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A red-emitting mitochondrial probe with ultra-high signal-to-noise ratio enables high-fidelity fluorescent images in two-photon microscopy Ge Zhang, Yuming Sun, Xiuquan He, Weijia Zhang, Minggang Tian, Ruiqing Feng, Ruoyao Zhang, Xuechen Li, Lifang Guo, Xiaoqiang Yu, and Shangli Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02807 • Publication Date (Web): 20 Nov 2015 Downloaded from http://pubs.acs.org on November 21, 2015

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Analytical Chemistry

A red-emitting mitochondrial probe with ultra-high signal-to-noise ratio enables high-fidelity fluorescent images in two-photon microscopy Ge Zhang,† Yuming Sun,‡ Xiuquan He,§ Weijia Zhang,† Minggang Tian,† Ruiqing Feng,† Ruoyao Zhang,† Xuechen Li,† Lifang Guo,† Xiaoqiang Yu,*,† Shangli Zhang*,ǁ †

Center of Bio & Micro/Nano Functional Materials, State Key Laboratory of Crystal Materials, Shandong University, Jinan 250100, P. R. China ‡ School of Information Science and Engineering, Shandong University, Jinan 250100, P. R. China § Department of Anatomy, Shandong University School of Medicine, Jinan 250012, P. R. China ǁ College of life science, Shandong University, Jinan 250100, P. R. China ABSTRACT: Herein, we reported a red-emitting probe (E)-4-(2-(8-hydroxy-julolidine-9-yl)vinyl)-1-methylpyridin-1-ium iodide (HJVPI) on rotor mechanism with ultra-high signal-to-noise ratio. HJVPI could give high-fidelity fluorescent images of mitochondria in living immortalized and normal cells and be suitable for IR excitation source of two-photon microscopy and various excitation sources of confocal microscopy. As a rotor, its single/two-photon fluorescence intensities directly depended on environmental viscosity. And, as a mitochondrial probe, it displayed much larger two-photon absorption cross-sections in comparison with commercial MitoTracker Green FM and MitoTracker Red FM. Moreover, the fact that living cells stained by HJVPI still possessed physiological function could also be confirmed: 1) MTT assay demonstrated that the mitochondria of cells stained remained their electron mediating ability, 2) Double assay of HJVPI and SYTOX Blue nucleic acid stain (S-11348) showed that the plasma membrane of cells stained was still intact. In addition, HJVPI possessed a number of beneficial properties in bioimaging such as good membrane permeability, high photostability, and excellent counterstain compatibility with Hoechst 33342. Related mechanism research suggested that its localization property was dependent on the mitochondrial membrane potential in living cells. All its remarkable properties can extend the investigation on mitochondria in a biological context.

For bioassay, high-fidelity fluorescent images of targets are very important, especially for mitochondria whose morphology and numbers are changing. For example, some researchers noted that mitochondria in a large number of cell lines unusually enlarged when exposed to long-term hypoxia.1 And the mitochondrial morphology can be controlled by a set of proteins, mutations of which will cause several human diseases including Parkinson’s and Alzheimer’s diseases.2 Therefore, only high-fidelity images can give the accurate information of mitochondria when imaging and especially tracking mitochondria. Improving signal-to-noise ratio (SNR) of fluorescent probes is an efficient method to improve the fidelity of images. To obtain ultra-high SNR mitochondrial probes, a molecular rotor is a good choice. Molecular rotors are sensors of microenvironmental restriction. The rotors may fluoresce only if their intramolecular rotational relaxation of a certain donoracceptor bond is constrained. In contrast, they do not possess fluorescence when free rotating.3,4 Generally, a rotor is a typical fluorophore with a rotatable conjugated moiety, namely, a certain part of whole molecule may rotate relatively to another part. Thus, in nonviscous environments, the intramolecular rotation relaxes the excitation energy resulting in significant quenching of fluorescence, whereas in viscous medium, the rotation is inhibited, thereby reducing the probability of nonradiating pathways and thus restoring fluorescence.5,6 So rotors

