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A phosphorescent silver(I)–gold (I) cluster complex that specifically lights up the nucleolus of living cells with FLIM imaging
Biomaterials 34 (2013) 4284e4295
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
A phosphorescent silver(I)egold (I) cluster complex that specifically lights up the nucleolus of living cells with FLIM imaging Min Chen a, Zhen Lei b, Wei Feng a, Chunyan Li a, Quan-Ming Wang b, *, Fuyou Li a, * a b
Department of Chemistry & Institute of Biomedicine Science, Fudan University, Shanghai 200433, PR China State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemistry, Xiamen University, Xiamen 361005, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2012 Accepted 10 February 2013 Available online 11 March 2013
The phosphorescent silver(I)egold(I) cluster complex [CAu6Ag2(dppy)6](BF4)4 (N1) selectively stains the nucleolus, with a much lower uptake in the nucleus and cytoplasm, and exhibits excellent photostability. This AgeAu cluster, which has a photoluminescent lifetime of microseconds, is particularly attractive as a probe in applications of time-gated microscopy. Investigation of the pathway of cellular entry indicated that N1 permeates the outer membrane and nuclear membrane of living cells through an energydependent and non-endocytic route within 10 min. High concentrations of N1 in the nucleolus have been quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and transmission electron microscopy coupled with an energy dispersive X-ray analysis (TEM-EDXA), which also helped to elucidate the mechanism of the specific staining. Intracellular selective staining may be correlated with the microenvironment of the nucleolus, which is consistent with experiments conducted at different phases of the cell cycle. These results prove that N1 is a very attractive phosphorescent staining reagent for visualizing the nucleolus of living cells. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Silver(I)egold (I) cluster complex Nucleolus Selective staining FLIM imaging
1. Introduction As the most prominent structure in a cell nucleus, the nucleolus plays a central regulatory role in cell growth and proliferation [1,2]. In particular, the nucleolus is the key site in which ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins [3e5]. Therefore, the visualization of nucleolus-related events is a valid demand. In light of the sub-cellular resolution and high sensitivity of fluorescence microscopy, the visualization of nucleolus by fluorescence microscopy has attracted increasing attention. Nowadays, there are several fluorophores used for staining the nucleolus specifically in living cells. For example, Wong et al. developed a 2,7-carbazole-based dicationic salt for two-photon fluorescence imaging of RNA in the nucleolus and cytoplasm [6]. Liu et al. have demonstrated the utility of a bidentate 1,2,3-triazole-based ligand modification of CdS quantum dots (QDs) for targeting the nucleolus of cells [7]. However, their potential toxicity of CdS QDs is still an issue, which limits their utility. To date, Syto RNASelect is the only commercially available dye for live cell RNA imaging [8].
* Corresponding authors. E-mail addresses: [email protected] (Q.-M. Wang), [email protected] (F. Li). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.02.032
When applied in real biological samples, the fluorescent probes often suffer from the interference from autofluorescence of biosamples and scattered excitation light, reducing the signal-to-noise ratio (SNR). To avoid such an interference, one effective solution is to use time-gated luminescence imaging based on long-lifetime emissive materials as probes [9,10]. The advantage of measuring the lifetime of photoluminescence emission is that this parameter is directly dependent upon excited-state reactions but independent of compound concentration, light intensity and photobleaching, so it is an ideal parameter for monitoring the local environment of the materials. Fluorescence lifetime imaging microscopy (FLIM) [11e15] is a technique used to study the nanosecond decay kinetics of the electronic excited state. To date, however, no example of FLIM imaging has been reported for nucleolus staining of living cells. For fluorescence lifetime imaging, a long-lifetime emissive materials for nucleolus staining should be developed. Heavy-metal complexes and lanthanide complexes are the ideal candidates as long-lifetime emissive probes [16e24]. Recently, Lo et al. reported that iridium(III) dipyridoquinoxaline complexes could stain the nucleoli of MDCK cells [19], and Parker et al. reported several europium and terbium complexes selectively stained the nucleoli of cells [25e27]. For these small-molecule stains, cytoplasm staining often accompanies staining of the nucleolus and interferes with the observation of nucleolus staining.
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Scheme 1. Possible pathway of nucleolus staining with the Ag(I)eAu(I) cluster complex [CAu6Ag2(dppy)6](BF4)4 (N1).
