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Interface-targeting strategy enables two-photon fluorescent lipid droplets probes for high-fidelity imaging turbid tissues and detecting fatty liver Lifang Guo, Minggang Tian, Ruiqing Feng, Ge Zhang, Ruoyao Zhang, Xuechen Li, Zhiqiang Liu, Xiuquan He, Jing Zhi Sun, and Xiaoqiang Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00278 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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ACS Applied Materials & Interfaces

Interface-targeting strategy enables two-photon fluorescent lipid droplets probes for high-fidelity imaging turbid tissues and detecting fatty liver Lifang Guo,† Minggang Tian,† Ruiqing Feng,† Ge Zhang,† Ruoyao Zhang,† Xuechen Li,† Zhiqiang Liu,*† Xiuquan He,*§Jing Zhi Sun,*# and Xiaoqiang Yu*† †

Center of Bio & Micro/Nano Functional Materials, State Key Laboratory of Crystal Materials, Shandong University. Jinan 250100, P. R. China, email: [email protected] and [email protected]. §

Department of Anatomy, Shandong University School of Medicine, Jinan 250012, P.R. China. [email protected] MoE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University. Hangzhou 310027, P. R. China. Email: [email protected]. #

ABSTRACT: Lipid droplets (LDs) with own interface architecture not only play crucial roles in protecting cell from lipotoxicity and lipoapoptosis but also closely relate with many diseases such as fatty liver and diabetes. Thus, as one of important applied biomaterials, fluorescent probes with ultrahigh selectivity for in-situ and high-fidelity imaging LDs in living cells and tissues are critical to elucidate relevant physiological and pathological events as well as detect related diseases. However, available LDs probes only utilizing their waterless neutral cores but ignoring the unique phospholipid monolayer interfaces exhibit low selectivity. Particularly, current probes cannot discriminate neutral cores of LDs from intracellular other lipophilic microenvironments, so that extensively cloud-like background fluorescence diffusing in cytoplasm, which severely limited their applications. Herein, in order to design LDs probes with ultrahigh selectivity, the exceptionally interfacial architecture of LDs is considered adequately and thus an interface-targeting strategy is proposed for the first time. According to the novel strategy, we have developed two amphipathic fluorescent probes (N-Cy and N-Py) by introducing different cations into a lipophilic fluorophore (NBD). Consequently, their cationic moiety precisely locates the interfaces through electrostatic interaction and simultaneously NBD entirely embeds into the waterless core via hydrophobic interaction. Thus high-fidelity and background-free fluorescence imaging of LDs is expectably realized in living cells in-situ. Moreover, LDs in turbid tissues like skeletal muscle slices have been clearly imaged (up to 82 µm depth) in two-photon microscope. Importantly, using N-Cy, we not only intuitively monitored the variations of LDs in number, size and morphology, but also clearly revealed their abnormity in hepatic tissues resulted from fatty liver. Therefore, these unique probes provide excellent imaging tools for elucidating LDs-related physiological and pathological processes, and the interface-targeting strategy possesses universal significance for designing probes with ultrahigh selectivity. Key words: Lipid droplets (LDs), Interface-targeting, Amphipathic probes, Ultrahigh selectivity, Two-photon, turbid tissue imaging, Fatty liver detection.

pids is the most prominent component in LDs6, various lipophilic fluorescent dyes have been developed to image LDs. Nile Red, as the first lipophilic probe, can preferentially accumulate in LDs with highly lipophilicity.7,8 But simultaneously it stains intracellular other lipophilic regions, causing severe background noise. In order to increase the selectivity and remove the background noise, BODIPY with higher lipophilicity than Nile Red is employed.9 However, the selectivity of BODIPY is still insufficient for many biological researches.10,11 Additionally, various LDs probes with lipophilicity have been delivered recently, which however still cannot address the concerns well.12-18 This is mainly because the nuance in lipophilicity between LDs and intracellular lipophilic environments is hard to discriminate. To break this bottleneck, exploring probes ultrasensitive to this nuance may be a possi-

INTRODUCTION Lipid droplets (LDs), long unappreciated as simple and inert globules for neutral lipids storage, are earning increasing recognition as complex and dynamic organelles.1,2 They involve in various physiological processes, such as regulating cellular energy homeostasis, protecting cells from lipotoxicity and lipoapoptosis, even possibly functionally interacting with other organelles.1,3,4 Moreover, their abnormality is a critical hallmark of related diseases, including fatty liver, type II diabetes, atherosclerosis and obesity.3-5 Therefore, to in-situ reveal LDs’ biological roles and the impact of various biological events on LDs, many efforts have been contributed to exploiting fluorescent probes for selectively imaging LDs. Based on the fact that a lipophilic and waterless core rich in neutral li-

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reported rarely23. Delightfully, N-Cy had appropriate TPEF properties and could clearly image LDs in turbid skeletal muscle tissues (up to 82 µm depth) in a two-photon microscope (TPM). (3) More importantly, using N-Cy, the abnormity of LDs in size, number and volume in hepatic tissues were unequivocally revealed and fatty liver could be clearly detected under TPM. Therefore, amphipathic probes (N-Cy and N-Py) could serve as powerful tools for revealing LDs-related biological events and importantly the interface-targeting strategy is valuable for designing probes with ultrahigh selectivity.

ble way, but comparatively, it is more feasible and necessary to develop novel fluorescent probes based on new mechanisms by fully utilizing LDs’ unique biological structural and physicochemical properties inherently different from all other intracellular organelles and lipophilic microenvironments. According to recent reports, The structural character of LDs is that the lipophilic and waterless core is exactly encased by an phospholipid monolayer.1,2 Moreover, to maintain the stability of this structure, water is efficiently excluded from lipophilic cores of LDs.19 Particularly, according to Thiam19 and Beller20, different from phospholipid bilayer of other intracellular membranous organelles such as mitochondria and lysosomes, the long alkyl chains of phospholipid constituting monolayer of LDs embed into waterless cores of LDs. And thus, a LD is actually a particularly amphipathic architecture, with negatively charged polar shell and entirely hydrophobic core, which is exceptional among all known organelles.20,21 Inspired by the exceptionally interfacial architecture of LDs, an interface-targeting strategy is proposed for the first time. According to the novel strategy, we design two amphipathic probes (N-Cy and N-Py) by introducing different cations into a lipophilic fluorophore (NBD) and a comparative lipophilic molecule (N-C6). As shown in Scheme 1, for N-Cy and N-Py, NBD part can totally embed into the waterless core of LDs via strong hydrophobic interaction, and simultaneously the additional cationic moiety can precisely locate at the polar shell of phospholipid monolayer through electrostatic interaction. The interface-targeting model endows the two amphipathic probes with ultrahigh selectivity to LDs. At the same time, the selectivity of N-Cy and N-Py has been confirmed by multiple methods: (1) The fluorescent entities stained by N-Cy and NPy were spherical and situated in cytoplasm, which coincide with the morphological features of LDs1,2,12; while in microscopic photos from N-C6 and Nile Red, cloud-like background signal widely existed in cytoplasm although spherical objects could be observed. (2) Further, all fluorescent dots stained by N-Cy and N-Py can overlap perfectly with the intracellular black dots representing LDs with higher refractive index in phase contrast microscopic photos1,7; comparatively, in cells dyed with N-C6 and Nile Red, cloud-like background signal could not overlap the intracellular black dots. (3) The in-situ fluorescence spectra of N-Cy and N-Py in the intracellular spherical dots were identical with that in sunflower seed oil (abbreviated as Oil, a reagent usually used to simulate the environment inside LDs21) and low polar 1,4-Dioxane, indicating that the staining location should be LDs with highly lipophilic cores; however, for N-C6 and Nile Red, the in-situ spectra in spherical dots were apparently different from that in cloud-like background regions, moreover only the former were identical with ones in Oil and 1,4-Diox. (4) After removing LDs with nonpolar xylene7,22, we found that fluorescent dots stained by N-Cy and N-Py also correspondingly disappeared, which powerfully demonstrated their specificity to LDs; but to N-C6 and Nile Red, cloud-like background noise still presented in cytoplasm after LDs were washed out. Upon the ultrahigh selectivity of such amphipathic probes, three important developments have been obtained: (1) the LDs in living cells have been high-fidelity imaged without background noise. (2) As known well, it is very important to image living thick and turbid tissue using a fluorophore with twophoton exciting properties, but, so far, relevant research is

