Conformationally Induced OffâOn Two-photon Fluorescent Bioprobes

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Conformationally Induced Off–On Two-photon Fluorescent Bioprobes for Dynamically Tracking the Interactions among Multiple Organelles Huihui Zhang, Xiaojiao Zhu, Gang Liu, Xinzhi Ding, Junjun Wang, Mingdi Yang, Ruilong Zhang, Zhongping Zhang, Yupeng Tian, and Hongping Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00806 • Publication Date (Web): 19 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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Analytical Chemistry

Conformationally Induced Off–On Two-photon Fluorescent Bioprobes for Dynamically Tracking the Interactions among Multiple Organelles Huihui Zhang,‡ Xiaojiao Zhu,‡* Gang Liu, Xinzhi Ding, Junjun Wang, Mingdi Yang, Ruilong Zhang, Zhongping Zhang, Yupeng Tian, Hongping Zhou* College of Chemistry and Chemical Engineering, Anhui University and Key Labotatory of Functional Inorganic Materials Chemistry of Anhui Province, Anhui Province Key Laboratory of Chemistry for Inorganic/Organic Hybrid Functionalized Materials, 230601, Hefei, P.R. China.

*Prof. Hongping Zhou, Email: [email protected] and Xiaojiao Zhu, Email: [email protected] ABSTRACT: Unveiling the synergism among multiple organelles for fully exploring the mysteries of the cell has drawn more and more attention. Herein, we developed two two-photon fluorescent bioprobes (Lyso-TA and Mito-QA), of which the conformational change triggered “off–on” fluorescent response. Lyso-TA can real-time monitor the fusion and movement of lysosomes, as well as unveil the mitophagy process with the engagement of lysosomes. Mito-QA was transformed from Lyso-TA by one-step ambient temperature reaction, visualizing the dysfunctional mitochondria through shift from mitochondria to nucleoli. With superior twophoton absorption cross-section, good biocompatibility and greater penetration depth, two small bioprobes were both applied in vivo bio-imaging of brain tissues and zebrafishes.

Membrane-bound organelles such as lysosomes, mitochondria or endoplasmic reticulun are integrated into reticular networks in eukaryotic cells, which are engaged in various cellular activities rather than act as isolated entities.1,2 The networks of dynamic integration enable them to regulate sophisticated cellular signaling pathways and maintain normal operation of the cell.3 Particularly, lysosomes and mitochondria as dynamic organelles with higher activity, extensively participate in cellular catabolic process and mitophagy through the interaction between the lysosome itself or the mitochondria.4-7 The recent advances of two-photon (TP) fluorescent bioprobes for specifically targeting individual organelle, such as lysosomes or mitochondria, have been widely reported.8-12 However, the interactive activities between lysosomes and mitochondria monitored by the twophoton fluorescent bioprobes are rarely reported,13 hampering the further insight into the metabolism process in cells. “Off-on” fluorescent bioprobes with high specificity and photo-stability holds great in visually tracking the interactive activities as well as individual dynamic behavior of multiple organelles, which is attributed to the reduced signal-to-noise ratio.14-16 Intracellular viscosity, as a cellular environmental factor that widely exists in various organelles, has high dynamic stability, which was usually used as a mediator for triggering the conformational change, realizing the “off-on” fluorescent response by hampering probe molecule’s free rotation. For instance, Krishnan reported that substrate accumulation in lysosomal storage disorders was monitored by the changes of viscosity17 and Tang reported a fluorescence probe (PIP–TPE) to realize the real-time monitor of lysosomal migration process with “off-on” response to viscosity.18 However, the further insight into the synergistic functioning

among diversified organelles is somehow neglected. In this regard, developing viscosity-induced conformational responsive fluorescence probes to dynamically track the interactions among multiple organelles is challenging but noteworthy. Herein, we developed two conformationally induced “off– on” TP bioprobes (Lyso-TA and Mito-QA) to not only dynamically track lysosomes and mitochondria, respectively, but vividly visualize the synergism between lysosomes and mitochondria. As for Lyso-TA, the probe could accomplish the real-time monitor of lysosomal fusion and migration process, as well as mitophagy process in which the lysosome is involved as “scavenger”. Mito-QA could serve a valid tool for visualizing the dysfunctional mitochondria through shift from mitochondria to nucleoli. In addition, Lyso-TA is designed based on integration of molecular rotor moiety and lysosomes-targeting tertiary amine, which can be switched into Mito-QA by one-step facile nucleophilic substitution reaction at room temperature. Both of these two small bioprobes with superior TP absorption cross-section delivered good biocompatibility and greater penetration depth, endowing them to be applied in vivo bio-imaging of brain tissues and zebrafishes.