It is well known that nucleolus organizing regions (NORs) contains a lot of argyrophilic proteins which have a high affinity of silver (AgNOR proteins) [28,29]. Such NORs can be identified by silver staining, and some silver-staining methods have been developed for immunolocalization of nucleolus proteins [30e33]. To data, however, these reported silver stains are non-emissive. In this present study, we demonstrated a phosphorescent silver(I)egold(I) cluster complex [CAu6Ag2(dppy)6](BF4)4 (N1, Scheme 1), which has a long lifetime of w32 ms, to be an excellent luminescent stain for nucleolus-specific bioimaging. Not only a red emission contrasting with the green fluorescence of commercial SYTO RNASelect, but also well photostability, allow the complex N1 for its continuous tracking studies. Of special note, complex N1, can specifically light up the nucleolus of a living cell by using the fluorescence lifetime imaging method. 2. Experimental section 2.1. Materials and general instruments Phosphate buffered saline (PBS), Fetal bovine serum (FBS), DMSO, 2-deoxy-Dglucose, oligomycin, Cytochalasin D, Filipin, NH4Cl, Lealanine (Ala), Learginine (Arg), Leasparagine (Asn), Leaspartic acid (Asp) Leglutamine (Gln), Leglycine (Gly), Leisoleucine (Ile), Leleucine (Leu), Lelysine (Lys), Lephenylalanine (Phe), Leproline (Pro), Leserine (Ser), Lethreonine (Thr), Letryptophan (Try), Letyrosine (Tyr), Levaline (Val), Leglutamic acid (Glu), Lecysteine (Cys), Lemethionine (Met), Lehistidine (His), bovine serum albumin (BSA), deoxyribonucleotide triphosphate (dNTP) and CT DNA were obtained from Acros. Syto RNASelect, DAPI, DiI, and cell culture reagents were purchased from Invitrogen. Metabolic inhibitors (oligomycin, deoxyglucose) and endocytosis inhibitor (Cytochalasin D, Filipin) were obtained from SigmaeAldrich. UVevisible spectra were recorded on a Shimadzu UV-2550 spectrometer. Steadyestate emission experiments at room temperature were measured on an Edinburgh instrument FL-900 spectrometer with Xe lamp as excitation source. Luminescence lifetime studies were performed with an Edinburgh FL-900 photocounting system with a hydrogenefilled lamp as the excitation source. Luminescence quantum yield (f) of N1 in aerated solution were measured with reference to terpyridine ruthenium Ru (bpy)3 (f ¼ 0.028). The cluster complex N1 was synthesized according to our previous literature [34].
24 h at 37 C under 5% CO2. The cluster complex N1 (100 mL/well) at different concentrations (1.6, 3.1, 6.2, 12.5, 25, 50, 100 mM, diluted in RPMI 1640) was added to the wells of the treatment group, and RPMI 1640 to the negative control group. The cells were then incubated for 24 h at 37 C under 5% CO2. Thereafter, MTT (20 mL; 5 mg/mL) was added to each well, and the plate was incubated for an additional 4 h at 37 C under 5% CO2. After changing the culture medium to 100 mL DMSO, the assay plate was allowed to stand at room temperature for 10 min. An enzyme-linked immunosorbent assay (ELISA) reader (infinite M200, Tecan, Austria) was used to measure the OD570 (Absorbance value) of each well with background subtraction at 690 nm. The following formula was used to calculate the viability of cell growth: Cell viability ð%Þ ¼ ðmean of absorbance value of treatment group=mean of A value of controlÞ 100
2.4. Luminescence imaging of live cells incubated with N1 Luminescence imaging including xy-scan, lambda-scan, T-scan, and time-lapseimaging, was performed with an Olympus FluoView FV 1000 confocal fluorescence microscope and a 60 oil-immersion objective lens. Cells incubated with N1 were excited at 405 nm with a semiconductor laser, and the emission was collected at 600 20 nm. Quantization by line plots was accomplished using the software package provided by Olympus instruments. DAPI was excited using a laser at 405 nm, and the emission was collected at 460 20 nm. 2.4.1. Luminescence imaging of live cells after treatment with metabolic and endocytic inhibitors Cells were detached from the culture and were preincubated with 50 mM 2-deoxy-D-glucose and 5 mM oligomycin, with 5 mM Cytochalasin D, or with 5 mM Filipin, or 50 mM NH4Cl in PBS, for 1 h at 37 C. The cells were then washed with PBS and incubated solely with 10 mM in DMSO/PBS （pH 7.4, 1:99, v/v） for 10 min at 37 C. Before imaging, the cells were washed three times with PBS.