are particularly attractive when used as the viscosity-sensitive fluorescent biosensors that need high SNR,7 and doublemembrane bounded mitochondria just can provide highly viscous microenvironment. From this, we imagine that if a rotor can selectively target mitochondria, it should exhibit little background emission in their unbinding states and strong emission once gathered by the mitochondria. Thus, fluorescent mitochondrial probes based on rotor mechanism will possess ultra-high SNR. In various fluorescent biosensors, the red-emitting twophoton fluorescent probes are very suitable for imaging living cells.8,9 Firstly, red-emitting can minimize the background noises and increase SNR due to avoiding intracellular autofluorescence in blue/green regions when imaging.10,11 And red fluorescence in optical window of biological tissue with low Rayleigh scattering can penetrate the thick specimen.12-14 Secondly, two-photon fluorescence microscopy (TPM) possesses high detection sensitivity, deep penetration, no image distortion, low photodamage, and reduced photobleaching in comparison with confocal microscopy.15-20 Although endogenous fluorophores could produce fluorescence under two-photon excitation, their intensities are always weak due to very small two-photon absorption cross-sections (δ). If the δ value of a probe is much larger than that of the endogenous fluorophores, the probe can provide a high SNR when imaging under TPM

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on the premise keeping the advantages above.21 However, as single-photon excitation and two-photon excitation obey different selection rules,22,23 the δ values of conventional singlephoton probes are too small to be applied in TPM. For example, commercially available red-emitting mitochondrial probes do not show detectable two-photon excited fluorescence (TPEF) intensity due to their small δ values, such as MitoTracker Red FM (MTR), although MitoTracker Green FM (MTG), TMRM, TMRE, and JC family have been sold.21 So far, design and develop red-emitting probes which can give high-fidelity images of mitochondria under TPM are still challenging. Recently, a red-emitting probe AC with large δ was synthesized for exclusively imaging mitochondria.24 At the same time, a pyrene-based probe PY whose excitation and fluorescence in the biological optical window was reported for HEK 293 cells.25 By extending π-conjugations, Xiao et al. synthesized a near-infrared fluorescent probe for imaging mitochondria.26 Unfortunately, under TPM, fidelities of fluorescent images obtained by afore-mentioned probes are low. Most recently, Wong et al. reported two mitochondrial probes SPBN and SPHP with large δ.27 And Kim et al. reported a TPEF probe CMT-red and obtained clear images of mitochondria in cortical neurons.9 In this work, we synthesized a red-emitting mitochondrial probe (HJVPI) on rotor mechanism. Compared with other redemitting TPEF mitochondrial probes reported, HJVPI could give higher fidelity fluorescent images under TPM due to ultra-high SNR (Figure 1). The D-π-A type rotor was synthesized by modifying 9-(dicyanovinyl)julolidine (DCVJ), a classic rotor whose julolidine-vinyl bond is rotatable.7,28-31 In

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EXPERIMENTAL SECTION Apparatus and general methods. The UV-visible-near-IR absorption spectra of dilute solutions were recorded on a HITACH U-2910 spectrophotometer using a quartz cuvette having 1 cm path length. SPEF spectra were obtained on a HITACH F-2700 spectrofluorimeter equipped with a 450-W Xe lamp. TPEF was measured on a SpectroPro300i and the pump laser beam came from a mode-locked Ti:sapphire laser system at the pulse duration of 5 s, a repetition rate of 76 MHz (Coherent Mira900-D). The double-stranded DNA-specific dye Hoechst 33342, ideal dead-cell stain SYTOX Blue nucleic acid stain (S-11348), MTG, MitoTracker Deep Red FM (MTDR) and MTR were purchased from Molecular Probes. PBS buffer solution: 10 mM NaCl, Na2HPO4•12H2O, NaH2PO4•2H2O, pH = 7.40. Viability of the cells was assayed using cell proliferation Kit I with the absorbance of 492 nm being detected using a Perkin-Elmer Victor plate reader. Mitochondrial fluorescence detection was carried out on flow cytometry (ImageStreamX MarkII). The data were obtained using INSPIRE software and analyzed using IDEAS Application v6.0 software. Measurement of δ. Quantum yield (Φ) can be calculated by means of Eq. (1):34

(1)

where the subscripts s and r refer to the sample and the reference materials, respectively. Φ is the quantum yield, F is the integrated emission intensity, A stands for the absorbance, and n is the refractive index. In this work, the quantum yields were calculated by using Rhodamine B (Φ = 0.97 in EtOH) as a standard. The δ values have been measured using the two-photon induced fluorescence method, and thus the δ can be calculated by means of Eq. (2):35

(2)

Figure 1. Structure of HJVPI and the rotation around the julolidine-vinyl bond (a), and TPEF images in SiHa cells stained with HJVPI (b, 2 µM, 30 min), Ex = 900 nm, Em < 720 nm.