EXPERIMENTAL SECTION Materials. S. cerevisiae RNA (RNA), calf thymus DNA (DNA) and serum albumin for human (HSA) and bovine (BSA) were purchased from commercial sources (SigmaAldrich). HeLa (human cervical cancer cell) and HeGp2 (human liver cancer cell) were purchased from BeNa culture collection. MEF (mouse embryonic fibroblasts) and HUVECs

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(human umbilical vein endothelial cells) were obtained from American type culture collection. Instrumentations. The NMR spectra were measured on a Bruker AVANCE-600 MHz (1H) and 150 MHz (13C) NMR instruments. Mass analysis was recored using a LTQ Orbitrap XL mass spectrometer. IR spectra were obtained on a Vertex80+Hyperion2000 spectrometer. The pH values were measured in a Leici pH3c pH meter and the viscosity were obtained using a Brook field Rheo3000 R/S plus Rheometer. The UV-vis absorption spectra were recorded on a UV-265 spectrophotometer. Fluorescence spectra were measured using a Hitachi F-4500 fluorescence spectrophotometer. One- and two-photon confocal fluorescence imaging was performed on Olympus FV 1200 MPE-share and ZEISS710 confocal laserscanning microscope. Cell culture and cytotoxicity test. The cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented in humidified atmosphere with 5% CO2/95% air at 37 oC. Two day before imaging, the cells were seeded in 15 mm glass bottom cell culture dishes, and incubated at cell incubator. The cytotoxicity was performed through using MTT assays. Hela cells were cultured in DMEM in 96-well culture plates for 24 hours at 5% CO2/95% air. And then MTT assays were used. Preparation of fresh mouse brain slices and zebrafishs. The brain of a 10-day-old mouse was removed, and they were cut with Leica Ultracut EM microtome by using a in 10 mM PBS buffer (pH = 7.4). 4-day-old zebrafishs were prepared. After fed with the probes, the fishes were terminally anaesthetized using MS222. Synthesis of Lyso-TA. Lyso-TA was synthesized by twostep facile routes depicted in Scheme S1. 4-aminobenzyl cyanide (0.82 g, 6.21 mmol) and M (1.0 g, 5.18 mmol) were added to 25 mL ethanol solution with 10 drops of tetrabutylammounium. The mixture was stirred for 5 h in refluxing condition. After it cooled at rt, the yellow solid was filtered, wash with ethanol solution. The crude product was purified by column chromatography using petroleum ether/ethyl acetate (1/2, v/v) as eluent to give the yellow product (1.05 g, 65.6%). 1H NMR (600 MHz, DMSO-d6), δ (ppm): 7.84-7.83 (J = 8.88, d, 2H), 7.61 (s, 1H), 7.41-7.39 (J = 8.64, d, 2H), 7.07-7.06 (J = 8.82, d, 2H), 6.65-6.63 (J = 8.64, d, 2H), 5.55 (s, 2H), 4.13-4.11 (J = 5.76, t, 2H), 2.66-2.64 (J = 5.76, t, 2H), 2.23(s, 6H). 13C NMR (150 MHz, DMSO-d6), δ (ppm): 160.17, 147.59, 141.61, 138.02, 130.02, 129.93, 126.86, 124.62, 118.69, 114.28, 108.84, 66.30, 58.06, 45.59. FT-IR (KBr, cm−1): 3455 (m), 3365 (s), 3207 (s), 2950 (s), 2829 (m), 2775 (m), 2212 (m), 1605 (m), 1515 (m), 1456 (m), 1299 (m), 1249 (m), 1172(m), 1024(m), 821(m), 533(m). MS (ESI) m/z: calcd for [M+H]+, 308.1763; Found, 308.1756. Synthesis of Mito-QA. Mito-QA were synthesized by onestep facile nucleophilic substitution reaction with CH3I at room temperature depicted in Scheme S1. Methyl iodide (0.46 g, 3.25 mmol) and Lyso-TA (0.2 g, 0.65 mmol) were added to 5 mL dry dichloromethane solution. The mixture was stirred for 2 h at room temperature (rt), and the yellow solid was filtered as the pure pruduct (0.27 g, 93.0%). 1H NMR (600 MHz, DMSO-d6), δ (ppm): 7.88-7.87 (J = 8.58, d, 2H), 7.66 (s, 1H), 7.42-7.40 (J = 8.40, d, 2H), 7.15-7.13 (J = 8.58, d, 2H), 6.65-6.64 (J = 8.40, d, 2H), 5.57 (s, 2H), 4.55 (s, 2H), 3.833.82 (J = 4.29, t, 2H), 3.20(s, 9H). 13C NMR (150 MHz, DMSO-d6), δ (ppm): 158.72, 150.34, 141.15, 136.76, 129.75,