2.2. Cell culture (Human epithelial cervical cancer cell line) Hela, (Human nasopharyngeal carcinoma cell line) KB and (Human normal hepatocyte) LO2 cell lines were provided by the Institute of Biochemistry and Cell Biology, (The Chinese Academy of Sciences) CAS. Hela cells were grown in (Dulbecco’s modified Eagle’s medium) DMEM supplemented with 10% v/v FBS and 1% antibiotic/antimycotic solution (penicillin and streptomycin, Invitrogen). KB and LO2 cells were grown in RPMI 1640 supplemented with 10% v/v FBS and with and 1% antibiotic/antimycotic solution. All cells cultures were kept at 37 C in a humidified incubator with 5% CO2. 2.3. Cytotoxicity assay of the cluster N1 The in vitro cytotoxicity was evaluated by performing the methyl thiazolyl tetrazolium (MTT, SigmaeAldrich) assay in Hela cells. Cells growing in log phase were seeded into 96-well cell-culture plate at 5 103/well and then incubated for
Fig. 1. Absorption (black line) and photoluminescence (red line) spectra of the N1 solution in DMSO/PBS (1:99, v/v), lex ¼ 405 nm. Inset: photographs showing the brightfield and luminescence photos of the N1 solution in DMSO/PBS (1:99, v/v) under excitation from UV lamp (lex ¼ 365 nm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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2.4.2. Colocalization imaging of live cells incubated with N1 and Syto RNASelect The living cells were plated on 14 mm glass coverslips and allowed to adhere for 24 h. After washing with PBS, the cells were incubated with 10 mM N1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 37 C, and then further incubated with Syto RNASelect for another 20 min before imaging. 2.4.3. Luminescence imaging of fixed cells incubated with N1 The cells were detached from the culture and were fixed with 4% paraformaldehyde at 4 C for 30 min. After washing with PBS, the fixed cells were incubated with 10 mM N1 in DMSO/PBS （pH 7.4, 1:99, v/v） for 10 min at 37 C. After washing with PBS, the coverslips were separated from the chamber, and the cells were mounted with 10% glycerol and sealed with nail varnish on a glass substrate. 2.5. Fluorescent lifetime imaging microscopy (FLIM) After incubation of N1, the cells were fixed by addition of 4% paraformaldehyde/2.5% glutaraldehyde in PBS (pH 7.4, 1 mL) for 1 h. An inverse
time-resolved laser scanning confocal microscope was employed for FLIM experiments (Nikon, A1R-si). The fixed Hela cells attached onto a slide glass were covered with a thin cover glass, on which an excitation beam was focused. A 488 nm ps pulsed diode laser with 20 MHz repetition rate was used for excitation. The instrumental response function of the system was 30 ps at fwhm. A filter of FITC (515-30) and a single photon avalanche diode were used to collect emissions from the Hela cells. The time-gated emission signals were obtained with a TCSPC technique. Typically, an 80 mm 80 mm sample area consisting of 256 256 pixels was scanned with an acquisition rate of 0.83 ms/pixel. FLIM images and their exponential fits were analyzed using NIS-Elements C software provided by manufacturer. 2.6. Flow cytometry analysis Cellular uptake of N1 under different conditions was analyzed by flow cytometry (Beckman Counter). Cells were filtered through 41 mM nylon mesh in preparation to analyze at a rate of 200e500 cells/s using FACS flow as at the sheath fluid and 10,000 events were recorded for each sample.
Fig. 2. Confocal luminescent images of living LO2 cells (a), Hela cells (b), and KB cells (c) incubated with 10 mM N1 in PBS (pH 7.4) for 10 min at 25 C. (d) Corresponding to extracellular region (1 and 4), cytoplasm (2), and nuclear region (3). (e) Quantification of the luminescence intensity profile of N1-treated KB cells shown in panel d. (lex ¼ 405 nm, lem ¼ 600 20 nm). Scale bar: 20 mm.
M. Chen et al. / Biomaterials 34 (2013) 4284e4295 2.7. Cellular uptake and mechanism 2.7.1. Analysis of inhibitor experiment Hela cells preincubated with or without 50 mM 2-deoxy-D-glucose and 5 mM oligomycin in PBS for 1 h at 37 C were incubated with 10 mM N1 in PBS (pH 7.4) for 10 min at 37 or 4 C. After incubation, adherent cells were detached from the surface of culture dishes by treatment with trypsin-EDTA solution (Hangzhou Genom Biomedical & Technology Co. Ltd). The mean fluorescence was measured for 10,000 cells by using flow cytometry. 2.7.2. Cell cycle analysis Hela cells were grown for 24 h in culture (DMEMþ10% FBS) and then incubated DMEM containing colchicine at the final concentrations of 1.0 mg/mL to synchronize cells in the metaphase (M phase). After 7 h, suspended cells which were in M phase were collected by shake off to centrifugate at 1000 g for 10 min. The supernatant solution was removed after centrifugation, and then one-quarter of cells were fixed with cold methanol (70%) at 20 C until analysis. The other three quarters were equally released in three dishes containing fresh culture medium (DMEM) and collected at the indicated time points, then fixed with 20 C cold methanol (70%) for further analysis. To prepare for flow cytometry analysis, all fixed cell suspensions were centrifuged at 1000 rpm for 10 min, and cells were resuspended in 0.25 mL PBS containing 200 mM/mL RNase (SigmaeAldrich) and incubated at 37 C for 30 min. Then, 100 mg/mL of propidium iodide solution was added. After incubating at 37 C for 10 min, cells were already for further analysis by flow cytometry. 2.7.3. Interaction of N1 and biomolecules The interaction of N1 with amino acids, bovine serum albumin (BSA), deoxyribonucleotide triphosphate (dNTP), and CT DNA has been investigated by luminescent emission titration. Herein, L-alanine (Ala), L-arginine(Arg),L-asparagine (Asp), L-glutamine (Gln), L-glycine (Gly), L-isoleucine (Ile),L-leucine (Leu),L-lysine (Lys),L-pheneylalanine (Phe), L-proline (Pro), L-serine (Ser), L-threonine (Thr), L-tryptophan (Try), L-tyrosine (Tyr), L-valine (Val), L-glutamic acid (Glu), L-cysteine (Cys), L-methionine (Met), L-histidine (His) were used as examples of amino acids.
