Sensing Mechanism and Excited-State Dynamics of a Widely Used Intracellular Fluorescent pH Probe: pHrodo

The pHrodo with an “off–on” response to the changes of pH has been widely used as a fluorescent pH probe for bioimaging. The fluorescence off–on mechanism is fundamentally important for its application and further development. Herein, the sensing mechanism, especially the relevant excited-state dynamics, of pHrodo is investigated by steady-state and time-resolved spectroscopy as well as quantum chemical calculations, showing that pHrodo is best understood using the bichromophore model. Its first excited state (S1) is a charge transfer state between two chromophores. From S1, pHrodo relaxes to its ground state (S0) via an ultrafast nonradiative process (∼0.5 ps), which causes its fluorescence to be “off”. After protonation, S1 becomes a localized excited state, which accounts for the fluorescence being turned “on”. Our work provides photophysical insight into the sensing mechanism of pHrodo and indicates the bichromophore model might be relevant to a wide range of fluorescent probes.


Theoretical calculation method
All of the simulations were performed using the Gaussian 09 program package. 1 Ground state (S0) geometries were initially optimized using density functional theory (DFT) with the Cam-B3LYP functional 2 and 6-31g(d) basis set 3 in acetonitrile with polarizable continuum model (PCM) using integral equation formalism variant (IEF-PCM) 4,5 .All the excited states were optimized based on S0 geometries using time-dependent density functional theory (TDDFT) under Cam-B3LYP/6-31g(d) theoretical level in acetonitrile with PCM.

Root-mean-squared deviation (RMSD) calculation
RMSD value is a typical parameter to characterize the discrepancy between two structures, which can be expressed by the following formula: Where x i , y i , z i ,and x i ′ , y i ′ , z i ′ , are the x, y, z axis coordinate of i th atom in the first and second structure, respectively, and N is the total number of atoms in the structure.

Dihedral angle calculation
Dihedral angle is defined by the planes between arylpyrylium and diamion-xanthium.In our analysis, we drew two planes, which cross arylpyrylium and diamion-xanthium, and we measured the angle between the two planes.

S3
Steady state absorption spectra were measured by Cary 5000 UV/Vis/NIR spectrophotometer.
Steady state fluorescence spectra and fluorescence decay were measured by FluoTime 300.
Fluorescence quantum yield (FQY) was calculated at the excitation wavelength of 510 nm, with respect to Rhodamine 6G (FQY=0.95) as the reference. 6 Where m is the slop of the plot of integrated fluorescence intensity against absorbance (Figure S1

S4
fs-TA experiments were performed by using a femtosecond pump-probe setup.Laser pulses (796 nm, 60 fs pulse length, 4 kHz repetition rate) were generated using a regenerative amplifier (Solstice Ace) seeded by a femtosecond oscillator (Mai Tai SP, both Spectra Physics).For the pump, we used the TOPAS C (Light Conversion) to obtain pulses with a central wavelength located at 525 nm or 640 nm.The pump pulse energies were set to 0.

c
and f); n is the refractive index of solvent, ethanol (ns=1.363)for Rhodamine 6G and acetonitrile (nr=1.325)for pHrodoH.The subscripts 'r' and 's' refer to Rhodamine 6G and pHrodoH, respectively.

Figure S3 .
Figure S3.The optimized geometries and dihedral angle of pHrodo for (a) S0 state and (b) S1 state.

Figure S4 .
Figure S4.The optimized geometries and dihedral angle of pHrodoH protonated at meta-position

Figure S5 .
Figure S5.The optimized geometries and dihedral angle of pHrodoH protonated at para-position

Figure S8 .
Figure S8.(a) The fs-TA spectra of pHrodo TM as a function of time delay with 640 nm excitation.(b) The kinetics traces at 440 and 550 nm, respectively.
3 µJ and 2.0 µJ per pulse for 525 nm and 640 nm, respectively.The spot size was approximately 0.2 mm 2 for both pump wavelengths.

Table S2 .
Dipole moment of pHrodo and pHrodoH at S0 and S1.

Table S3 .
Summary of calculated data for pHrodoH with different protonated positions.
a Root Mean Square Deviation (RMSD) between the optimized structure of S0 and S1.S12

Table S4 .
Optimized geometry based on DFT calculation for pHrodo in S0 state.

Table S5 .
Optimized geometry based on DFT calculation for pHrodo in S1 state.

Table S6 .
Optimized geometry based on DFT calculation for pHrodoH (Meta-Position) in S0 state.

Table S7 .
Optimized geometry based on DFT calculation for pHrodoH (Meta-Position) in S1 state.

Table S8 .
Optimized geometry based on DFT calculation for pHrodoH (para-Position) in S0 state.

Table S9 .
Optimized geometry based on DFT calculation for pHrodoH (para-Position) in S1 state.