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127.00, 119.13, 114.34, 109.17, 64.50, 62.23, 53.60. FT-IR (KBr, cm−1): 3337(m), 3284(s), 3198(s), 3013(s), 2208(m), 1600(m), 1510(m), 1474(m), 1420(m), 1281(m), 1171(m), 1046(m), 958(m), 821(m), 709(m), 533(m). MS (ESI) m/z: calcd for [M+H]+, 322.1919; Found, 322.1905. The structures of Lyso-TA and Mito-QA were comprehensively confirmed by 1H and 13C NMR spectroscopy, as well as FTIR and MS spectrometry (Figure S1, S2 and S3).

RESULTS AND DISCUSSION Design and synthesis. Smaller molecule bioprobes are more susceptible to the resistance induced by intracellular viscosity, which would change their conformation and be conducive to realization of “off-on” fluorescence response. As shown in Scheme 1, the probe (Lyso-TA) composed of a molecular rotor moiety and a targeted group was constructed with the small conjugate system of two benzene rings and the bridging double bond. Without external resistance, this molecular rotor moiety in Lyso-TA could efficiently rotate, forming the non-planar conformation and resulting in the fluorescence “off” because of the non-radiative decay process of excited states19. Interestingly, in cellular microenvironment with viscosity-induced resistance, the rotation is hindered, enabling it to stay the planar conformation and forming a strong ICT system, which could show a fluorescence “on” emission and even increase TP absorption efficiency. Therefore, Lyso-TA holds great promise in realizing the realtime monitoring of the interaction between specific targeted organelles with viscosity-induced conformational responsive fluorescence probes.

Scheme 1. The structures and proposed mechanisms of LysoTA Conformationally induced “off-on” fluorescence. To explore the fluorescence "off-on" induced by conformational changes, in vitro experiments of the fluorescence responsive to different solvents with various viscosity were conducted. Firstly, the spectroscopic properties of Lyso-TA with common solvents were obtained (see the details in Figure S4 and Table S1), which showed Lyso-TA response to glycerol (viscosity) with higher fluorescence intensity, fluorescence quantum yield (Φf = 0.0788) and fluorescence lifetime (Τ = 1.25 ns) than those of other solvents. Then the fluorescence emission spectra of Lyso-TA in water-glycerol mixture of different viscosity were carried out (Figure 1a), which displayed ratiometric changes to viscosity with the good linear relationships by fitting the Förster-Hoffmann equation, and the obvious “off-on” fluorescence response were observed by the naked eye (Figure S5). Likewise, the fluorescence lifetime (τf) of Lyso-TA increased gradually along with increase of viscosity (ƞ), and the good linear relationships were depicted in Figure 1b and Figure. S5b. Meanwhile, the structures of Lyso-TA were calculated by TD-DFT with a B3LYP/6-31G (d) basis set using Gaussian 09 (see the details in Figure S6). All of the above results indicated that Lyso-TA can realize “off-on” fluorescence response to viscosity-induced conformational change.