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2.7.4. Determination of gold and silver content After digestion by trypsin-EDTA solution, Hela cells were divided into two parts. When the cells adhered to the culture flask, the culture medium was changed to 10 mL of PBS with 10 mM N1. The cells were incubated with N1 for 10 min at 37 C. Thereafter, they were carefully washed with PBS, and then the nuclei and cytoplasm were extracted using a nucleus extraction kit (Nanjing KeyGen Biotech. Co., Ltd.). The gold and silver concentration in the samples was determined by an inductively coupled plasma atomic emission spectroscopy (ICP-AEC Thermo Elemental Co., Ltd.). 2.7.5. Transmission electron microscopy and energy-dispersive X-ray analysis (TEM-EDXA) After incubation with 10 mM N1 for 10 min, the cells were thoroughly washed with PBS buffer. Cells were then scraped from the culture dish to centrifuge at 2000 g for 5 min, and the supernatant was removed. The cell pellets were fixed by addition 4% paraformaldehyde in PBS (pH 7.4, 1 mL) for 1 h. Then the cells were rinsed with PBS buffer, postfixed using 4% aqueous solution of OsO4 (Caution! Extremely toxic) for 1 h. Subsequently the cells were rinsed with distilled water, and stained with 0.5% uranyl acetate (0.5 mL, in 30% ethanol) for 1 h. Cells were then gradually dehydrated using a series of ethanol solutions (30, 60, 70, 80, and 100%) and embedded in epoxy resin.
3. Results and discussion 3.1. Photophysical properties of complex N1 Complex N1 [CAu6Ag2(dppy)6](BF4)4 was synthesized according to our previous method [34], and can be dissolved in a solution of DMSO/PBS (1:99, v/v). The electronic absorption and emission spectra of N1 in DMSO/PBS (1:99, v/v) solution at room
Fig. 3. Colocalization images of living KB cells incubated with 10 mM N1 in PBS for 10 min at 25 C and then incubated with Syto RNASelect. (a) Green channel for Syto RNASelect (lex ¼ 488 nm, lem ¼ 530 20 nm). (b) Red channel for N1 (lex ¼ 405 nm, lem ¼ 600 20 nm). (c) Bright-field image. (d) Colocalization of green and red luminescence is shown as yellow pixels. Scale bar: 30 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Three-dimensional luminescence images of live HeLa cells loaded with 10 mM N1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 25 C. The nucleus was stained blue with DAPI. Panel a is a xy image obtained at z ¼ 3.35 mm, while panels b and c display the yz and xz cross sections (z ¼ 12.25e18.35 um) taken at the lines shown in panel a, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Real-time monitoring of N1 uptake and intracellular transport in cultured Hela cells over a 700 s period. The views were monitored by confocal luminescence microscope. (a) Luminescence images of living HeLa cells incubated with 10 mM N1 in DMSO/PBS (pH 7.4, 1:99, v/v) at 37 C at selected time points (lex ¼ 405 nm, lem ¼ 600 20 nm; scale bar: 10 mm). T0 indicates a short time (<30 s) for cells entering the focal plane after N1 was added. (b) Luminescence image of Hela cells incubated with N1 for 1038 s. (c) Time course of luminescence intensity in the nucleolus (regions 3 and 4 in b), membrane (region 2 in b) and nucleus (region 1 in b).