Scheme 1 Schematic illustration of amphipathic probes (NCy and N-Py) targeting LDs with ultrahigh selectivity.

EXPERIMENTAL SECTION Apparatus and general methods. The UV-visible-near-IR absorption spectra of dilute solutions were recorded on a Hitachi U2910 spectrophotometer using a quartz cuvette of 1 cm path length. Fluorescence spectra were obtained on a HITACH F-2700 spectrofluorimeter equipped with a 450-W Xe lamp. The doublestranded DNA-specific dye Hoechst 33342, dead cell stain SYTOX Blue nucleic acid stain (S-11348), Nile red and Mito Tracker Deep Red FM (MTDR) were purchased from Molecular Probes. Viability of the cells was assayed using cell proliferation Kit I with the absorbance of 450 nm being detected using a PerkinElmer Victor plate reader. Calculation methods. The geometrically optimized structure and the frontier orbitals of the probe molecules were calculated with Gaussian 09 package. Chemical structures were optimized sequentially with the basic set of PM3, B3LYP/3-21g, B3LYP/631g, and cam-B3LYP/TZVP. The frontier molecular orbitals were obtained via TD-DFT calculation of the single point of the optimized structure on the basic set of cam-B3LYP/TZVP TD (nstate = 10, root = 1). Cell culture and staining. HeLa cells were cultured in HDMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Mesenchymal stem cells (MSC) were grown in alpha-MEM supplemented with 10% FBS and 1% penicillin and streptomycin. PC-3 cells were cultured in HDMEM supplemented with 10% FBS. All above cells were cultured in a 5% CO2 incubator at 37 oC. N-Cy, N-Py, N-C6, Nile red, Hoechst 33342 and S-11348 were dissolved in DMSO at a concentration of 1 mM and MTDR were prepared as 0.1 mM in DMSO. All cells were placed on glass coverslips and allowed to adhere for 24 h. For cells staining experiments, cells were treated with N-Cy (4 µM, 2 min), N-Py (10 µM, 30 min), N-C6 (4 µM, 5 min) or Nile red (4 µM, 20 min). For co-staining experiments, living cells were treated with Hoechst 33342 (5 µM, 30 min) or S11348 (5 µM, 30 min) or MDTR (200 nM, 20 min) followed by rinsed with PBS twice, then stained with N-Cy (4 µM, 2 min), NPy (10 µM, 30 min), N-C6 (4 µM, 2 min) or Nile red (4 µM, 20

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min). Then washing with PBS twice, the cells were observed under fluorescence microscopy.

Immunofluorescence staining. Hela cells were cultured on glass coverslips for 24 h, and then pre-treated with 40 µM oleic acid for 4h. Next, cells were fixed with 4% formaldehyde for 30 min and permeabilized with saponin and blocked with 10% goat serum. After this, primary (anti-human TIP47 rabbit polyclonal antiserum, abbreviated as anti-TIP47 antiserum) and secondary (Alexa Fluor 594-conjugated goat anti-rabbit IgG) antibodies were added sequentially to the cells. Finally, the cells were stained with 4 µM N-Cy for 2min. Images from the samples were obtained using a Zeiss LSM 780 confocal microscope.

Tissue extraction and staining. The mouse muscular and hepatic tissue slices or blocks were directly removed from just killed adult wistar mouse, and incubated at room temperature in HDMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. For tissue slices staining experiments, skeletal muscle tissue slices were treated with N-Cy (4 µM, 2 min). For tissue slices co-staining experiments, hepatic tissue slices were treated with Hoechst 33342 (5 µM, 30 min) followed by rinsed with PBS twice, and then treated with N-Cy (4 µM, 2 min). For tissue blocks staining, tissue blocks were treated with N-Cy (10 µM, 2 min). Then in absence of a washing step, these tissues were directly observed under fluorescence microscopy. All above experiments were performed according to the international, national and institutional rules considering animal experiments, clinical studies and biodiversity rights.

RESULTS AND DISCUSSION Synthesis and characterization of N-Cy, N-Py and N-C6. The synthetic routes of N-Cy, N-Py and N-C6 were showed in Scheme 2A. Their structural characterizations were Figure S1Figure S13. Scheme 2 The synthesis of N-Cy, N-Py and N-C6 (A) as well as frontier molecular orbitals of N-Cy (B).

The procedure of removing LDs in living cells and tissues. Living cells and tissues were firstly fixed with paraformaldehyde for 30 min, and then rinsed with PBS twice. After that, these biosamples were successively treated with the alcohol of different concentration gradient (70%, 80%, 90%, 95% and 100%) for 5 min and xylene for 5 min twice, then washed by the opposite different concentration gradient of alcohol for 5 min and PBS for two times. Finally, LDs were completely removed. Cell-viability assay. The study of the effect of N-Cy on viability of cells was carried out using Cell Counting Kit-8 (CCK-8), purchased from Dojindo. HeLa cells growing in log phase were seeded into 96-well plates (ca. 1 × 104 cells/well) and allowed to adhere for 24 h. N-Cy (200 µL/well) at concentration of 4 µM was added into the wells of the treatment group, and 200 µL/well DMSO diluted in DMEM at final concentration of 0.2% to the negative control group, respectively. The cells were incubated for 2, 10, and 24 h at 37 oC under 5% CO2, then 10 µL of CCK-8 was added into each well. Place it in the incubator for 1 h, and then measure the absorbance at 450 nm with a microplate reader. The cell survival rate can be calculated using the following equation:

Optical properties. The optical properties of three compounds have been shown in Figure 1, Figure S14, Figure S15and Table S1. In Figure 1A, the absorption spectra of NCy had little change in solvents with different polarity, but fluorescence intensity (Figure 1B) and quantum yield (Φ) (Table S1) were very sensitive to the polarity of solvents. With increasing the polarity of solvents, its fluorescence intensity dramatically decreased and the Φ value in low polar solvent MeCN reached 13~75 folds of that in solvents with higher polarity (Table S1). Meanwhile, the emissive spectra redshifted from 510 nm to 535 nm, as shown in Figure 1C. These phenomena could be well explained by the intramolecular charge transfer (ICT) principle.24 To illustrate this effect, the frontier orbitals have been calculated with Gaussian 09 package.25 According to the calculation results, the oscillator strength (f) for the electron transition between HOMO and LUMO is 0.2704, and the transition between the two orbitals is responsible for the absorption and emission behaviors of NCy. In Scheme 2B, according to the molecular orbitals, the imino group serves as strong electron donor and nitro severs as electron acceptor in the fluorophore structure, which contributes to transferring electron from imino group to nitro in the excited state. Moreover, in Figure 1D, the two-photon excitation fluorescence (TPEF) properties of N-Cy excited by 840 nm exhibited also ICT characteristics similar to one-photon

Survival rate = (ASample - ADMSO)/( ASample -ABlank) Fluorescence imaging. Confocal fluorescence imaging was obtained with Olympus FV 1200 laser confocal microscope. In twophoton experiments, excitation wavelength was 840 nm 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. In-situ spectra acquirement. The in-situ emission spectra inside cells were obtained by means of the spectral imaging function of Olympus FV1200 confocal microscope. With the excitation by 473 nm, a series of fluorescent images were acquired sequentially with the emission wavelength range of 10 nm. Dealing with these fluorescent images by pixels, the in-situ emission spectra can be finally obtained with the wavelength gap of 10 nm. Antibodies. The anti-human TIP47 rabbit polyclonal antiserum was purchased from Affinity Biotech and Alexa Fluor 594conjugated goat anti-rabbit IgG was purchased from proteintech group.

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excitation fluorescence (OPEF). Furthermore, because N-Py and N-C6 utilized the same fluorophore (NBD) with N-Cy, thus their fluorescence also exhibited ICT features as displayed in Figure S14, Figure S15, Figure S16. In addition, the fluorescence of Nile Red also showed ICT features (Figure S17). Therefore, the four compounds had the potential for distinguishing intracellular areas with different polarities by means of fluorescence spectra. In addition, the fluorescence responses of three probes (N-Cy, N-Py and N-C6) to various pH also have been measured. As shown in Figure S18, their absorption (1) and emission spectra (2) all did not show significant change, and meanwhile the total fluorescence intensity only showed slight fluctuation in different pH media (3), which fully implied that the three probes were not affected by pH.

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other cells were intact. When excited by 473 nm, all cells emitted green fluorescence in (3) of Figure S20A, indicating that N-Cy had perfect membrane permeability to both living and dead cells. The same results also appeared in the costaining experiments of S-11348 and N-Py as well as N-C6 in Figure S20B and Figure S20C, implying that the two probes also could stain living cells. In-situ and high-fidelity imaging of lipid droplets achieved by amphipathic probes (N-Cy and N-Py). As immortalized model cells, HeLa cells are often used to detect the imaging ability of a fluorescent probe.26,27 Herein, living HeLa cells were also used to check the imaging performance of NCy and N-Py as well as N-C6 and Nile Red. In (1) of Figure 2, the DIC photos of cells incubated by the four dyes were shown respectively. To definitely indicate spatial position of the cells, their cell nuclei were labeled by Hoechst 33342 with blue fluorescence as shown in (2) of Figure 2. In (3) of Figure 2A and Figure 2B, these intracellular entities stained by N-Cy and NPy presented round “droplets”, and all “droplets” distributed randomly in cells and did not contacted one another. In a cell, these “droplets” formed a natural population and every “droplet” in this population exhibited itself individually. Although the sizes of these “droplets” were different, a diameter of 1 µm was their classical size which corresponded to the size character of LDs. According to the related literatures5,6, the diameters of LDs are between 0.1 µm and 100 µm in different cell types. Furthermore, the merged image in (4) of Figure 2A and Figure 2B demonstrated that these “droplets” all dispersed in cytoplasm, and there were not fluorescent “droplets” in extracellular regions as well as nucleic space. Taken together, the morphology, size and distribution of these “droplets” stained by N-Cy and N-Py were all in line with the characteristics of LDs reported in many related researches1,2,5,6,11. For cells stained with N-C6 and Nile Red in (3) in Figure 2C and Figure 2D, although many fluorescent “droplets” also corresponded to the characteristics of LDs including morphology, size and distribution, they were closely surrounded by non-circular and cloud-like background signal dispersing in cytoplasm as shown in (4) in Figure 2C and Figure 2D. Especially in areas with strong background noise, some LD could not exhibit itself individually and presented as a blurred mass, which severely interfered the observation of LDs. Therefore, N-Cy and N-Py could label LDs with ultrahigh selectivity, but N-C6 and Nile Red could not. To further analyze the imaging results, we randomly magnified a small part in (4) of Figure 2 and the magnified images were shown in (5), (6) and (7) of Figure 2. In generally, LDs presented black spots in DIC images in a DIC due to their higher refractive index than intracellular other structures1,7. Thus, black dots shown in (5) of Figure 2 should be LDs. In (7) of Figure 2A and Figure 2B, the fact that the green fluorescent dots from N-Cy and N-Py point by point respectively merged with these black dots indicated the specificity of N-Cy and N-Py to LDs. In contrast, in cells stained with N-C6 or Nile red shown in (5-7) of Figure 2C and Figure 2D, the black dots in DIC could be hazily covered by the fluorescent dots, but there existed serious background fluorescence signals.

Figure 1. Absorption spectra (A), actual (B) and normalized (C) OPEF spectra of N-Cy in solvents with different polarity. Insert in B: Photos of N-Cy in solvents (from 1 to 6: MeCN, Ace, MeOH, DMF, DMSO, H2O) under a hand-held UV lamp (λex: 365 nm). (D) TPEF spectra of N-Cy in solvents with different polarity. Concentration: 10 µM; λex (OPEF) = 473 nm; λex (TPEF) = 840 nm.