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Analytical Chemistry High selectivity is an essential requirement for fluorescence probes. The selectivity of Lyso-TA for viscosity over competing species was implemented. As shown in Figure S7a, the addition of the disturbed species only caused negligible perturbation compared with that of 99% glycerol. Notably, the addition of RNA or DNA to the solution of the probes led to a weak enhancement of fluorescence emission compared with that of other competitive species, which manifested Lyso-TA could intercalate into the helical strands of DNA or RNA, hampering its free rotation.20 (see the details in Figure S7b). Meanwhile, the effect of pH on the probe was investigated, which exhibited the weak fluorescence emission at pH 2-13 in water (see the details in Figure S8). These results suggested that the probe Lyso-TA delivered specific response to viscosity in a complex biological environment. Then TP fluorescence spectra and TP cross-section of Lyso-TA in PBS buffer solutions were implemented which showed superior TP absorption cross-section compared with the reported small molecular bioprobes, respectively21-22 (Figure 1c, Figure S9). The results indicated that the probes Lyso-TA can be an effective tool for fluorescence imaging in living cells and in vivo on account of the fluorescence "off-on" induced by conformational changes by a two-photon laser confocal fluorescence microscopy (TPFM).

Figure 1 (a) Fluorescence spectra of Lyso-TA (10 µM) in water/glycerol system. (b) Fluorescence lifetime spectra at 518 nm for Lyso-TA (10 µM) at different viscosities. (c) Twophoton excitation action cross-secction in the water-glycerol systems with different viscosities of Lyso-TA, Two-photon excitation: 680-900 nm. (d) MTT assay of Hela cells treated with Lyso-TA at different concentrations for 12 h, 24 h, 36 h, 48 h and 60 h, error bar represents standard deviation (SD). Cytotoxicity and co-localization assays. The cytotoxicity and photostability of Lyso-TA was investigated using MTT assays and TPFM in HeLa cells, respectively. (Figure 1d and Figure S10), which showed the probes had the low cytotoxicity, good bio-compatibility, superior photostability in the cell. For evaluating the targeting abilities of Lyso-TA, the subcellular localization experiments of Lyso-TA (10 µM) with Mito-Tracker Deep Red dye, CellMask Deep Red, ER-Tracker Red and Lyso-Tracker Red were conducted in Hela cells for 30 min at 37 oC, respectively. As shown in Figure 2, the LysoTA could target lysosomes in Hela cells with high synchronization of the intensity profile and high pearson correlation factors value (Pr = 0.9226). The high targeting ability of Lyso-TA was further confirmed by systematic control experiments of three different cells and various concentrations (Figure S11, S12). Then 3-D confocal

fluorescence imaging and 3-D fluorescence intensity of the probes (Lyso-TA) were performed, which exhibited the spatial distribution morphology and fluorescence intensity of lysosomes (Figure S13). To further investigate the change of probe conformation induced by the viscosity of lysosomal microenvironment, the high concentrated sucrose solution inducing lysosomal viscosity enhancement23 were added to the cells in the presence of Lyso-TA. The HUVCEs incubated with only the probe Lyso-TA (10 µM, 30 min) exhibited poor fluorescence. Then the high concentrated sucrose solution (80 mM, 15 min) were further added to the cells, resulting in obvious increment of fluorescence intensity in lysosomes. And the fluorescence intensity quantification of Lyso-TA and Lyso-TA + Sucrose were measured (Figure S14). These studies clearly suggested that the probes Lyso-TA could achieve the enhancement of fluorescence by lysosomal microenvironmental viscosityinduced conformational responsive in living cells consisted with in vitro experiments.