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Fig. 6. Effect of incubation temperature and endocytic inhibitors on cellular uptake of N1 in Hela cells. (a) The cells were pretreated with 50 mM 2-deoxy-D-glucose and 5 mM oligomycin in PBS for 1 h at 37 C, and then incubated with 10 mM N1 at 37 C for 10 min (bec) The cells were incubated with 10 mM N1 for 10 min at 4 C, 37 C, respectively. (def) The cells were pretreated with endocytic inhibitors Cytochalasin D (5 mM), Filipin (5 mg/mL) and NH4Cl (50 mM), respectively, and then incubated with 10 mM N1 at 37 C for 10 min (lex ¼ 405 nm, lem ¼ 600 20 nm).
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temperature are shown in Fig. 1. N1 has intense absorption bands in the ultraviolet region of 280e480 nm. Moreover, upon excitation at 405 nm, N1 exhibits visible emission at 650 nm. With reference to terpyridine ruthenium Ru(bpy)3 as a standard (f ¼ 0.028) in deionized water, the luminescence quantum yield of N1 in DMSO/ PBS solution was measured to be 1.11% (f) at room temperature and 1.44% in a degassed solution. Compared with the luminescence quantum yield of 30% in chloroform solution, the photoluminescence emission of N1 shows a significant dependence on solvent polarity. In addition, the luminescent lifetime of N1 in DMSO/PBS solution was measured to be w32 ms (Fig. S1 in the Supporting Information). Such a long lifetime on the microsecond scale suggests the emission has a phosphorescent nature. 3.2. Selective nucleolus staining Three kinds of cells, including two cancer cells (Hela and KB) and one normal cell (LO2), were employed for bioimaging testing. Typically, a cell sample was incubated with 10 mM N1 for 10 min and then cell images were recorded by confocal fluorescence microscopy (Fig. 2). The emission spectrum of living cells incubated with N1 is shown in Fig. S2. Interestingly, an intense luminescence signal was detected in the nucleolus region, while the luminescence in the remainder of the cells was very weak (Fig. 2c). Quantification of the luminescence intensity profile of N1-treated KB cells revealed an extremely high signal intensity ratio for the nucleolus (region
3, z4000) compared to both the nucleus and cytoplasm (regions 1, 2 and 4, z0). Such high nucleolus/nucleus and nucleolus/cytoplasm luminescence intensity ratios suggest an exclusive staining of the cell nucleolus with N1 (Fig. 2d,e). Similarly, upon incubation with N1, the normal cell line LO2, also exhibited intense luminescence in the nucleolus region (Fig. 2a). These facts indicate that nucleolus-specific staining by N1 occurs regardless of the type of cell line. To confirm the luminescence spots observed in the nucleolus (Fig. 2), colocalization experiments were carried out with a confocal fluorescence microscope (Fig. 3). Living cells were loaded with N1 and the commercial nucleolus stain Syto RNASelect. The luminescence (red) of N1 (excitation at 405 nm, emission at 600 20 nm) was completely colocalized with that (green) obtained at the wavelength for Syto RNASelect (excitation at 488 nm, emission at 530 20 nm). The colocalization was evident with the observation of bright yellow spots in the region of the nucleolus (Fig. 3d), suggesting that the targeted organelles of N1 are nucleoli. To further verify that the localization of N1 is internalized within the nucleolus, three-dimensional (3D) imaging of live cells was carried out. With the advantage of the confocal technique, which allows filtering by depth of field, the 3D image is built up through separately recorded images of optical slices (Z-scans) of the sample. In this experiment, Hela cells were coloaded with N1 and the commercial nucleus stain DAPI, and were then imaged by serially scanning at increasing depths along the z-axis. As shown in Fig. 4a,
Fig. 7. Flow cytometric histogram profile (a) and the ICP-MS analysis (b) of cellular uptake of N1 in Hela cells. Hela cells were incubated with 10 mM N1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 37 C, 4 C, and 37 C after cells had been preincubated with 50 mM 2-deoxy-D-gluscous and 5 mM oligomycin in PBS for 1 h, respectively.
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the nucleus was stained blue with DAPI, while the nucleolus regions were clearly lighted up by N1. The nucleoli of Hela cells were perfectly visualized in the yz and xz cross-sectional images (Fig. 4b,c) through 3D reconstruction of serial xy sections, which indicates that N1 stains the nucleolus rather than other locations in the cell. In addition, the presence in the nucleolus of the Ag-Au cluster N1 was further confirmed by transmission electron microscopy (TEM) of the cells incubated with N1 (Fig. S3).
3.3. Kinetic processing of cellular uptake of N1 To monitor the dynamic process of nucleolus staining by N1 in real time, continuous imaging of living Hela cells was performed (Fig. 5 and movie in Supporting Information). In the cells prior to incubation with N1 (<500 s), luminescence was observed near the plasma membranes. After incubation with the N1 solution for 500 s, the luminescence signal of N1 appeared in the nucleolus. At 700 s, the red luminescence became strong in the nucleolus, indicating more and more N1 was transported to and accumulated in the nucleolus.
