Controlling Two-Photon Action Cross Section by Changing a Single Heteroatom Position in Fluorescent Dyes

The optimization of nonlinear optical properties for “real-life” applications remains a key challenge for both experimental and theoretical approaches. In particular, for two-photon processes, maximizing the two-photon action cross section (TPACS), the figure of merit for two-photon bioimaging spectroscopy, requires simultaneously controlling all its components. In the present Letter, a series of difluoroborates presenting various heterocyclic rings as an electron acceptor have been synthesized and their absorption, fluorescence, photoisomerization, and two-photon absorption features have been analyzed using both experimental and theoretical approaches. Our results demonstrate that the TPACS values can be fine-tuned by changing the position of a single heteroatom, which alters the fluorescence quantum yields without changing the intrinsic two-photon absorption cross section. This approach offers a new strategy for optimizing TPACS.

Absorption spectra were recorded at room temperature with a Shimadzu UV-vis Multispec-1501 spectrophotometer (Japan), using quartz cells with a path length of 1.0 cm. The concentration of the compounds was ca. 1.0 ´ 10 -5 M.
Emission spectra were taken on a Hitachi F-7100 spectrometer (Japan) in ca. 1.0 ´ 10 -6 M solutions. Fluorescence quantum yields were determined based on the equation 1 following a literature protocol. [4] (eq. 1) where Φ is the fluorescence quantum yield, A is the absorbance at the excitation wavelengths, I is the integrated emission intensity and n is the refractive indices of the solvents used. The "dye" and "ref" index refer to the dye and standard samples, respectively. As a standard [5] we used Coumarin 153 in anhydrous ethanol, Ex=450 nm or 420 nm, Fref = 0.38 and Fluorescein in 0.1N NaOH, Ex=470 nm, Fref = 0.91.
Time-correlated single-photon counting measurements were performed with an Edinburgh Analytical Instruments F920P spectrometer. Samples were excited at 466.6 nm using a laser diode with a pulse width of about 81.5 ps, and maximal average power 5 mW. The emission intensity was recorded at fluorescence maximum wavelength. A solution of colloidal silica was used to obtain the instrument response function (IRF). Fluorescence lifetimes were calculated using the FAST software package by fitting an exponential decay curve to the obtained data.
The average lifetime, tav was calculated as (eq. 2) were ai and ti are the amplitudes and lifetimes.
The radiative (kr) and nonradiative (knr) rate constants were calculated based on the average lifetime of the S1 excited state (τav) and the fluorescence quantum yield (Φ), using following equations: , (eq. 3) .
(eq. 4) The measurements of bleaching process studied by UV-Vis spectroscopy were performed in a quartz cuvette with dimensions 4´1´1 cm. In order to ensure complete absorption of light, the cuvette was placed in a horizontal position and irradiated with diode-pumped solid state (DPSS) laser light (lEM=457 nm; intensity 50 mW) through the bottom wall (the optical path length = 4 cm). The solution was stirred during irradiation.  a -Absorption maximum (lmax), extinction coefficient (e), emission maximum (lflu), Stokes shift (DS) and fluorescence quantum yield (FQY), b -vibrational features, c -values very close to the benzoate derivative [6] Table S2. The time-resolved fluorescence data a of 1-5 in chosen solvents

S18
Time-correlated single-photon counting measurements                  The multiphoton absorption properties of 2-5 were investigated in broad range (650 -1700 nm) using the Z-scan technique with a laser system consisting of a Quantronix Integra-C Ti:sapphire regenerative amplifier operating as an 800 nm pump and a Quantronix-Palitra-FS BIBO crystal-based optical parametric amplifier. This system allows for wavelength tuning between 530 and 2000 nm pulses of ∼130 fs length and operates at the repetition rate of 1 kHz. The solutions of 2-5 in chloroform (with concentration of ca. 0.3% w/w) were placed in 1 mm path length Starna glass cuvettes, which were moved along focused femtosecond laser beam in the Z axis typically from -20 to 20 mm. To determine the value of the laser intensity, scans of a 4.6 mm thick silica glass plate (used as a reference) and a cuvette filled with chloroform were investigated together with scans of the samples. The solvent measurement was conducted to eliminate the influence of the cuvette walls and the solvent itself to the closed aperture (CA) Z-scan traces. The open aperture (OA) scans for the solvent revealed absence of 2PA. To monitor the laser input, the CA signal and OA signal, the data were collected using three Si and InGaAs photodiodes (Thor Laboratories Inc.) for VIS and NIR range, respectively. Studying wavelength dependences by Z-scan requires aligning the system and determining the properties of the laser beam at each wavelength separately. Employment of appropriate expressions, introduced by Sheik-Bahae et al. [7], allows us to fit the transmittance traces corresponding to closed-aperture (CA) and open-aperture (OA) Z-scan signals, which were analyzed with the help of a custom fitting program. The detailed description of experimental setup can be found in our previous paper. The real and imaginary parts of the second hyperpolarizability γ of the solutes were computed assuming additivity of the nonlinear contributions of the solvent and the solute and the applicability of the Lorentz local field approximation [8].

Quantum-chemical calculations
The ground state geometries of 5 molecules (both trans and cis isomers were optimized using the density functional theoretical method with the M06-2X functional [9]and the 6-31G* basis set [10,11]. In doing so, we employed IEF-PCM method. The minima on potential energy hypersurface were confirmed by evaluation of hessian. Subsequently, the minimum-energy geometries were used for electronic-structure calculations

Molecular dynamics simulations
All structures were solvated by adding about 470 chloroform molecules to form 40x40x40 Å box (density of such system is close to chloroform density at room temperature (1,49 g/cm 3 )). Total charge for each system was zero. Molecular dynamics simulations program NAMD was used to perform simulations [12]. The partial charges were obtained from M06-2X/6-31G* calculations emplying IEF-PCM model at the equilibrium geometry using the CHELPG method. The CHARMM force field and chloroform force field of Dietz and Heinzinger were used to describe the system [13][14][15]. The system was minimized during 10000 steps followed by constant temperature NVT dynamics for 2 ns (1 step = 2 fs) at 300 K using a Langevin thermostat. MD simulations were performed for the rigid geometry of solute. Periodic boundary conditions were applied. After hydrogen bonds balance and energy equilibrium had been achieved, 50 snapshots were taking from the resulting trajectory for subsequent electronic-structure calculations.

Electronic-structure calculations
Based on rigid-body MD simulations, for each molecule (1-5) we performed we performed ab initioquantum-chemistry calculations using resolution-of-identity coupled-cluster CC2 model [16] and the aug-cc-pVDZ basis set [17]. In particular we calculated one-and twophotontransition strengths using electrostatic embedding to account for discrete solvent representation. The choice of RI-CC2 method is dictated by recent reports that all studied density functional theory approximations show serious limitations as far as predictions of twophoton absorption strengths are concerned, i.e. hybrid functionals with fixed amount of HF exchange are burdened by cancellations of significant errors, while range-separated functionals systematically underestimate two-photon transition strengths due to their inability to correctly reproduce excited-state density distributions [18,19]. RI-CC2 calculations were performed with the TURBOMOLE-~7.3 program [20]. Moreover, to shed light on ground-and excitedstate density distribution we have determined the electron density difference plots at the M06-2X/6-31G* level of theory. The calculations in question were performed using GAUSSIAN 16 program.