HJVPI, two nitriles in DCVJ were replaced by the cationic pyridinium unit which can exclusively accumulate in the mitochondria due to its membrane potential up to -180 mV.32 The extended conjugation structure efficiently increased δ and redshifted its emission, and particularly the enlarged ionic radius would facilitate its localization at mitochondria.33 At the same time, the hydroxyl in HJVPI improved its hydrophilicity. A series of photophysical properties tests showed that HJVPI had viscosity-dependent fluorescence similar with DCVJ. HJVPI was non-luminous in nonviscous medium due to the free rotation around the julolidine-vinyl bond (Figure 1a), but both its single-photon excited fluorescence (SPEF) and TPEF emissions were dramatically enhanced in viscous glycerol (Gly), providing high SNR. The probe could enter a living cell only by simple incubation. And the high co-localization coefficients with commercially available mitochondrial probes demonstrated HJVPI could selectively image mitochondria. More importantly, it could be excited by long excitation wavelengths of TPM and could clearly image mitochondria in immortalized and normal cells.

F is TPEF integral intensity. Φ is the quantum yield. δr is the two-photon absorption cross-section of fluorescein in sodium hydroxide aqueous solution (pH = 13.0). Cell culture and staining. All cells were grown in a 5% CO2 incubator at 37 oC. SiHa and HeLa cells were grown in H-DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. HUVEC cells were grown in M199 medium supplemented with 10% FBS and 2 ng/mL FGF-2. BMSC cells were grown in L-DMEM medium supplemented with 10% FBS and 2 ng/mL FGF-2. MC3T3-E1 cells were grown in αMEM medium supplemented with 10% FBS. Cells were placed on glass coverslips and allowed to adhere for 24 h. For living cells imaging experiments of probes, cells were incubated with probes (HJVPI: 2 µM; MTG: 0.2 µM; MTR: 0.2 µM) for 30 min. To co-staining experiments, living cells were incubated with HJVPI firstly for 30 min, then stained with 5 µM Hoechst 33342 for 30 min, or stained with 5 µM S-11348 for 30 min, or stained with 0.2 µM MTG for 30 min, or stained with 0.2 µM MTDR for 30 min. Every time the cells were washed to remove the unbound probes before staining with another probe. After rinsing with PBS twice, cells were imaged immediately. Cell-viability assay: the study of the effect of HJVPI on viability of cells was carried out using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. SiHa cells growing in log phase were seeded into 96-well plates (ca. 1 × 104 cells/well) and allowed to adhere for 24 h. HJVPI (200


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Figure 2. Photos under UV light (a, d) and SPEF (b, e)/TPEF (c, f) spectra of HJVPI (10 µM) in different solvents (a, b, and c) or in H2OGly mixtures (d, e, and f). Ex: b, the corresponding maximum absorption wavelengths; c and f, 900 nm; e, 530 nm.