Cytotoxicity and the ability of staining living cells. Firstly, the cytotoxicity of the three proebs (N-Cy, N-Py and N-C6) was assessed with Cell Counting Kit-8 (CCK-8, a commercial reagent for determining the cell viability). As depicted in Figure S19A, the viability of HeLa cells after cultured with 4 µM N-Cy and N-Py for 2 h, 10 h and 24 h held above 92%, indicating their negligible damage to living cells. Further, the influence of different concentrations of N-Cy and N-Py to HeLa cells also have been tested in Figure S19B, and the results showed above 90% viability. In comparison, N-C6 displayed slightly higher toxicity when cells were incubated with larger concentrations (10 µM) or for longer time (24 h). However, when stained with low concentration (4 µM) within 2 h, the cells kept 92% viability, indicating that N-C6 also could be used in living cells. In addition, their ability of staining living cells were further evaluated. For this, SYTOX Blue (S-11348), a cell-impermeant nucleic acid dye, was used to testify whether the three probes (N-Cy, N-Py and N-C6) could stain living cells with intact plasma membrane. There were five cells in (1) of Figure S20A. When excited by 405 nm, two cells emitted blue fluorescence and others did not show any fluorescence signal as shown in (2) of Figure S20A, which indicated that plasma membrane of two cells were broken and that of

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Figure 2. DIC, fluorescence and merged images of HeLa cells stained by corresponding probes and Hoechst 33342 as well as intracellular in-situ fluorescence spectra of probes (A: N-Cy, B: N-Py, C: N-C6 and D: Nile Red). (1): DIC images; (2): Confocal fluorescence images of Hoechst 33342; (3): Confocal fluorescence images of every corresponding probe; (4): Merged images of (1), (2) and (3); (5): Amplified DIC images of yellow rectangular areas in (4); (6): Amplified fluorescence images of yellow rectangular areas in (4); (7): Merged images of (5) and (6); (8): The in-situ spectra of corresponding probes in the circle areas with digital annotation in (6). Hoechst 33342: 5 µM, 30 min; λex = 405 nm, λem = 410-440 nm. N-Cy: 4 µM, 2 min; λex = 473 nm, λem = 510-540 nm. N-Py: 10 µM, 30 min; λex = 473 nm, λem = 510-540 nm. N-C6: 4 µM, 5 min; λex = 473 nm, λem = 510-540 nm. Nile Red: 4 µM, 20 min; λex = 473 nm, λem = 550-650 nm. Bar (1 - 4): 20 µm, Bar (5 - 7): 1µm.

Investigation of LDs’ polarity in vivo and in vitro. In the same cell type, sizes of LDs may be different, but their chemical constitution remains highly identical1,20. Thereby, for probes with specificity to LDs, their emission profile of in-situ fluorescence spectra collected in multiple points should remain unanimous, which representing the polarity of LDs. Conversely, if the in-situ fluorescence spectra exhibit different emission profiles, they respectively represent the polarity of LDs and other intracellular regions. Given that the polarity of LDs is lower than intracellular other regions, the emissive peaks of probes in LDs should blue-shift and the intensities enhance compared to that in other regions. Therefore, to investigate the polarity of LDs, in-situ fluorescence spectra of N-Cy, N-Py, N-C6 and Nile Red at intracellular different regions with digital annotation in (6) of Figure 2 were collected. In (8) of Figure 2A, the in-situ spectral profiles of N-Cy in the areas of white circles ((6) of Figure 2A) kept coincident and all peaks were at 525 nm. Meanwhile, N-Py showed similar results and the fluorescence peaks were at 530 nm in (8) of Figure 2B. The results above indicated that LDs has identical polarity in cells. Comparatively, in (8) of Figure 2C, the insitu spectral profiles and peaks of N-C6 in the white circle areas (510 nm) were obvious different from that in the pink circle areas (530 nm). Very clearly, the fluorescent intensities in the former were higher than the latter. Similarly, as shown in (8) of Figure 2D, the fluorescent peaks of Nile Red in white regions and pink regions were respectively at 570 nm and 620

nm, and intensities in white regions were also higher. Since the photophysical properties of N-C6 and Nile Red conform to ICT mechanism, thus the red-shift of peak and decrease of fluorescence intensity of N-C6 and Nile Red in the pink areas indicated that these regions had higher polarity than the LDs.

Figure 3. The emission spectra of probes (A: N-Cy, B: N-Py, C: N-C6, D: Nile Red) in different environments under same confocal microscope. LDs dots: intracellular LDs; Oil: sunflower seed oil; 1,4-Diox: 1,4-dioxane; Other regions: intracellular other regions; Mixed solvents: 1,4-Diox/water (V/V = 8:2). λex = 473 nm.

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Figure 4. Confocal fluorescence images of HeLa cells stained with Hoechst 33342 and N-Cy (A), N-Py (B), N-C6 (C) and Nile Red (D) after treated by different ways: (1) - (4) The cells were fixed; (5) - (8) The cells were firstly fixed, and then LDs were removed with xylene. (1) and (5): DIC; (2) and (6): Hoechst 33342; (3) and (7): Corresponding probes; (4): Merged images of (2) and (3); (8): Merged images of (6) and (7). (3a) and (7a): The in-situ spectra of corresponding probes in the circle areas with digital annotation in (3) and (7), respectively. Hoechst 33342: 5 µM, 30 min; λex = 405 nm, λem = 410-440 nm. N-Cy: 4 µM, 2 min; λex = 473 nm, λem = 510-540 nm. N-Py: 10 µM, 30 min; λex = 473 nm, λem = 510-540 nm. N-C6：4 µM, 5 min; λex = 473 nm, λem = 510-540 nm. Nile Red: 4 µM, 20 min; λex = 473 nm, λem = 550-650 nm. Bar = 20 µm.

As is known, sunflower seed oil (abbreviated as Oil) with similar polarity to LDs is often used to simulate internal environment of LDs21. Thereby, the fluorescence spectra of the four probes in Oil were collected under the confocal microscope, as shown in Figure 3, Figure S21, and Table S2. Obviously, the fluorescence spectra of four probes (A: N-Cy, B: NPy, C: N-C6, D: Nile Red) in Oil were very similar to their respective in-situ spectra in intracellular LDs. The results indicated that the polarity of LDs was very low, therefore we next used 1,4-dioxane with very low polarity to simulate internal environment of LDs. Meanwhile, considering higher polarity of intracellular other regions stained by N-C6 and Nile Red, various 1,4-Diox-H2O mixtures with different polarities were

also used to simulate these regions. As concluded in Table S2, the polarity of LDs was very similar to 1,4-Diox. While the polarity of intracellular other regions was similar to the mixture of 1,4-Diox-H2O with 20% volume of water. These in vitro results not only revealed that LDs possessed lower polarity than intracellular other regions, but also confirmed that NCy and N-Py could exclusively recognize low polar LDs while N-C6 and that Nile Red could not discriminate low polar LDs from intracellular other regions with higher polarity. Fully demonstrating the specificity of amphipathic probes with a novel chemical method of removing LDs. To confirm the specificity of N-Cy and N-Py, we extracted intracellular LDs and then contrasted the staining results of four

