulse from the diode laser was ca. 75-90 ps with an instrument response function (IRF) of ca. 230 ps FWHM. The IRFs were measured from the scatter of an aqueous suspension of Ludox at the excitation wavelength. Decays were recorded to 10 000 counts in the peak channel with a record length of 8192 channels. The band pass of the emission monochromator and a variable neutral density filter on the excitation side were adjusted to give a signal count rate of <60 kHz. Iterative reconvolution of the IRF with one decay function and non-linear leastsquares analysis were used to analyse the data. The quality of all decay fits was judged to be satisfactory, based on the calculated values of the reduced χ 2 and Durbin-Watson parameters and visual inspection of the weighted residuals.
Two-photon absorption measurements. The two-photon absorption cross-section of the compounds was determined by the two-photon induced fluorescence technique. In detail, the fundamental laser source used is an amplified Ti: sapphire laser (Solstice, Spectra Physics) operating at 1 KHz repetition rate with 100 fs pulses at 800 nm. 70% of the of the available energy seeds a computer-controlled optical parametric amplifier (TOPAS-C, Light Conversion), which produces pulses in the range of 290 -2600 nm. Excitation of the samples was achieved using a protected silver parabolic mirror, using vertically polarized light with the excitation energy varying in the 0.2 -3 μJ range. The latter conditions were established by using a series of three thin broadband polarizers and a mechanical rotational mount.
Maintenance of identical excitation conditions for both reference and samples was achieved using a high-precision motorized rotational stage to ensure that the unknown compounds and the secondary reference standard are subjected to the same excitation conditions. Coumarin 540A in CCl4 and Styryl 9M in CHCl3 were used as reference compounds. [4] The emitted fluorescence signal was detected at 90° with respect to the excitation beam, and recorded using a compact CCD spectrometer. Two-photon excitation was verified by log-log plots of fluorescence intensities vs. excitation power at various wavelengths, all giving slopes of 2.
Transition Dipole Moment Calculation. The squares of the transition dipole moment |μ mi | 2 and |μ fm | 2 were calculated from the following equations: where h is the Planck´s constant, is the speed of light, ε 0 is the vacuum permittivity, n is the refractive index of the medium, N is Avogadro´s number and ε ge (k) the extinction coefficient at the wavenumber k; The integral is over the 0 → 1 absorption band, and |μ fm | 2 = 5h 2 n 2 c 2 32π 4 L 4 [ where L is the local field factor, v is the transition frequency of the 2PA cross-section maximum σ max (v), the bracket (v mi − v ) is the detuning frequency and can be calculated from the absorption peaks in the one-and two-photon spectra. Finally, the term G vib. is a Gaussian line shape function that is centered at the energy maximum for the two-photon allowed state in order to include the vibrational transitions. The magnitudes of the 2PA crosssections 2 were estimated using a three-level model. Thus, the product of the square of the transition dipole moments are analogous to the 2PA cross-section: ( 2 ∝ |μ ⃗ mi | 2 |μ ⃗ fm | 2 ).
Theoretical studies. All calculations (DFT and TD-DFT) were carried out with the Gaussian 09 (Rev. D/E.01) [5] program package and were performed on a parallel cluster system. GaussView 6.0.9 was used to visualize the results, to measure calculated structural parameters, and to plot orbital surfaces (isovalue: ±0.03 [e a0 -3 ] 1/2 ). The ground-state geometries were optimized using the B3LYP functional [6] in combination with the 6-31G(d) basis set. [7] The optimized geometries were confirmed to be local minima by performing frequency calculations and obtaining only positive (real) frequencies. Where optimized structures of a higher symmetry (>C1) were determined as local minima by frequency calculation, the symmetry was included in the subsequent calculations as well. Stability analysis showed the wavefunction to be stable in each case. Based on these optimized structures, the lowest-energy gas-phase vertical transitions were calculated (singlets, 25 states) by TD-DFT, using the Coulomb-attenuated functional CAM-B3LYP [8] in combination with the 6-31G(d) basis set. [6a, 6b] The CAM-B3LYP functional has been shown to be effective for the ICT systems, hence its selection here. [9] The polarizable continuum model (PCM) was used to include solvent effects in the TD-DFT calculations. [10] Natural transition orbitals were calculated for the lowest three energy transitions in each case using Multiwfn. [11] The ultrafine integration grid was used throughout.