µL/well) diluted in DMEM at concentration of 2 µM was added into the wells of the treatment group, and DMSO (200 µL/well) diluted in DMEM at final concentration of 0.2% to the negative control group, respectively. The cells were incubated for 2, 6, 12, 24, and 48 h at 37 oC under 5% CO2, then MTT (5 mg/mL in PBS) was added into each well. After 4 h incubation, DMSO (200 µL) was added to dissolve the purple crystals. After 20 min incubation, the optical density readings at 492 nm were taken using a plate reader. Each individual cytotoxic experiment was repeated for three times. Fluorescent imaging. Wide-field fluorescent images were acquired with an Olympus IX71 inverted microscope coupling with a CCD and display controller software. The fluorescence of S-11348 and HJVPI were excited and collected through the blue channel and the red channel, respectively. Confocal fluorescent images were obtained with an LSM 780 confocal laser scanning microscope or an Olympus FV 300 laser Confocal Microscope. The overlap coefficients and fluorescence intensity of the images were determined by the software with the LSM 780 confocal microscope. TPEF imaging was obtained with Olympus FV 300 laser Confocal Microscope. In twophoton experiments, different excitation wavelengths (800-900 nm) were from a Ti:sapphire femtosecond laser source (Coherent Chamelon Ultra), and the incident power on samples was modified by means of an attenuator and examined with Power Monitor (Coherent). A multiphoton emission filter (FF01-750; Semrock) was used to block the IR laser. The DIC images were taken with 488 nm Ar+ ion laser. Cell mitochondrial isolation and flow cytometry analysis. Mitochondria were isolated from cultured HeLa cells (5 × 107 cells). The cells were first incubated with DMSO (control, 30 min), HJVPI (2 µM, 30 min), and MTDR (0.2 µM, 30 min). The medium was removed from the cells and the cells were washed once with PBS. Then cells were centrifuged (800 g for 10 min) in PBS (pH 7.4). The ice-precooling Lysis buffer (1.0 mL) was added to resuspend the cells. And the cell suspension was transferred to the glass homogenizer and was grinded (40 times) at 0 oC. The resulting material was transferred to the centrifuge tube and centrifuged again (1000 g, 4 oC for 5 min).

The resulting supernatant was centrifuged at 1000g for 5 min (4 oC) and then the resulting supernatant was centrifuged at 12000g for 10 min (4 oC) to pellet the crude mitochondria. The crude mitochondria were resuspended in Wash buffer (0.5 mL) and centrifuged again (1000 g, 4 oC for 5 min). The resulting supernatant was centrifuged at 12000g for 10 min (4 oC). Discard the suspernatant and mitochondria were suspended in Store Buffer and analyzed by flow cytometry. Excitation wavelengths were 488 and 642 nm, and the corresponding collected wavelengths were 595-642 nm and 642-745 nm, respectively. Statistical analyses. Statistical analyses were performed using Spss Statistics 17.0. Data are presented as mean values ± standard errors (SD) from three independent experiments. Unless otherwise stated, statistical discriminations were performed with paired samples t test, and p < 0.05 was regarded as significant.

RESULTS AND DISCUSSION The rotor character of HJVPI. The absorption, SPEF, and TPEF spectra of HJVPI in various low-viscosity solvents and high-viscosity Gly were shown in Figure 2 and Figure S1. Expectedly, SPEF and TPEF intensities of HJVPI were very low in H2O. And in H2O and Gly mixed solvents, the SPEF and TPEF intensities obviously increased with increasing Gly volume fraction. In pure Gly, HJVPI showed the strongest and naked eye visible red emission peaking at about 620 nm. For SPEF rotors, the ratio of Φ in Gly and that in low-viscosity solvents can represent their imaging SNR in laser scanning confocal microscopy (LSM). Thus, in order to acquire high SNR, the low-viscosity Φ of a rotor the best is as low as possible, and the high-viscosity Φ the best is as high as possible.36-46 Among the rotors reported, the lowest Φ in H2O and highest Φ in Gly were 0.000336 and 1.0,37 two values coming from two different rotors. The low and high Φ values of HJVPI were 0.00084 and 0.046, respectively. As shown in Table S1, the δ value of HJVPI in H2O was 2.96 GM, equivalent to 2.1 GM of SPEF probe Indo-1 (high CaII).47 Delightfully, the δ in Gly was up to 35.67 GM. And thus the TPEF action cross-sections (δ × Φ) ratio of HJVPI attained 660, while the sole ratio re-



