The chemicals were obtained from commercial sources and used as received. Water was twice distilled and passed through a Millipore apparatus. All solvents (spectroscopic or equivalent grade) were used without further purification.

2.1 pH sensor Ir52.1.1 Synthesis and characterization

General comments. NMR of newly synthesized starting materials (dimeric iridium complexes Ir1 and Ir2 as well as pH-sensitive N^N ligand L1, see Scheme S2) and target complex Ir5 are shown in Figures S1-S5 in Supplementary Information file. HR ESI+ mass-spectrum of pH sensor Ir5 is shown in Figure S6 in Supplementary Information file.

NMR spectra (1D 1H, 2D 1H-1H COSY and NOESY) were recorded on a Bruker 400 MHz Avance; chemical shift values were referenced to the solvent residual signals. Mass spectra were recorded on a Bruker maXis HRMS-ESI-QTOF in the ESI+ mode.

Reagents: 14-amino-10-((2-(2-methoxyethoxy)ethoxy)methyl)-N-(2,5,8,12,15,18-hexaoxanonadecan-10-yl)−13-oxo-2,5,8-trioxa-11-azapentadecan-15-amide trifluoroacetate [DOI: https://doi.org/10.3390/molecules26102898] and 1,10-phenanthroline-5,6-dione [DOI: https://doi.org/10.1134/S1070363219050281] were obtained according to the published procedures. Other solvents and reagents were received form BLD Pharmatech (Shanghai, China), Merck (Darmstadt, Germany), Vekton (St. Petersburg, Russia) and used without additional purification.

Synthesis of iridium dimeric complex Ir1. In a 25 mL round-bottom flask (equipped with condenser) were placed IrCl3·6H2O (100 mg, 0.246 mmol), 2-phenylquinoline-4-carboxylic acid (129 mg, 0.516 mmol), dioxane (9 mL), and distilled water (3 mL). The reaction mixture was stirred under reflux for 24 h. The resulting suspension was centrifuged to provide the precipitate, which was washed with water (1 × 5 mL), methanol (2 × 5 mL), and diethyl ether (1 × 5 mL), and then vacuum-dried. Brown-red solid, 163 mg, yield 92%. 1H NMR (CD3OD, 400 MHz, 323 K, δ): 9.60 (br s, 1H), 8.85 (br m, 1H), 8.68 (br m, 1H), 7.89 (br m, 1H), 7.75 (br m, 2 H), 6.93 (br m, 1H), 6.61 (br m, 1H), 6.16 (br m, 1H).

Synthesis of iridium dimeric complex Ir2. In a 5 mL vial were placed dimeric complex Ir1 (82 mg, 0.057 mmol), 14-amino-10-((2-(2-methoxyethoxy)ethoxy)methyl)-N-(2,5,8,12,15,18-hexaoxanonadecan-10-yl)−13-oxo-2,5,8-trioxa-11-azapentadecan-15-amide trifluoroacetate (224 mg, 0.279 mmol), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (124 mg, 0.238 mmol), N,N-Diisopropylethylamine (ca. 110 mg, 0.851 mmol), and dry dimethyl sulfoxide (2 mL). The reaction mixture was stirred at room temperature for 48 h. The resulting solution was vacuum dried. The residue was dissolved in 6 mL of toluene, isolated by centrifugation and vacuum-dried to give the oily substance. The oily residue was dissolved in 6 mL of water, and centrifuged. The resulting solution was dried, dissolved in 0.5 mL of CHCl3 and then precipitated by adding 8 mL of 3:1 Et2O/n-hexane mixture. The solution was decanted and the residue was evaporated. This step was repeated three times. Additional purification was done by dissolving in CHCl3 followed by column chromatography (sequentially eluted with CHCl3 and CHCl3/MeOH 20:1, 10:1 mixture). The target fractions were combined, to give the desired product. Brown viscous substance, 98 mg, yield 42%. 1H NMR (CD3OD, 400 MHz, 323 K, δ): 9.43 (br s, 1H), 8.69 (br m, 1H), 8.35 (br m, 1H), 7.93 (br m, 1H), 7.71 (br m, 2 H), 6.94 (br m, 1H), 6.59 (br m, 1H), 6.20 (br m, 1H), 5.17 (br m, 1H), 4.27 (br m, 2 H), 3.60 (br m, 40 H), 3.32 (br m, 12 H), 2.97 (br m, 1H), 2.85 (br m, 1H).

Synthesis of 4-(2-(4-hydroxyphenyl)−1H-imidazo[4,5-f] [1, 10]phenanthrolin-1-yl)benzoic acid – diimine ligand L1. 1,10-phenanthroline-5,6-dione (100 mg, 0.476 mmol), 4-aminobenzoic acid (65 mg, 0.476 mmol), 4-hydroxybenzaldehyde (58 mg, 0.476 mmol), ammonium acetate (108 mg, 1.400 mmol), and 10 mL of glacial acetic acid were stirred at reflux in a 25 mL round-bottom flask. After 18 h, the yellow precipitate was formed. It was washed and centrifuged two times with 10 mL of methanol. Yield: 140 mg, 85%. 1H NMR ((CD3)2SO, 400 MHz, 298 K, δ): 9.89 (br s, 1H), 9.09 (d, 1H), 9.01 (d, 1H), 8.95 (d, 1H), 8.23 (d, 2 H), 7.87 (m, 3 H), 7.50 (m, 1H), 7.39 (m, 3 H), 6.77 (d, 2 H).