Figure 2 Subcellular colocalization assays of Lyso-TA (10 µM) with commercial organelle dyes in Hela cells. Commercial organelle dyes: Mito-Tracker Deep Red dye (λex = 644 nm, λem = 658-678 nm), CellMask Deep Red (λex = 649 nm, λem = 659-685 nm) and ER-Tracker Red (λex = 587 nm, λem = 607-627 nm), Lysosome-tracker Red (λex = 577 nm; λem = 590-610 nm). Lyso-TA: λex =800 nm; λem = 490-540 nm, scale bar = 20 µm. Fluorescence imaging of lysosomal fusion, migration as well as mitophagy process. As is known, the mutual fusion and space-time distributions of lysosomes in living cells are the key indicators for diagnosis of the lysosomal storage diseases.24 In particular, the HUVCEs were easily utilized to observe the active situations of the lysosomes owing to the isolated distributions of lysosomes. As shown in Figure 3a, the HUVCEs were incubated with chloroquine (5 µM, 30 min), which could drive lysosomal migration without inducing any other apparent disturbance in the cells,25 and then were stained with the probe Lyso-TA (10 µM) for another 30 min. The “Run-and-Kiss” process of lysosomes were monitored in real-time by TPFM at random time interval, respectively. Meanwhile, the observed diameters of lysosome-1 and -2 was approximately 1.0 µm and 0.8 µm at 0 second (s) and 0.9 µm, and 0.7 µm at 68 s, respectively (Figure 3b). Then the slight movements of lysosomes were observed and unambiguously traced in HUVCEs at different time (Figure 3c). In addition, fluorescence intensity of the lysosomal fusion and movement



Figure 3 (a) The TP imaging of the HUVCEs stained with Lyso-TA (10 µM, 30 min) and stimulated using chloroquine (5 µM, 30 min) at different stimulation time (0–85 s), different color channels on behalf of different time. Lyso-TA: λex =800 nm, λem = 490540 nm. Scale bar: 20 µm (b) The simulated diagram of run-and-kiss process. (c) The simulated diagram of migration process. Num (1, 2, 3, 4, 5): lysosome-1, 2, 3, 4, 5. (d) The changes of fluorescence intensity during the fusion of lysosomes. (e) The changes of fluorescence intensity during the movement of lysosomes.

Figure 4 (a) TP fluorescence imaging of Hela cells incubated Lyso-TA (10 µM, λex =800 nm, λem = 490-540 nm) and Mito-Tracker Deep Red dye (10 µM) for different times. (b) TP fluorescence imaging incubated further rapamycin (50 µM) for different times. Scale bar: 20 µm.


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Analytical Chemistry were collected, which showed the viscosity of lysosomal fusion increased obviously (Figure 3d), while the viscosity of lysosomal migration did not show significant change (Figure 3e). As shown above, the probe Lyso-TA could realize the real-time monitor of lysosomal fusion and migration process and measure qualitatively the relationship between viscosity and probe conformational change in lysosomes, showing a great promise in applying for diagnosis of the lysosomal storage diseases. Lysosome, not only undergo fusion and migration to maintain the health, but also is responsible for scavenging various cell wastes, for instance, useless biomacromolecules, damaged and senescent organelles as well as dead cells.2 Mitophagy, the specific autophagic elimination of mitochondria, maintains efficient cellular metabolism process in which the lysosome is involved as “scavenger”.26 Therefore, the dynamic physical interactions between mitochondria and lysosomes during mitophagy were examined using our lysosomal-targeted fluorescence probe (Lyso-TA). As shown in Figure. 4a, the Hela cells were treated with Lyso-TA (10 µM) and Mito-Tracker Deep Red dye (10 µM) for different times (0-60 min), respectively. The yellow fluorescence (the merged region of lysosomes that stained green fluorescence and mitochondria that stained red fluorescence) is almost nonexistent because 1) Lyso-TA could specifically target lysosomes, 2) the dynamic physical interactions between mitochondria and lysosomes during mitophagy in living cells were unnoticeable during a short period of time. So rapamycin (50 µM) that induces obvious mitophagy by inhibiting mTOR (mammalian target of rapamycin) in living cells,27 were further