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ported was only 20.8.3 In addition, the δ × Φ of HJVPI at different excitation wavelengths and solvents were plotted in Figure S2. From Figure S3, in Gly, the SPEF of HJVPI at 75 o C was much weaker than that at -10 oC, because the viscosity of Gly in high temperature is low. Furthermore, SPEF, TPEF, Φ, and δ × Φ of HJVPI in organic solvents with different polarities were very small (Figure 2, Figure S1 and Table S1). And in low-viscosity buffers with different pH (from 3.0 to 8.0), its SPEF still was weak (Figure S4b). All results demonstrated that HJVPI could exhibit strong fluorescence only in high-viscosity environments restricting free rotation of julolidine-vinyl bond, and other factors which could not affect bond rotation, such as polarity and pH, did not alter its fluorescence. These experiment phenomena and big δ × Φ ratio declared that HJVPI was a two-photon molecular rotor with red-emitting. In order to confirm better the rotor character of HJVPI, the DNA and RNA titrations were carried out. Usually, a cationic compound can incorporate into the hydrophobic grooves of DNA or RNA with anions due to the static interaction.10,48 This interaction should be able to restrict the free rotation of intramolecular bonds. As expected, the fluorescence intensity of HJVPI in buffer solution (pH = 7.4) evidently increased with the addition of DNA or RNA (Figures S5a and S5b). Selectivity to mitochondria. To demonstrate fluorescent imaging ability of HJVPI, cell imaging was firstly performed with SiHa cells under wide-field microscopy. As shown in Figure S6, it was clear that intracellular HJVPI emitted bright fluorescence and no fluorescence was observed in extracellular regions. This implied that HJVPI exhibited qualified permeability to plasma membranes and could enter into living cells by simple incubation.

Figure 3. Confocal fluorescent images in SiHa (a, d) and BMSC cells (b, e) stained with HJVPI (2 µM, 30 min) and MTG (0.2 µM, 30 min), or stained with HJVPI (2 µM, 30 min) and MTDR (0.2 µM, 30 min). The co-localization coefficients of HJVPI and MTG (c) or that of HJVPI and MTDR (f) in SiHa and BMSC cells. a, b: HJVPI: Ex = 561 nm, Em = 580-679 nm; MTG: Ex = 458 nm, Em = 484-546 nm. d, e: HJVPI: Ex = 488 nm, Em = 580-630 nm; MTDR: Ex = 633 nm, Em = 689-740 nm. Bar = 10 µm (Data are expressed as mean ± SD from three independent experiments).

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The co-staining experiment with a commercial probe is a common method to detect the selectivity of a new probe,49 and the prerequisite of the co-localization experiment is that no optical and chemical interferences exist between two probes.50,51 Herein, we selected MTG and MTDR, two commercial mitochondrial probes, as the co-staining regents to detect the selectivity of HJVPI. Accordingly, the absorption and fluorescence spectra of HJVPI, MTG or MTDR, and their mixture have been carefully studied. As shown in Figure S7a and Figure S8a, the absorption spectrum of HJVPI-MTG or HJVPI-MTDR mixed solution was merely linear superposition of the absorption spectra of HJVPI and MTG or MTDR, indicating no chemical interaction between them. Very importantly, when excited by 458 nm or 633 nm, SPEF spectrum of the mixed solution only contained the SPEF band of MTG (Figure S7b) or MTDR (Figure S8b). Moreover, when the excitation wavelength was 561 nm (Figure S7c) or 488 nm (Figure S8c), the SPEF spectrum of both mixed solution was consistent with that of pure HJVPI. These results demonstrated that no optical interferences exist between them. Subsequently, the co-staining experiments of HJVPI and MTG or MTDR have been performed in immortalized and normal cells. The confocal fluorescent images of SiHa and BMSC cells incubated with HJVPI and MTG in sequence are shown in Figure 3a and 3b. The red and green color fluorescent pictures were obtained when excited by 561 nm and 458 nm, respectively. And the red fluorescence dedicated the distribution of HJVPI, and green represented MTG. From the merged images, the red image merged well with the green one. The mean co-localization coefficients of HJVPI and MTG from SiHa cells and from BMSC cells were 0.91 and 0.89, respectively (Figure 3c). At the same time, as shown in Figure 3d and 3e, the red fluorescence excited by 488 nm dedicated the distribution of HJVPI, and green excited by 633 nm represented MTDR. The mean co-localization coefficients of HJVPI and MTDR from SiHa cells and from BMSC cells were 0.93 and 0.96, respectively (Figure 3f). This showed that HJVPI could stain mitochondria with high selectivity. Moreover, flow cytometry analysis of mitochondrial isolation from HeLa cells has been performed as secondary evidence of the fluorescent signal targeted in mitochondria.12 From Figure S9, the fluorescence intensity of isolated mitochondria stained with HJVPI increased compared with that of isolated mitochondria unstained. Very similarly, isolated mitochondria stained with MTDR also showed the same results.