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ACS Applied Materials & Interfaces circle regions were respective at 510 nm and 530 nm, and intensities in the former were also stronger than the latter. Similarly, (3a) of Figure 4D showed that the fluorescence peaks of Nile Red in pink regions had an apparent red-shift from 570 nm to 620 nm compared with the white regions, and intensities in the former were lower than that in the latter. Thereby, N-C6 and Nile Red could not specifically stain LDs in fixed cells. Further, after treated with xylene, although LDs have been removed, intracellular other regions stained by N-C6 or Nile Red before still existed, as shown in (7) and (8) of Figure 4C and Figure 4D. The phenomenon above about Nile Red conformed well to the previous report7. In addition, (7a) of Figure 4C and Figure 4D showed that the in-situ spectra profiles of N-C6 and Nile Red respectively remained the red-shifted emissions pecked at 530 nm and 620 nm. All results above fully demonstrated that N-Cy and N-Py were specific to LDs while N-C6 and Nile Red not. Notably, tomography enables the deep observation of the internal structures of biological samples, and thus has become a significant tool for researching biological targets. Herein, we selected N-Cy as a representative to investigate the morphology and distribution of LDs in a whole cell by tomography. As shown in Video S1, the tomography pictures at different depth of the cell showed that LDs presented as “droplets” and mainly distributed in cytoplasm, which agreed to the previous reported1,2,11 and also demonstrated the specificity of N-Cy to LDs. Demonstrating the LDs identity of amphipathic probes based on biological methods. We selected one of amphipathic probes (N-Cy) as a representative to examine the LD identity by colocalization experiments with various intracellular components. Firstly, cationic dyes are usually known to target mitochondria due to large mitochondrial membrane potential.28 Given that amphipathic probes (N-Cy and N-Py) contain cationic moieties, it is essential to eliminate the possibility of staining mitochondria. Therefore, we chose N-Cy to stain with Mito Tracker Deep Red FM (MTDR), a commercial mitochondria probe. As shown in Figure 5, the cell nucleus, LDs and mitochondria were labeled by blue fluorescence from Hoechst 33342, green from N-Cy and red from MTDR, respectively. From morphology, LDs and mitochondria were different. LDs presented round “droplets” and mainly distributed around the nucleus, while mitochondria exhibited filaments shape and distributed throughout the cytoplasm. Moreover, the co-location coefficient of N-Cy and MTDR was very low (only 30%), which declared that N-Cy could not stain mitochondria. In fact, another vital factor affecting the location of a cationic probe is its hydrophilicity-lipophilicity (parameterized by logP).29,30 The greater the value of logP, the stronger lipopgilicty. For mitochondrial staining, logP value of probes should distribute in the range of 0～5, while for LDs staining, the corresponding probes request a higher logP in range of 5.5-7.4. 29,30 The logP of N-Cy was 6.243 falling in the scope of LDs staining, which indicated N-Cy had stronger binding force with LDs than mitochondrial. Hence, it is reasonable for N-Cy to selectively target LDs. Secondly, substantial studies have suggested that LDs stem from endoplasmic reticulum (ER)20. Thus it is also necessary to investigate the possibility of amphipathic probes targeting ER. Herein, N-Cy has been chosen to co-stain with ERTracker red (abbreviated as ER red, a commercial ER probe), and the results were showed in Figure S23 (1-6). LDs stained

Figure 5. Confocal fluorescence images of HeLa cells stained with Hoechst 33342, N-Cy and MTDR. (1): DIC; (2): Hoechst 33342; (3): N-Cy; (4): MTDR; (5): Merged image of (2), (3) and (4); (6): Merged image of (1) - (4). Hoechst 33342: 5 µM, 30 min; λex = 405 nm, λem = 410-440 nm; N-Cy: 4 µM, 2 min; λex = 473 nm, λem = 510-540 nm. MTDR: 200 nM, 20 min; λex = 633 nm; λem = 650-700 nm. Bar = 20 µm.

Figure 6. Illustration of chemical structure of lipophilic probes (1) and amphipathic probes (2) as well as their bioimaging results.

probes in normal and treated cells. According to related reports7,22, some nonpolar solvents can efficiently remove intracellular LDs with low polarity. Thus, if a probe is specific to LDs, there will be not its fluorescence signal in the cells which LDs have been extracted. Conversely, if a probe is not specific to LDs, its fluorescence signal will still be observed in intracellular other regions even if LDs have been removed. Considering that this experiments need to be performed in fixed cells, the ability of these probes to stain LDs in fixed cells should be investigated. As shown in (1-4) of Figure. 4A, LDs in the fixed HeLa cells could be exclusively stained by N-Cy. Furthermore, in (3a) of Figure 4A, the in-situ spectra profiles of N-Cy collected in white circles ( (3) of Figure 4A) kept identical, and all fluorescence peaks were all at 530 nm which were accordant with the in-situ spectra profiles of LDs in living cells as shown in Table S2. The results manifested that NCy also specifically stain LDs in fixed cells. After treated with xylene (a nonpolar solvent), LDs disappeared and no fluorescence could be observed as shown in (7) of Figure 4A. Meanwhile, (7a) of Figure 4A implied that no any fluorescence signal could be detected. The results above manifested that N-Cy could specifically stain LDs. Meanwhile, the same conclusions with respect to N-Py were also drawn from Figure 4B. Comparatively, N-C6 and Nile Red could not discriminate LDs from intracellular other regions in fixed cells, as shown in (3) and (4) of Figure 4C and Figure 4D (corresponding magnified images in Figure S22). Moreover, in (3a) of Figure 4C, the fluorescence peaks of N-C6 in white circle regions and pink