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Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2013.02.032. Quantification of the photoluminescence intensity in the microscopic image of the Hela cells revealed that the addition of N1 triggered a continuous increase in luminescence intensity at the nucleolus, while the signal from the cytoplasm remained negligible (Fig. 5b, c). In addition, the fluorescence signals of the membrane shown in Fig. 5b are much stronger than those in Fig. 2. It is probable that the cells in Fig. 5b were continuously incubated with N1, whereas the medium containing the AgeAu cluster which were not taken up by the cells in Fig. 2 was washed by the PBS buffer. Those results indicate that a 700 s incubation of N1 at 37 C is sufficient to detect a significant intensity increase in the nucleoli and that specific nucleolus staining can be effectively performed by N1 even in the absence of a molecular transporter. 3.4. The pathway of cellular uptake of N1 The internalization of extracellular material can occur through energy-independent (facilitated diffusion, passive diffusion) and
Fig. 8. The histogram of the cell cycle obtained using flow cytometry and the confocal images in specific cell cycle phase cells treated 10 mM N1 for 10 min. (a) Histogram of the cell cycle obtained from Hela cells cultured under different collected time after synchronization. (b) Bright-field images of Hela cells in cell cycle images. (c) Luminescence images. (d) Overlay of luminescence image and their corresponding brightfield images.
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energy-dependent (endocytosis, active transportation) pathways [35]. Herein, a detailed investigation of the cellular internalization mechanism and uptake pathway of N1 was carried out, at different temperatures with various inhibitors of endocytic pathways (Fig. 6). The effect of temperature on the cellular uptake of N1 was investigated by confocal luminescence microscopy. As shown in Fig. 6b, luminescence was hardly observed for N1-incubated cells at 4 C, in contrast to the case at 37 C (Fig. 6c). Cell metabolism and physiological activity would be suppressed under low-temperature conditions. Hence, N1 is likely to enter the cells by an energydependent pathway. To confirm this deduction, we investigated the effect of metabolic inhibitors 2-deoxy-D-glucose and oligomycin on cellular uptake of N1. As shown in Fig. 6a, when the cells were preincubated with metabolic inhibitors, the intracellular luminescence was significantly suppressed. To quantitatively analyze the cellular uptake effect for the AgeAu cluster, flow cytometry and ICP data were used to study the intracellular fluorescence and metal contents (Fig. 7). The mean fluorescence of Hela cells incubated at 37 C was higher than that of cells incubated at 4 C or preincubated with metabolic inhibitors. The ICP-MS data confirmed this result. These results imply that energy plays a very important role during the process by which N1 cross the plasma membrane and eventually reach the nucleolus. Next, we investigated the possibility of cellular entry by N1 through an endocytosis or positive pathway that is an energy-
dependent route. As shown in Fig. 6def, when cells were preincubated with an endocytic inhibitor such as cytochalasin D, filipin or NH4Cl, which block different endocytic pathways, no significant reduction in luminescence signal in the nucleolus was observed. That is, there was no measurable decrease in the cellular uptake of N1 under endocytosis-inhibiting conditions. Therefore, the nucleolus-specific staining of living cells by N1 is related to an energy-dependent non-endocytic pathway across the cell membrane and nuclear membrane. The cell membrane controls the movement of substances in and out of cells. When the cells were fixed, the plasma membrane and the nuclear membrane became permeable. Interestingly, when the fixed cells were incubated with N1, an intense luminescence signal from N1 (lex ¼ 405 nm, lem ¼ 600 20 nm) was detected in the whole fixed cells, including the nucleus and cytoplasm (Fig. S4). The generalized, diffuse whole-cell staining pattern for the fixed cells was significantly different from the specific nucleolus staining seen in living cells. Therefore, it can be safely deduced that N1 is transported by an active mechanism rather than passive diffusion. 3.5. The mechanism of selective nucleolus staining of N1 Considering that N1 is red emissive in specific staining of N1 in the nucleoli may possible causes: either N1 is specifically nucleolus or N1 is distributed throughout
solution (Fig. 1), the be attributed to two concentrated in the the whole cell, and