Figure 4. TPEF images in SiHa (a) and BMSC (b) cells stained with HJVPI (2 µM, 30 min). Ex = 900 nm, Em < 720 nm. Bar = 20 µm.


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TPEF/SPEF imaging ability. As displayed in Figure 4, living SiHa (Figure 4a) and BMSC (Figure 4b) cells incubated with HJVPI were imaged in TPM using a 10 mW beam at 900 nm. Fluorescent and merged photos demonstrated intracellular HJVPI clearly stained mitochondria with bright fluorescence and no fluorescence was observed in extracellular regions, indicating that HJVPI was a TPEF probe with excellent SNR. By comparison, SiHa cells incubated with MTG or MTR did not show detectable TPEF intensity due to their small δ values.21 In addition, the imaging ability of HJVPI in LSM was also examined (Figure 5). Interestingly, HJVPI could be excited by various excitation sources of LSM (458 nm, 488 nm, 514 nm, 543 nm, and 561 nm), and mitochondria could be seen clearly in every fluorescence image. Its ability to stain HeLa and various normal cells (HUVEC, BMSC, and MC3T3-E1 cells) was precious. As shown in Figure 6 and Figure S10, the red fluorescence from HJVPI clearly showed the mitochondrial granular or filamentous morphology, indicating HJVPI was a qualified mitochondria probe for various cells. Moreover, Figure

Figure 6. Confocal fluorescent images in HeLa (a), MC3T3-E1 (b), HUVEC (c), and BMSC (d) cells stained with HJVPI (2 µM, 30 min). Ex = 488 nm, Em > 565 nm. Bar = 20 µm.

Figure 5. Confocal fluorescent images in SiHa cells stained with HJVPI (2 µM, 30 min) by different excited wavelengths: a, Ex = 458 nm, Em = 580-679 nm; b, Ex = 488 nm, Em > 565 nm; c, Ex = 514 nm, Em = 580-679 nm; d, Ex = 543 nm, Em > 565 nm; e, Ex = 561 nm, Em = 580-679 nm. Bar = 10 µm.

S11 showed a sequence of optical sections obtained at different depths in SiHa cells. At the same time, a 3D reconstruction based on the optical sections was presented in Figure S12. Cell viability after incubation. A key consideration is whether the cells remain healthy after incubation with HJVPI. Therefore, S-11348, a cell-impermeant nucleic acid dye that can only penetrate the broken plasma membrane of dead cells, was used to testify whether plasma membrane of cells stained by HJVPI was still intact. From Figure S13, when upon binding DNA, S-11348 has an absorption band in the region of 380-470 nm with emitting at 450-550 nm. From Figure 2 and Figure S1, the absorption of HJVPI is about 450-600 nm and the emission is about 550-675 nm. Although the absorption spectra of HJVPI and S-11348 overlapped in the wavelength range from 450 nm to 470 nm, red fluorescence (above 570 nm) excited by 510-550 nm should only come from HJVPI, while blue fluorescence (467-499 nm) excited by 426-450 nm should be attributed to S-11348. From the DIC picture (Figure 7a), there were three cells in this field. When three cells were radiated by 426-450 nm light, only one cell pointed by the red arrow emitted blue fluorescence and others did not show any fluorescence signal (Figure 7b), which indicated that plasma membrane of the cell pointed by the red arrow was broken and that of other two cells should be intact. When excited by 510-



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Figure 7. Wide-field fluorescent images in SiHa cells stained with HJVPI (2 µM) and S-11348 (5 µM) in sequence. Each incubation 30 min. (a) DIC picture; (b) Ex = 426-450 nm, Em = 467-499 nm; (c) Ex = 510-550 nm, Em > 570 nm; (d) Overlay images of a, b and c. Bar = 20 µm.