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by N-Cy showed bright green puncta around the nucleus (Figure S23 (2)), while ER distributed in the whole cytoplasm (Figure S23 (3)). And in Figure S23 (4) and (5), their fluorescence exhibited almost no overlap, and the colocation coefficient was only 42%. Moreover, the fluorescence distributions of N-Cy and ER red in Figure S23 (6) were also almost nonsynchronized, which efficiently ruled out the possibility of staining ER. Thirdly, we all know that the neutral core of LDs is surrounded by a phospholipid monolayer and a specific set of proteins. These proteins include the adipose differentiationrelated protein (ADRP; also called adipophilin), perilipins and TIP47.5,31,32 Amongst, perilipins are restricted in adipocytes and steroidogenic cells, while ADRP and TIP47 are widely distributed in various non-adipocytes cells. If the fluorescence of N-Cy can overlap with the LD protein, it means that N-Cy has excellent specificity to LDs. Given that this colocalization experiment was performed in HeLa cells, we selected TIP47 as the native LD protein to co-stain with N-Cy, and the results were displayed in Figure S24 (1-6). LDs in cells could be strongly stained with N-Cy in green (Figure S24 (2)) and antiTIP47 antiserum in red (Figure S24 (3)). Moreover, in Figure S24 (4), the fluorescence from N-Cy merged well with that of anti-TIP47 antiserum and their colocalization was up to 88%, indicating the identity of N-Cy to LDs. Meanwhile, along the white arrow in Figure S24 (4), the fluorescence distributions of N-Cy and anti-TIP47 antiserum were almost synchronized in Figure S24 (6). The above results fully demonstrated that N-Cy could specifically identify intracellular LDs. As above, we have roundly proved that amphipathic probes N-Cy and N-Py could give high-fidelity images of intracellular LDs. And the comparison experiments with lipophilic dyes (N-C6 and Nile Red) further showed that the selectivity of amphipathic N-Cy and N-Py to LDs was obviously superior. In particular, N-Cy, N-Py, and N-C6 have same fluorophore (NBD) yet exhibited rather different imaging performances. As shown in Scheme 2A, only difference between amphipathic N-Cy and N-Py and lipophilic N-C6 in chemical structure is an additional cation group. As envisioned in Scheme. 1, N-Cy and N-Py with cationic part and lipophilic NBD possess hydrophobic interaction and electrostatic interaction with LDs composed of a phospholipid monolayer and a lipophilic core. In comparison, N-C6 and traditional LDs probes such as Nile Red have only a lipophilic interaction with the LDs core. Therefore, as shown in Figure 6, it is reasonable to assume that the cationic moieties in N-Cy and N-Py play determinative effect on designing LDs probes with ultrahigh selectivity. Determination of the optimal staining concentration of amphipathic probes. In order to employ amphipathic probes as useful imaging tools in further biological researches and medical diagnosis, the optimal concentration for LDs staining should be investigated. Above cell experiments have showed that N-Cy possessed excellent permeability and could fast stained LDs within 2 min. However, N-Py was hard to bind LDs within 2 min (Figure S25A) and it always needed larger staining concentration and longer incubation time. Therefore, in terms of bioapplication prospect, N-Cy is a better choice. Next, the optimal staining concentration of N-Cy were further determined by detailedly comparing the staining results of different concentrations from 0.5 µM to 30µM under the same

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incubated time (2 min) and collecting conditions. As shown in Figure S25B, when the concentration was lower than 4 µM, the fluorescence in LDs was relatively weak to detect. When the concentration increased to 4 µM, LDs began to be clearly observed with bright fluorescence. Continuing to enlarge the staining concentration, N-Cy also could well stain LD and emitted brighter fluorescence. Moreover, the co-colocation experiments of N-Cy and ER red in Figure S26 fully manifested that even if the concentration reached to 30 µM, N-Cy still accurately labeled punctate LDs. Moreover, the counterstaining rate was as low as 45%, suggesting that it would be not off target to ER. However, it should be noticed that increasing staining concentration will inevitably cause greater damage to living cells. Thus under the premise of desired fluorescence images, the staining concentration should be as low as possible. All things considered, the staining concentration of 4 µM should be optimal for N-Cy, which would be used in living cell imaging.

Figure 7. (A) Confocal fluorescence images of HeLa cells untreated and treated with 40 µM oleic acid for different time of 2 h, 4 h, 6 h and then stained with N-Cy. (B) The ratio of fluorescence intensity in the cells treated with oleic acid for different time to that in untreated cells. Data are expressed as mean ± SD. Experiment times n = 3. N-Cy: 4 µM, 2 min; λex = 473 nm, λem = 510540 nm. Bar = 20 µm.

Analysis of LDs variation induced by oleic acid using NCy. With the function in lipid storage, LDs play prominent role in common pathologies that are associated with lipid accumulation, such as fatty liver, obesity, diabetes and atherosclerosis. Since oleic acid is known to induce cell to produce LDs,16,33 we used N-Cy to monitor LDs variation stimulated by oleic acid. As shown in Figure 7A, LDs in untreated cells

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ACS Applied Materials & Interfaces diagnosis of fatty liver36. As mentioned before, N-Cy could monitor variations of intracellular LDs in size, number and morphology, so we use N-Cy to investigate fatty liver. As shown in Figure 8B, N-Cy was used to respectively stain hepatic tissues of mouse raised by two different ways (generally feeding and high-fat feeding). LDs in generally feeding mouse hepatic tissues distributed uniformly, whereas in high-fat feeding mouse hepatic tissues, LDs gathered to form many clusters (pointed by red arrows in Figure 8B). The results implied the potential of N-Cy in diagnosing fatty liver.

individually dispersed closely to the plasma membrane. Upon treatment with oleic acid for 2 h, LDs increased in number and size, meanwhile they gradually distributed around the nucleus. After 4 h, intracellular LDs further enlarged in size. Finally, after 6 h, some enlarged LDs aggregated into several large clusters like grapes. Also, as shown in Figure 7B, the ratio of fluorescence intensity in the cells treated with oleic acid for different time to that in untreated cells have been calculated and plotted. With increasing the treatment time, the ratio increased linearly, indicating that N-Cy could monitor LDs’ accumulation and had huge potential in analyzing LDs’ abnormity in relevant diseases3-5. Two-photon imaging applications of N-Cy in live cells and turbid tissues. Compared with confocal microscopy, TPM has unique superiorities in biological investigations, such as reduced photodamage, low photobleaching and especially deep turbid tissue penetration.23,34,35 Thus, to assess the TPEF imaging capability of N-Cy, TPEF pictures of HeLa cells treated with N-Cy were captured. As shown in Figure S27A, intracellular LDs have been clearly observed when excited by 840 nm and the corresponding in-situ spectra showed only one emission peaked at 530 nm, indicating that N-Cy could specifically detect LDs in two-photon microscopy. As a significant evaluation criterion for two-photon imaging, tissue-imaging ability of N-Cy should also be investigated. Up to date, TPEF probes for imaging LDs in tissues are very rare. In this work, we made an attempt at visualizing the morphology and distribution of LDs in tissues especially thick and turbid tissues under TPM. In this case, it is important to first manifest whether N-Cy could still specifically target LDs in tissues. To this end, we adopted the previous method of removing LDs with xylene. Thus, various tissue slices (the cross and vertical sections of skeletal muscle slices as well as hepatic slices) from vista mice were chosen as experimental samples. In the control groups in Figure S27B, after being fixed, muscle slices and hepatic slices were stained with Hoechst 33342 for 30 min and N-Cy for 2 min, respectively. From the magnified muscle cross section image in (4) of Figure S27B, these approximate spherical dots with green emission of N-Cy were supposed to be LDs. And in turbid skeletal muscle vertical section images, LDs exhibited a regular mesh arrangement due to the presence of numerous muscle fibers, as displayed in (10) of Figure S27B. And in liver, the main site for lipid storage, sandy LDs widely distributed in the cytoplasm, as shown in Figure S27B (16). In contrast, in experimental groups without LDs, no green signals were obtained at the same detective condition in (6), (12), and (18) of Figure S27B, indicating that N-Cy also could specifically stain LDs in tissues. Therefore, we next used N-Cy to stain LDs in thick tissue block. As shown in Figure 8A, LDs in both turbid skeletal muscle and hepatic tissues could be clearly observed in different depth (up to 82 µm). The applications of N-Cy in revealing abnormity in hepatic tissues. Fatty liver is a LDs-related disease that usually expressed as the accumulation of LDs in hepatocyte36. In the early stage of fatty liver, a timely treatment can restore liver function. But if allowed to continue, fatty liver will further evolved into cirrhosis. In this case, the liver is very difficult to return to normal even if there is more treatment37. Therefore, direct observation of LDs is extremely crucial for the early

Figure 8. (A): TPEF images of live tissues (the cross section and vertical section of muscle tissues as well as hepatic tissues) stained with N-Cy (10 µM, 2 min) at different depth. (B): The TPEF images of hepatic tissues from mouse raised by different ways (general feeding and high-fat feeding) stained with N-Cy (10 µM, 2 min) as well as their corresponding magnified images in yellow rectangular areas; the red arrows pointed to LDs clusters formed in high-fat feeding mouse hepatic tissues. N-Cy: λex (TPEF) = 840 nm, λem = 495-540 nm.