Fig. 9. Comparison of photobleaching of N1 and Syto RNASelect in confocal luminescence microscopy imaging. The living Hela cells were incubated with DAPI (blue) and N1 (red), respectively. (a) Confocal luminescence microscopy images of 10 mM N1-pretreated Hela cells incubated with Syto RNASelect for another 20 min. Green channel for Syto RNASelect (lex ¼ 488 nm, lem ¼ 530 20 nm), Red channel for N1 (lex ¼ 405 nm, lem ¼ 600 20 nm). (b) Quantitative analysis of the changes in fluorescence intensities of N1 (red line) and Syto RNASelect (black line) in panel a. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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enhanced luminescence is generated in the nucleolus. To verify the real reason, the amounts of N1 in the nucleolus and the remaining parts of the cell should be measured. Herein, a quantitative method utilizing transmission electron microscopy coupled with an energy dispersive X-ray analysis (TEM-EDXA) was used to determine the concentrations of N1 in the nucleolus and cytoplasm. As shown in Fig. S5, gold was detected in the nucleolus at a concentration of 3.85% dry weight, which is 30 times higher than the concentration in the cytoplasm, which was approximately 0.10% dry weight. Furthermore, the amounts of Au in the cytoplasm and nuclei of N1 stained cells were quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AEC). The results of quantitative distribution revealed similar proportions in the nuclei (0.38 pg Au/ cell) and cytoplasm (0.32 pg Au/cell), indicating that more than 54.29% of Au accumulated in the nuclei. Considering the very small volume of the nucleolus compared with the cytoplasm and nuclei, the concentration of N1 in the nucleolus is much higher than that in the cytoplasm. These results proved that N1 is concentrated in the nucleolus instead of being distributed over the whole cell. It was unclear whether the interactions between N1 and the biomolecules affect the luminescence characteristics of N1 or not. To address this question, we investigated the luminescence change of N1 upon addition of numerous substances (including CT DNA, BSA, RNA, various amino acids and triphosphates) by luminescence analysis techniques. As shown in Fig. S6, the emission intensity of N1 in BSA increased nearly three times compared with that of N1 in PBS, and two-fold enhancement was observed in DNA. Fig. S7 shows that the quantum yield of N1 is strongly influenced by the solvent polarity. So, it can be deduced that N1 in nonpolar (or hydrophobic) microenvironment will display intense luminescence emission, which is relative to the increase in luminescence quantum yield of N1 in BSA and DNA. But there was nearly no change in the RNA solution. This is quite different from the case of Syto
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RNASelect, which gives rise to an enhanced fluorescence on binding to RNA [8]. The N1 in solutions of various amino acids and triphosphates behaves similarly to that in TriseHCl/NaCl. These results indicate that high concentration in nucleolus may be very important for specific staining. As we know, the nucleolus is a dynamic structure that is disassembled and reassembled at different phases of the cell cycle. Therefore, the distribution of N1 during the different phases of the cell cycle was examined by flow cytometry and confocal microscopy imaging (Fig. 8). For convenience, the cycle has been classified into four phases (G1/S/G2/M). Between the mitosis (M) phase and the S phase is the G1 phase, which has a normal diploid (2N) DNA content, whereas the G2 (between the S phase and M phase) cells have tetraploid (4N) DNA content, the same as the M phase. S phase cells contain intermediate amounts of 2N and 4N [36e39]. In practice, Hela cells were collected at the indicated time point in accordance with the lengths of the individual phases of the cell cycle after the cells were synchronized by colchicine. The collection of cells was carried out by monitoring the content of DNA intercalated with propidium iodide (PI) dye to identify the different phases. As shown in Fig. 8a, the percentages of cells existing within the various phases of the cell cycle were calculated by gating on G0, G1, S, and [G2 þ M] cell populations (it is difficult to differentiate between cells in the G2 and M phases, as both have double DNA), and the prominent peak represents the specific cell cycle state in the histogram. Fluorescence microscopy provides a sensitive means of acquiring information about the organization and dynamics of complex cellular structures. Confocal images of stained cells clearly illustrate the distribution of N1 compared with cycling cells (Fig. 8bed). The M phase cells have no nucleoli, whereas cells in other phases all have nucleoli. Interestingly, N1 stains whole cells in the M phase but only lighted up the nucleoli in the other three phases. The special environment of the nucleolus enables N1 to
Fig. 10. Fluorescence lifetime microscope images of Hela cells treated with 10 mM N1 for 10 min (aed) Images of N1 luminescence which was measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. (e) False color fluorescence lifetime images of N1 in Hela cells under TCSPC FLIM equipment, excited at 488 nm.