Synthesis of pH-sensitive iridium complex Ir5. The following components were placed in a 5 mL vial: iridium dimeric complex Ir2 (48 mg, 0.012 mmol), diimine ligand L1 (12 mg, 0.028 mmol) and acetone (2 mL). The reaction mixture was stirred for 18 h at room temperature. After that, it was centrifuged and evaporated. The solid residue was dissolved in water (1.5 ml), centrifuged, and the resulting solution was evaporated. The dried residue was dissolved in toluene (3.0 ml), centrifuged and evaporated again. The obtained substance was dissolved in 1.0 mL of acetone and then precipitated by adding 10 mL of 4:1 Et2O/n-hexane mixture. The resulting solid material was additionally purified by column chromatography (eluted with CHCl3/MeOH 10:1 mixture). Red-brown viscous solid, 58 mg, yield 54%. 1H NMR (CD3OD, 400 MHz, 298 K, δ): 9.22 (d, 1H), 8.62 (m, 3 H), 8.49 (dd, 1H), 8.37 (m, 2 H), 8.22 (d, 1H), 8.18 (d, 1H), 8.03 (m, 3 H), 7.64 (m, 2 H), 7.59 (d, 1H), 7.49 (dd, 1H), 7.43 (d, 1H), 7.31 (m, 7 H), 6.89 (m, 4 H), 6.66 (m, 4 H), 5.10 (m, 2 H), 4.26 (m, 4 H), 3.57 (m, 80 H), 3.35 (m, 12 H), 3.27 (m, 12 H),2.91 (m, 2 H), 2.87 (m, 2 H). HRMS (ESI) m/z: 1239.0360 calcd. for C118H158IrN13O332+ [M + NH4]2+, found 1239.0386; 1242.0147 calcd. for C118H154IrN12NaO332+ [M + Na]2+, found 1242.0142.

2.1.2 Photophysical experiments

Absorption, excitation and emission spectra of complex Ir5 are shown in Figure S7 and Table S1, lifetime values in water and other aqueous medium at different pH and other conditions are shown in Tables S1 and S2 in Supplementary Information file.

Photophysical measurements were performed in aqueous media. Deaeration was performed by passing argon through the studied solution for 30 min, still complete deaeration was not achieved and the residual oxygen content was determined using the mentioned below oxygen meter. Absorption spectra were measured with a Shimadzu UV-1800 spectrophotometer. The emission spectra were registered using an Avantes AvaSpec-2048 × 64 spectrometer. The absolute emission quantum yield was determined in solution by a comparative method. LED (365 nm) was used for pumping and [Ru(bpy)3] [PF6]2 water solution (Φ = 0.040 air-saturated, 0.063 Ar-saturated) was used as the reference. Pulse laser DTL-375QT (wavelength 355 nm, pulse width 5 ns, repetition frequency 1000 Hz), Hamamatsu (H10682-01) photon counting head, FASTComTec (MCS6A1T4) multiple-event time digitizer and Ocean Optics monochromator (Monoscan-2000, interval of wavelengths 1 nm) were used for lifetime measurements. An oxygen meter (PyroScience FireStingO2, equipped with an oxygen probe OXROB10 and a temperature sensor TDIP15) was used to determine partial pressure and concentration of molecular oxygen in aqueous solutions. Temperature control was performed by using a Quantum Northwest Qpod-2e cuvette sample compartment. pH control was provided using pH-150MI pH-meter.

2.2 Fluorescence/Phosphorescence lifetime imaging microscopy

To simultaneously record FLIM of NADH and PLIM of Ir5 in living cells we used multiphoton laser scanning microscopy (LSM 710, Zeiss, Germany) in combination with advanced multi-dimensional time-correlated-single-photon-counting (TCSPC) techniques. The luminescence signal was obtained after excitation with two photons of a femtosecond pulsed Ti: Sa laser (Spectra Physics) at 740 nm.

For simultaneous FLIM and PLIM measurements, excitation was provided by a two-photon laser with an 80 MHz repetition rate. An Acousto-Optic Modulator (AOM) was used to modulate the laser beam, creating distinct ‘on’ and ‘off’ phases. During the laser ‘on’ phase (~ 2.8 µs), fluorescence photons were detected for FLIM while the phosphorescence signal build up. In the subsequent laser ‘off’ phase (~ 3.6 µs), the delayed phosphorescence photons were detected to generate the PLIM signal. This technique was previously described by our group [14].