550 nm, other two cells emitted red fluorescence. Combining with Figure 7b, this showed that the plasma membrane of two cells stained by HJVPI indeed was intact (Figure 7c). Interestingly, only the dead cell did not emit in Figure 7c. This implied that once the cell died, HJVPI would not accumulate in the mitochondria without membrane potential. The free rotation of julolidine-vinyl bond of HJVPI in cytoplasm would restore and thus HJVPI did not emit fluorescence, and the mechanism will be discussed in the following. Based on a large number of experimental data, the S-11348 staining rates in different samples were displayed in Figure S14d. From Figure S14, when staining dead cells fixed by 4% paraformaldehyde, the staining rate of S-11348 was about 100% (positive control). When staining normal living cells, the staining rate was 1.188% (negative control), while the rate is 1.281% when staining living cells prestained with HJVPI. The two staining rates in living cells were very similar, indicating that the cells stained by HJVPI were living. Moreover, cytotoxicity of HJVPI was evaluated in SiHa cells by MTT assays. The cell viability assay data of SiHa cells treated with HJVPI (2 µM) at different incubation time were quantified (Figure S15). The SiHa cells showed >90% viability after 12 h incubation. Thus the concentrations at a micromolar order of magnitude and short incubation time (such as < 30 min) for HJVPI did not damage the cellular

Figure 8. Comparison of photobleaching of HJVPI (2 µM, 30 min), MTR (0.2 µM, 30 min), and MTG (0.2 µM, 30 min) in SiHa cells under TPM or LSM. HJVPI-TP: Ex = 900 nm, Em < 720 nm; HJVPI-SP: Ex = 488 nm, Em > 565 nm; MTR-SP: Ex = 543 nm, Em > 565 nm; MTG-SP: Ex = 488 nm, Em = 510-540 nm (Data are expressed as mean ± SD from three independent experiments).

electron mediating ability. The results proved the cellular viability after incubation. Photostability and other advantages. Under high-intensity illumination conditions in LSM and TPM, the irreversible destruction or photobleaching of the excited fluorophore often becomes the primary factor limiting fluorescence detectability. The two-photon photostability of HJVPI was examined under TPM. Upon excitation at 900 nm with femtosecond pulses and 10 mW average power at the focal plane, TPEF images were collected (Video S1). In Figure 8, the TPEF intensity of SiHa cells stained underwent little decrease at exposure time of 12 minutes. On the other hand, SPEF photostability of HJVPI, MTG, and MTR was also detected. The unchanged time of HJVPI SPEF intensities were 15 minutes (Video S2), but that of MTR and MTG only were 0.5 min. To validate the ability of HJVPI for imaging mitochondrial viscosity, confocal fluorescence images of SiHa cells incubated by HJVPI were acquired in the presence of nystatin, an ionophore that could increase the mitochondrial viscosity.4 Figure S16 gives a series of fluorescent images of SiHa cells treated with different doses of nystatin under the same imaging conditions. With increasing the dose of nystatin, fluorescence enhancement of HJVPI in mitochondria was observed. According to Figure S16, under the same incident power (just lower than that used by Figure 5b), the relative intensity of HJVPI in SiHa cells treated with different doses of nystatin was showed in Figure S16f. It is obvious that the fluorescence intensity increased with increasing the dose of nystatin. Therefore, HJVPI is capable of sensing mitochondrial viscosity change (Figure S16f). A multicolor labeling experiment entails the deliberate introduction of two or more probes to simultaneously target different entities. This technique has major applications in fluorescence microscopy. In multicolor imaging, probes that have strong absorption at a coincident excitation wavelength and well-separated emission spectra will be ideal. From Figure 5, we know HJVPI could be excited by various excitation sources of LSM (458 nm, 488 nm, 514 nm, 543 nm, and 561 nm). Due to its flexibility in selecting excitation sources of LSM, HJVPI is particularly useful in multicolor application.21 For example, both BODIPY FL DHPE (D-3800, a membrane probe) and HJVPI could be excited by 488 nm, and their multicolor imaging could be easily obtained when collecting green fluorescence (500-540 nm) for D-3800 and red fluorescence (600-650 nm) for HJVPI. In addition, 458 nm could excite both SYTO 45 (S-11356, a blue fluorescent nucleic acid stain) and HJVPI in multicolor labeling experiment, and S-11356 emits blue fluorescence (460-500 nm), while HJVPI emits red fluorescence (600-650 nm). In this work, the double-staining