added into the Hell cell in the presence of Lyso-TA (10 µM) and Mito-Tracker Deep Red dye (10 µM). The yellow fluorescence (identified with white arrowheads) appeared in the cytoplasm at 40 min due to the fusion of damaged mitochondria and lysosomes (mitophagy) and mitophagy increased obviously with the extension of the incubation time (Figure 4b and Figure S15). Obviously, these results indicate that Lyso-TA could be uesd to monitor mitophagy, showing promise in the diagnosis of disease such as Parkinson's disease.28 Fluorescence imaging of shift from mitochondria to nucleoli. Combined with all the results, the designed Lyso-TA bioprobe could be used to real-time monitor the fusion, movement of lysosomes and unveil the mitophagy, which lured us to wonder whether we can realize the targeting of mitochondria based on the framework of Lyso-TA bioprobe. Interestingly, tertiary amine (TA) unit of Lyso-TA containing a lone pair on the nitrogen could be easily transformed into quaternary ammonium (QA), which is a well-recognized mitochondria targeting group due to the positive charge.29 Thus, Mito-QA was designed and synthesized by one-step facile nucleophilic substitution reaction with CH3I at room temperature, accomplishing switch of targeted organelles from lysosomes to mitochondria. Photochemical property tests and theoretical calculations showed that the Mito-QA also can realize “off-on” fluorescence response to conformational change, and can be the excellent probes based on sensitivity of small molecules to microenvironment (Figure S16-20, Table S2). The subcellular localization experiments of Mito-QA

Figure 5 (a) Two-photon colocalization imaging of Hela cells stained with Mito-QA (20 µM) and Mito-Tracker Deep Red dye (20 µM) for 30 min at 37 oC. (b) The TP imaging of HeLa cells incubated in the absence or presence of 10 μM CCCP or H2O2, then stained with Mito-QA (20 μM). (c) The structure of probe Mito-QA and the schematic illustration of Mito-QA that was released from mitochondria and migrated to the nucleoli. Mito-QA: λex = 780 nm, λem = 480-530 nm, Mito-Tracker Deep Red dye (λex = 644 nm, λem = 658-678 nm), scale bar = 20 µm.



(20 µM) with Mito-tracker Deep Red (10 µM) manifested the Mito-QA could accumulate on the mitochondria with the high pearson correlation factors (Pr = 0.9224) as shown in Figure 5a. As is well known, mitochondria are the principal energyproducing compartments in cells which play essential roles in transmitting cellular energy and signals, and maintaining the cellular redox homeostasis, etc.30 However, the dysfunctional mitochondria could induce diverse diseases, such as neurodegenerative diseases, cardiovascular diseases and atherosclerosis.31 Therefore the visualization of mitochondrial dysfunction by TPFM was investigated. As shown in Figure 5b, the carbonyl cyanide m-chlorophenyl-hydrazone (CCCP, 10 μM, 20 min) and H2O2 were added to the Hela cells, respectively, which resulted in mitochondrial dysfunction. Afterwards, the cells were stained with the probe Mito-QA (20 µM) for 30 min, of which the TP imaging were displayed. Interestingly, with the addition of CCCP and H2O2, the dysfunctional mitochondria caused by abnormal mitochondrial membrane potential and mitochondrial oxidative damage, Mito-QA was released from mitochondria and migrated to the nucleoli with green emission. In order to explore whether the migration of Mito-QA and the mitophagy process occur simultaneously, the migration of Mito-QA with adding the lysosomal commercial dyes to observe the movement of lysosomes were tested. The Hela cells were treated with MitoQA (20 µM), Lysosome-tracker Red (10 µM) for 30 min, and further treated with CCCP (50 µM) or H2O2 (200 µM) just right before the observation. As shown in Figure S21. it was clearly recorded that the gradual disappearance of fluorescence intensity in mitochondria was accompanied by a gradual increasing fluorescence intensity of the nucleoli throughout the entire process for 100 s, meanwhile the mitophagy process was not observed during the migration of Mito-QA, which maybe because Mito-QA was released from mitochondria and then migrated to the nucleoli fast. The Hela cells were further treated H2O2 (200 µM), which showed the similar fluorescence imaging (Figure S22). These results confirmed that Mito-QA could serve a valid tool for visualizing the dysfunctional mitochondria through shift from mitochondria to nucleoli and the mitophagy process not occur simultaneously (Figure 5c). Two-photon fluorescence imaging in Brain Tissues and Zebrafishes. In light of the excellent features of TP excitation of the probes, the viscosity environment in mouse brain tissues stained with the probes Lyso-TA and Mito-QA were further carried out via TPFM, respectively. As shown in Figure 6a-d and Figure S23, the tissues treated with Lyso-TA (20 µM, 30 min) and Mito-QA (30 µM, 40 min) exhibited bright green fluorescence. Moreover, the experiments of the tissues showed obvious fluorescence at different tissue depth (0-100 µm) utilizing the z-scan mode of TPFM, which elucidated the probes could image viscosity in deep-tissues by TPFM. Furthermore, the abilities of the probes Lyso-TA and MitoQA for the visualization of viscosity changes were further investigated in the 4-day-old zebrafishs larvae by TPFM. As shown in Figure 6e-h, the zebrafishs treated with the probe Lyso-TA (10 µM, 60 min) exhibited the weak fluorescence. However, with the additional treatment with the high concentrated sucrose solution (90 mM) for another 30 min, an apparent fluorescence enhancement could be observed. Similarly, as shown in Figure 6i-l, zebrafishs pretreated with the probe Mito-QA (20 µM, 60 min) were further treated with nystatin (10 µM) for 30 min, which showed a strong fluorescence. The phenomena was consistent with the