A three channel TCSPC system (Becker & Hickl GmbH, Berlin, Germany) was used to simultaneously observe the fluorescence of NADH (between 420 and 460 nm), the fluorescence of green emitting dyes (between 510 and 570 nm) and FLIM and delayed signals of indirubin derivatives between 610 and 690 nm [6, 8]. Both NADH-FLIM and PLIM were analyzed with SPCImage software (Becker & Hickl GmbH, Berlin, Germany). For microscopy an objective lens with 40x/1.3 oil, zoom 1 was used, excitation power of the laser at the objective lens was 3 mW. The scan area was 212.1 × 212.1 μm. The total scan time was 3 min. Pixel dwell time was 6.3 µs. Calculation of fluence rate in mW/cm2 and fluence rate in mJ/cm2 which was applied during two photon laser scanning microscopy is always a matter of discussion. Direct calculation by the power of the excitation laser and the scan area leads to values which seems much too high; however, one must consider the scanning procedure and the pixel dwell time. With this the fluence rate is in the µW/cm2 region and the fluence is a few mJ/cm2.

2.3 Cell culture studies

We performed this study in two different cell lines, Human oral squamous carcinoma cells SCC-4 (ATCC-No. CRL-1624) and the immortalized keratinocyte cell line HaCaT. SCC-4 were grown in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12, cat.-no. 110039-047, Thermo Fisher Scientific, Waltham, MA USA) medium supplemented with 1% GlutaMAX™ Supplement (Thermo Fisher Scientific, Waltham, MA USA), 0,4 µg/ml hydrocortisone (Sigma-Aldrich, St. Louis, Missouri) and 10% fetal bovine serum (FBS) (Biochrom GmbH, Berlin, Germany) at 37 °C and 5% CO2.

On the other hand, we cultivated the spontaneously transformed aneuploid immortalized keratinocyte cell line obtained from the adult healthy human skin HaCaT, which was grown in DMEM with 4,5 g/L glucose (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% GlutaMAX™ and 10% fetal bovine serum (FBS) at 37 ◦C and 5.0% CO2.

For microscopy, cells were seeded on glass bottom microwell dishes with a coverglass of 0.16 mm to 0.19 mm (Greiner Bio-one GmbH, Frickenhausen, Germany) at a density of 1,2 × 104 cells∕compartment and were allowed to grow for 48 h. Microscopic observations were performed immediately after removing the incubation medium and rinsing twice with medium without supplements. The cells were incubated for 16 h in medium with 150 µM of Ir5, before imaging. The samples were imaged under standard conditions for cell cultivation (37 °C, 5% CO2).

2.4 Intracellular pH calibration

For intracellular pH calibration, buffers from the Spexyte™ [21] - Intracellular pH Calibration Buffer Kit (AAT Bioquest, Cat# 21235) - were used, containing nigericin to equilibrate intracellular and extracellular pH. SCC4 and HaCaT cells were incubated with calibration buffers at pH 7.0, 7.5, and 8.0 [22]. The incubation was carried out at 37 °C for 10 min prior to fluorescence microscopy analysis.

2.5 Image analysis and segmentation

FLIM and PLIM images were analyzed using the SPCImage software (v8.9, Becker & Hickl GmbH). The fluorescence decay curve of NADH was fitted to an incomplete two-component decay model:

$$\:I\left(t\right)=_^_}}+\:_^_}}$$

\(\:I\left(t\right)\) is the fluorescence intensity, a1and a2 are the relative amplitudes, \(\:_\)and \(\:_\) are the fluorescence lifetimes of the two components. Fixed values of \(\:_\)= 400 ps and \(\:_\)= 2500 ps.

corresponding to the free and protein-bound fractions of NADH and an incomplete exponential model were used for the fluorescence decay fitting [19]. Incomplete means that the fluorescence does not completely decay within a single excitation period. MLE (maximum likelihood) estimation gives a better accuracy of multiexponential fitting. NADH lifetimes (τ₁ and τ₂) were fixed based on extensive literature evidence and over a decade of experience within our group using these parameters for metabolic FLIM analysis in various cell types [6, 7, 19, 20, 23]. These lifetimes are well-established and biologically consistent, allowing us to focus the analysis on the relative contribution (α₁/α₂) of each component, which reflects the metabolic state.

The value of the mean lifetime is given by \(\:_\):

Where \(\:_\)and \(\:_\) are the fluoerecence lifetime of component 1 and 2. a1and a2 represents the contributions of each component in the studied system.

In the case of the phosphorescence lifetime an incomplete two-component decay model was used however without a fixing the values.

Based on the fitting applied to each pixel, a colormap was generated where the fluorescence and phosphorescence lifetimes correspond to a color legend. The phasor plot was calculated with the SPCImage software (v8.9, Becker & Hickl GmbH).

Segmented images were generated with SPCImage Software, and pixel areas were quantified using FIJI [19, 20].

2.6 Statistical analysis

The statistical analysis of the data was performed using the software OriginPro (OriginLab Corporation, Northampton, MA, USA). For each measured condition (pH: 7.0; 7.5; 8.0 and medium), 12 independent images were analyzed (N = 12). A normativity test was used to evaluate whether the data sets were normally distributed. The one-way analysis of variance (ANOVA) test was used to test the significance of the population means between a pair of test conditions. All hypothesis tests were performed using a significance level of 5% (p = 0.05).