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Figure 9. Confocal fluorescent images of SiHa cells treated without (Control) or with CCCP (1 µM, 2 µM, 5 µM, 8 µM, 12 µM, 15 µM, 18 µM, 20 µM, 25µM, 30 min) and then stained with HJVPI (2 µM, 30 min). Ex = 488 nm, Em > 565 nm. Bar = 20 µm.

experiment of HJVPI and Hoechst 33342 (a commercial cell nucleus probe) was performed. The red channel was excited by 514 nm only suitable for HJVPI and not for Hoechst 33342 (Figure S17a). The blue channel was excited by 405 nm only for Hoechst 33342 and not for HJVPI (Figure S17c). From Figure S17a, the red fluorescence from HJVPI was distributed in mitochondria around the nuclei with blue emission. HJVPI and Hoechst 33342 kept their own labeling ability and did not interfere into each other, suggesting HJVPI had good counterstain compatibility with Hoechst 33342. Thus, HJVPI was a more ideal probe when utilized in fluorescent image. As shown in Figure S18, the Stokes shifts of HJVPI in high viscous media are about 90 nm at pH 7.0 and 60 nm at pH 8.0. Thus, as a mitochondrial probe, its Stokes shift at pH 8.0 (mitochondrial pH) is larger than that of MTG (18 nm) and a little larger than that of MTR (37 nm). Mechanism discussion. For discussing the mechanism by which HJVPI selectively images mitochondria, it is essential to understand the structural character of the target. Mitochondrion has a very large membrane potential up to -180 mV. Thus, cationic species tend to accumulate in the mitochondria rather than other organelles.21 To confirm this statement above, SiHa cells were pre-treated with CCCP, a type of protonophore that could decrease of membrane potential. In Figures 9 and S19, control cells and cells treated with CCCP were stained with HJVPI, MTR, and MTG, respectively. According to Figures S19a and S19b, membrane potential independent MTG49 still stained the mitochondria in cells treated with CCCP, while membrane potential dependent MTR9 no longer labeled the mitochondria in cells treated (Figures S19c and S19d). Quantitative analysis of the effect of CCCP within cells stained by HJVPI has been carried out and the results were showed in Figure 9 and Figure S20. With increasing the concentration of CCCP, the intensity of HJVPI within cells was weaker. And its intensity within cells treated with 8 µM CCCP was obviously weakened, while mitochondria almost could not

been seen clearly within cells treated with 25 µM CCCP. This suggested that the HJVPI was a membrane potential dependent probe.

CONCLUSIONS To summarize, we presented a red-emitting TPEF rotor, HJVPI, as a membrane potential dependent probe for specific mitochondrial imaging in living immortalized and normal cells with ultra-high SNR. As a rotor, its SPEF and TPEF emissions were very weak in low-viscosity medium, but both of them were very strong in high-viscosity Gly. The co-staining experiment of HJVPI and MTG or MTDR demonstrated HJVPI was capable of exclusively imaging mitochondria. And it could give high-fidelity SPEF/TPEF images due to little background noises in its unbinding states. Double assay of HJVPI and S11348 showed that HJVPI could stain mitochondria in living cells. Moreover, the new probe possessed good membranepermeability, low toxicity, high photostability, large Stokes shift, and excellent counterstain compatibility with Hoechst 33342. All these properties have ranked it as one of the best red-emitting mitochondrial TPEF probes.

ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Experiment details of synthesis of all compounds; photophysical properties of HJVPI; additional fluorescent images of HJVPI in immortalized and normal cells; mitochondrial flow cytometry analysis; optical sectional fluorescence images of SiHa cells stained by HJVPI; MTT assay for HJVPI; confocal fluorescent images of HJVPI in SiHa cells treated with nystatin; spectral characterization for HJVPI; Video S1 and Video S2.

AUTHOR INFORMATION Corresponding Author



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Xiaoqiang Yu Tel.: +86 0531 88366418; Fax: +86 0531 88366418; E-mail address: [email protected] Shangli Zhang E-mail address: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Our Laser confocal scanning imaging of living cells was performed at The Microscopy Characterization Facility, Shandong University. For financial support, we thank the National Natural Science Foundation of China (51273107 and 51303097), Natural Science Foundation of Shandong Province, China (ZR2012EMZ001), Shandong University 2011JC006, Open Project of State Key Laboratory for Supramolecular Structure and Materials (SKLSSM201524).

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