Photostability, cytotoxicity and biological universality of N-Cy. Photostability is a key factor of fluorescent probes for long-time monitoring the behaviors of targets. Hence, both OPEF and TPEF photostability of N-Cy as well as Nile red were evaluated, as shown in Figure S28, Figure S29, Figure S30 and Figure S31. Under the ceaseless laser exposure, OPEF images of N-Cy (Figure S28) maintained clear and the fluorescence signal had no significant loss within 20 min (Figure S31). By contrast, the OPEF images of Nile red became vague after 10 min in Figure S29, and the emission almost disappeared at 20 min under the same conditions (Figure S31). In addition, upon excitation at 840 nm with femtosecond pulses and 3 mW average powers at the focal plane, clear TPEF images of HeLa cells stained with N-Cy have also been captured in Figure S30, and the intensity remained stable in 20 min in Figure S31. In order to detect its universality, PC-3 cells and normal MSC have been stained. From Figure S32, LDs in PC-3 cells and MSC all exhibited bright green fluorescence dots with peaks of 525 nm, indicating that N-Cy was a universal LDsspecific probe for multiple cell types.

CONCLUSION In summary, based on the renewed cognition that LDs possess unique architecture with a phospholipid monolayer interface, we for the first time proposed an interface-targeting strategy to design LDs fluorescent probes for achieving high-fidelity bi-

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(3) Schaffer, J. E., Lipotoxicity: when tissues overeat. Curr. Opin. Lipidol. 2003, 14, 281-287. (4) Suzuki, M.; Shinohara, Y.; Ohsaki, Y.; Fujimoto, T., Lipid droplets: size matters. J. Electron Microsc. 2011, 60, Suppl 1, S101-116. (5) Walther, T. C.; Jr, F. R., The life of lipid droplets. Biochim. Biophys. Acta 2009, 1791, 459-466. (6) Thiele, C.; Spandl, J., Cell biology of lipid droplets. Curr. Opin. Cell Biol. 2008, 20, 378-385. (7) Greenspan, P.; Mayer, E. P.; Fowler, S. D., Nile Red: A Selective Fluorescent Stain for Intracellular Lipid Droplets. J. Cell Bio. 1985, 100, 965-973. (8) Levitt, J. A.; Chung, P. H.; Suhling, K., Spectrally resolved fluorescence lifetime imaging of Nile red for measurements of intracellular polarity. J. Biomed. Opt. 2015, 20, 096002. (9) Govender, T.; Ramanna, L.; Rawat, I.; Bux, F., BODIPY staining, an alternative to the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae. Bioresource Technol. 2012, 114, 507-511. (10) Shingo, I.; Naoko, S.; Wang, C. W.; Cheng, Y. H.; Hayato, I.; Gi-Wook, H.; Akira, N.; Shusuke, K., The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System. Plos One 2016, 11, e0159324. (11) Beller, M.; Sztalryd, C.; Southall, N.; Ming, B.; Jäckle, H.; Auld, D. S.; Oliver, B., COPI Complex Is a Regulator of Lipid Homeostasis. Plos Biology 2008, 6, e292. (12) Wang, Z.; Gui, C.; Zhao, E.; Wang, J.; Li, X.; Qin, A.; Zhao, Z.; Yu, Z. Q.; Tang, B. Z., Specific Fluorescence Probes for Lipid Droplets Based on Simple AIEgens. Acs Appl. Mater. Inter. 2016, 8, 10193-10200. (13) Santos, F. M. F.; Rosa, J. N.; Candeias, N. R.; Carvalho, C. P.; Matos, A. I.; Ventura, A. E.; Florindo, H. F.; Silva, L. C.; Pischel, U.; Gois, P. M. P., A Three‐Component Assembly Promoted by Boronic Acids Delivers a Modular Fluorophore Platform (BASHY Dyes). Chem. - Eur. J. 2016, 22, 1631-1637. (14) Gao, M.; Su, H.; Li, S.; Lin, Y.; Ling, X.; Qin, A.; Tang, B. Z., An easily accessible aggregation-induced emission probe for lipid droplet-specific imaging and movement tracking. Chem. Commun. 2017, 53, 921-924. (15) Listunov, D.; Mazã¨Res, S.; Volovenko, Y.; Joly, E.; Gã©Nisson, Y.; Maraval, V.; Chauvin, R., Fluorophore-tagged pharmacophores for antitumor cytotoxicity: Modified chiral lipidic dialkynylcarbinols for cell imaging. Bioorg. Med. Chem. Lett. 2015, 25, 4652-4656. (16) Wang, E.; Zhao, E.; Hong, Y.; Lam, J. W. Y.; Tang, B. Z., A highly selective AIE fluorogen for lipid droplet imaging in live cells and green algae. J. Mater. Chem. B 2014, 2, 2013-2019. (17) Goel, A.; Sharma, A.; Kathuria, M.; Bhattacharjee, A.; Verma, A.; Mishra, P. R.; Nazir, A.; Mitra, K., New fluoranthene FLUN-550 as a fluorescent probe for selective staining and quantification of intracellular lipid droplets. Org. Lett. 2014, 16, 756-759. (18) E, Ö.; Appelqvist, H.; Kpr, N., Non-fused Phospholes as Fluorescent Probes for Imaging of Lipid Droplets in Living Cells. Frontiers in Chemistry 2017, 5, 1-9. (19) Thiam, A. R.; Jr, R. V. F.; Walther, T. C., The biophysics and cell biology of lipid droplets. Nat. Rev. Mol. Cell Bio. 2013, 14, 775786. (20) Beller, M.; Thiel, K.; Thul, P. J.; Jäckle, H., Lipid droplets: a dynamic organelle moves into focus. Febs Lett. 2010, 584, 2176-2182. (21) Raudsepp, P.; Brüggemann, D. A.; Knudsen, J. C.; Andersen, M. L., Localized lipid autoxidation initiated by two-photon irradiation within single oil droplets in oil-in-water emulsions. Food Chemistry 2016, 199, 760-767. (22) Hara, A.; Radin, N. S., Lipid extraction of tissues with a lowtoxicity solvent. Anal. Biochem. 1978, 90, 420-426. (23) Zhang, Y. H.; Wang, J. J; Jia, P. f; Yu, X. Q; Liu, H; Liu, X; Zhao, N.; Huang, B. B. Two-photon fluorescence imaging of DNA in