observations in living cells, which further confirmed that Lyso-TA and Mito-QA were viscosity-induced conformational responsive fluorescence probes.

Figure 6 (a-b) The 3D TPFM of brain slides incubated with the probes Lyso-TA (20 µM) for 30 min, depth: 0-110 µm. (cd) The 3D TP imaging of brain slides incubated with the probes Mito-QA (30 µM) for 40 min, depth: 0-110 µm. (e-l) The TP imaging of the living 4-day-old zebrafishs larvae. (e,i) Contrast; (f) Zebrafishs were treated with Lyso-TA (10 µM) for 60 min; (g) Zebrafishs were treated with Lyso-TA (10 µM) for 60 min, then treated with the high concentration sucrose solution (90 mM) for 30 min; (h) fluorescence intensity quantification. (j) Zebrafishs were treated with Mito-QA (20 µM) for 60 min; (k) Zebrafishs were treated with Mito-QA (20 µM, 60 min) + nystatin (10 µM, 30 min) (l) fluorescence intensity quantification. Lyso-TA: λex =800 nm; λem = 490-540 nm. Mito-QA: λex =780 nm; λem = 480-530 nm. Scale bar: 500 μm

CONCLUSION In summary, two conformationally induced “off-on” responsive TP bioprobes (Lyso-TA and Mito-QA) with excellent specificity were developed. The probes not only dynamically track lysosomes and mitochondria, respectively, but visually image of the synergism between lysosomes and mitochondria. Lyso-TA realized the real-time monitor of lysosomal fusion and migration process, as well as mitophagy process in which the lysosome as for “scavenger” is involved, and Mito-QA monitored dynamic changes from mitochondria to nucleoli and mitochondrial dysfunction by TPFM. Meanwhile, Lyso-TA could measure qualitatively the relationship between viscosity and probe conformational change in lysosomes, The observed diameters of lysosomes were approximately 1.0 µm in HUVCEs. Furthermore, both of two small bioprobes were applied in vivo bio-imaging of brain tissues and zebrafishes. The above results indicated that the designed probes witness great advances for biological diagnosis and bio-imaging.

AUTHOR INFORMATION Corresponding Author * Prof. Hongping Zhou, Email: [email protected] * Xiaojiao Zhu, Email: [email protected]

Author Contributions ‡ Huihui Zhang and XiaoJiao Zhu contributed equally. All authors have given approval to the final version of the manuscript.


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Analytical Chemistry Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (51772002, 51432001 and 21805001), Science and Technology Plan of Anhui Province (1604b0602016), Natural Science Fund of Education Department of Anhui province (KJ2018A0024). Changjiang Scholars and Innovative Research Team in University, the Undergraduate Training Program for Innovation and Entrepreneurship of Anhui University (201610357007). Educational Commission of Anhui Province (KJ2018A0024, KJ2016JD14), Anhui Province Postdoctoral Science Foundation (2017B159).

ASSOCIATED CONTENT Supporting Information Experimental section, synthesis, theoretical calculations, photonphysical properties, standard MTT assays, photo-stability experiments, typical fluorescence images.

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