oimaging. Based on this strategy, amphipathic N-Cy and N-Py were successfully prepared by introducing additional cationic groups into lipophilic fluorophore, which show ultrahigh selectivity to LDs due to cooperation of the lipophilic interaction and electrostatic interaction. As expected, in-situ and highfidelity images of LDs in living and fixed cells were obtained with the two amphipathic probes. In addition, N-Cy exhibited two-photon excited fluorescence and could clearly visualize LDs at 82 µm depth in mouse turbid tissues. Importantly, it can be used to monitor the variations of LDs as well as their abnormity in hepatic tissues by TPM. Besides, N-Cy also showed other merits, including rapid stainability (within 2 min), excellent photostability, negligible cytotoxicity and abilities staining living and fixed cells. Furthermore, owing to its sensitivity to environmental polarity, N-Cy also revealed that LDs polarity is similar to 1,4-dioxane. Hence, not only N-Cy can serve as a powerful tool to specifically visualize LDs but also the interface-targeting strategy has universal significance for designing probes with ultrahigh selectivity.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The synthetic details and structural characterization of all compounds, photophysical properties of all compounds, the experiment of demonstrating the ability of staining living cells of N-Cy and N-Py, the fluorescence spectra of all compounds tested by confocal microscope, additional fluorescence images, photostability experiments of N-Cy compared with Nile Red and cell viability assay of N-Cy (PDF). Tomography scanning observation of LDs in HeLa cells stained with N-Cy at different depth (AVI).

AUTHOR INFORMATION Corresponding Author * Fax: +86 0531 88364263. E-mail: [email protected]. * Fax: +86 0531 88364263. E-mail: [email protected]. * Fax: +86 0531 88364263. E-mail: [email protected]. * Fax: +86 0571 87953734. E-mail: [email protected]. ORCID Xiaoqiang Yu: 0000-0002-4313-6464

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT For financial support, we thank National Natural Science Foundation of China (51773111, 21672130, 51273107 and 51273175), Natural Science Foundation of Shandong Province, China (ZR2017ZC0227), Fundamental Research Funds of Shandong University (2017JC011), Open Project of State Key Laboratory for Supramolecular Structure and Materials (SKLSSM201729). For calculation support, we thank Heng Zhang of School of Chemistry and Chemical Engineering, Shandong University.

REFERENCES (1) Jr, R. V. F.; Walther, T. C., Lipid Droplets Finally Get a Little RE-S-P-E-C-T. Cell 2009, 139, 855-860. (2) Beckman, M., Cell biology. Great balls of fat. Science 2006, 311, 1232-1234.

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living plant turbid tissue with carbazole dicationic salt. Org. Biomol. Chem., 2010, 8, 4582-4588. (24) Wu, J.; Liu, W.; Ge, J.; Zhang, H.; Wang, P., New sensing mechanisms for design of fluorescent chemosensors emerging in recent years. Chem. Soc. Rev. 2011, 40, 3483-3495. (25) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Men-nucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Son-ne D., Gaussian 09, Revision 1 A, Gaussian Inc., Wallingford, CT, 2009. (26) Zhang, G.; Sun, Y.; He, X.; Zhang, W.; Tian, M.; Feng, R.; Zhang, R.; Li, X.; Guo, L.; Yu, X., A red-emitting mitochondrial probe with ultra-high signal-to-noise ratio enables high-fidelity fluorescent images in two-photon microscopy. Anal. Chem. 2015, 87, 12088-12095. (27) Liu, Y.; Zhou, J.; Wang, L.; Hu, X.; Liu, X.; Liu, M.; Cao, Z.; Shangguan, D.; Tan, W., A Cyanine Dye to Probe Mitophagy: Simultaneous Detection of Mitochondria and Autolysosomes in Live Cells. J. Am. Chem. Soc. 2016, 138, 12368-12374. (28) Ross, M. F.; Kelso, G. F.; Blaikie, F. H.; James, A. M.; Cochemé, H. M.; Filipovska, A.; Da, R. T.; Hurd, T. R.; Smith, R. A.; Murphy, M. P., Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology. Biochemistry 2005, 70, 222-230. (29) Rashid, F.; Horobin, R. W., Interaction of molecular probes with living cells and tissues. Part 2. A structure-activity analysis of mitochondrial staining by cationic probes, and a discussion of the synergistic nature of image-based and biochemical approaches. Histochemistry 1990, 94, 303-308. (30) Horobin, R. W.; Stockert, J. C.; Rashid-Doubell, F., Fluorescent cationic probes for nuclei of living cells: why are they selective? A quantitative structure–activity relations analysis. Histochem. Cell Biol. 2006, 126, 165-175. (31) Wolins, N. E.; Rubin, B.; Brasaemle, D. L., TIP47 associates with lipid droplets. J. Biol. Chem. 2001, 276, 5101-5108. (32) Garcia, A.; Sekowski, A.; Subramanian, V.; Brasaemle, D. L. The central domain is required to target and anchor perilipin A to lipid droplets. J. Biol. Chem., 2003, 278, 625-635. (33) Guo, Y.; Walther, T. C.; Rao, M.; Stuurman, N.; Goshima, G.; Terayama, K.; Wong, J. S.; Vale, R. D.; Walter, P.; Farese, R. V., Functional genomic screen reveals genes involved in lipid-droplet formation and utilization. Nature 2008, 453, 657-661. (34) Chang, S. L.; Masanta, G.; Kim, H. J.; Ji, H. H.; Kim, H. M.; Cho, B. R., Ratiometric Detection of Mitochondrial Thiols with a Two-Photon Fluorescent Probe. J. Am. Chem. Soc. 2011, 133, 1113211135. (35) Denk, W.; Strickler, J. P.; Webb, W. W.; Inc, C. R. F., TwoPhoton laser scanning microscopy. Science. 1990, 248, 73-76. (36) Amacher, D. E., Strategies for the early detection of druginduced hepatic steatosis in preclinical drug safety evaluation studies. Toxicology, 2011, 279, 10-18. (37) Lin, J.; Lu, F.; Zheng, W.; Xu, S.; Tai, D.; Yu, H.; Huang, Z., Assessment of liver steatosis and fibrosis in rats using integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique. J. Biomed. Opt. 2011, 16, 11024.

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Table of Contents (TOC)

Interface-targeting strategy enables two-photon amphipathic lipid droplets probes high-fidelity imaging turbid tissues and detecting fatty liver.


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Interface-targeting strategy enables two-photon ... - ACS Publications

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