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distinguish the nucleolus from the other parts of the cell. These data distinctly verify that specific staining by N1 is closely related to the environment of the nucleolus. On the basis of the above discussion, we propose a mechanism for nucleolus staining by N1 as follows: (a) the specific staining is mainly associated with N1 concentration in the nucleoli; (b) the microenvironment of the nucleolus may account for N1 of the specific staining in the nucleolus. 3.6. Cytoxicity and photostability of N1 A key consideration is whether the cells studied remain healthy after incubation with N1. By using the MTT assay, cell viabilities were estimated to be greater than 80% in the presence of 10 mM N1. This indicates that N1 has low toxicity under luminescence nucleolus imaging conditions (Fig. S8). Moreover, the photostability of N1 was compared with that of the known fluorescent dye, DAPI. Hela cells were incubated with N1 (10 mM, 10 min), and thoroughly rinsed with fresh medium to remove floating cells. The cells were then treated with DAPI (50 mM, 15 min), and the extracellular solution was removed completely before imaging. Continuous illumination with a 405 nm laser with a power of 1.4 mW in the focal plane induced significant photobleaching of DAPI, whereas N1 exhibited a better photostability (Fig. S9). In addition, N1 is more photostable than Syto RNASelect in fixed cells (Fig. 9). The results indicate that N1 displays a better photostability than DAPI and Syto RNASelect do. 3.7. Fluorescent lifetime imaging microscopy (FLIM) As phosphorescent gold-silver cluster exhibit long lifetime, the N1 stained cells can be monitored with time-gated microscopy to minimize contributions from autofluorescence or coloaded dyes (Fig. 10). The photoluminescence lifetime images were acquired for an 80 mm 80 mm sample area consisting of 256 256 pixels at an acquisition rate 0.84 mm/pixel. A 488 nm ps diode laser was employed for excitation. Fig. 10a reflects the apparent excited-state lifetime of N1, fitted with one lifetime in each pixel. As seen from the colors in the FLIM image (scale range from 0 (blue) to 26.21 (red)), N1 has a longer apparent lifetime inside the cell than DiI dye. N1 displays the strongest emission intensity inside of the nucleolus (red), whereas the lifetimes of DiI staining in the membranes of nuclei and cells (green and blue, respectively) are much shorter than that of the nucleolus. The AgeAu cluster N1 possesses a long emission lifetime, which avoids interference from autofluorescence by biomolecules when using FLIM. The development of time-gated microscopy renders the AgeAu cluster N1 a good alternative to the commercial nucleolus stain [40,41]. 4. Conclusions In summary, we have presented the intensely luminescent silver (I)-gold (I) cluster as a new nucleolus stain that is specifically concentrated in the nucleolus of living cells. The complex is not only compatible with living cells, but also more photostable than DAPI and Syto RNASelect. Of special note, FLIM can be applied to discriminate the long-lived excited states of N1 from autofluorescence and conventional dyes, and this gives new insight into the interaction between N1 and the cellular microenvironment and into nucleolus accessibility. This makes this complex a new candidate as a selective nucleolus fluorescent probe. Moreover, we carried out a systematic investigation of the cellular internalization mechanism for N1 and concluded that N1 was highly concentrated in the nucleoli compared to the membrane and cytoplasm through an energy-dependent, non-endocytic pathway. Interestingly,
specific staining by this complex may be related to the microenvironment of the nucleolus. The experiments involving the cell cycles are consistent with this conclusion. N1 will be of particular interest for the development of new agents for living cell-related studies. Acknowledgment This work was financially supported by China National Funds for Distinguished Young Scientists from the NSFC (20825101 and 21231004), SSTC (11XD1400200 and 10431903100), the State Key Basic Research Program of China (2012CB932403), and SLADP (B108). Thanks to Prof. C. Yan and Dr. J. Zhou for help in FLIM experiment. Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2013.02.032. References [1] Boisvert FM, van Koningsbruggen S, Navascues J, Lamond AI. The multifunctional nucleolus. Nat Rev Mol Cell Biol 2007;8:574e85. [2] Lam YW, Trinkle ML, Lamond AI. The nucleolus. J Cell Sci 2005;118:1335e7. [3] Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JHD, et al. Crystal structure of the ribosome at 5.5 Å resolution. Science 2001;292:883e96. [4] Carmo FM, Mendes SL, Campos I. To be or not to be in the nucleolus. Nat Cell Biol 2000;2:E107e12. [5] Balakin AG, Smith L, Fournier MJ. The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions. Cell 1996;86:823e34. [6] Liu X, Sun Y, Zhang Y, Miao F, Wang G, Zhao H, et al. A 2,7-carbazole-based dicationic salt for fluorescence detection of nucleic acids and two-photon fluorescence imaging of RNA in nucleoli and cytoplasm. Org Biomol Chem 2011;9:3615e8. [7] Shen R, Shen X, Zhang Z, Li Y, Liu S, Liu H. Multifunctional conjugates to prepare nucleolar-targeting CdS quantum dots. J Am Chem Soc 2010;132: 8627e34. [8] Haulgland RP. Assays for cell viability, proliferation and function. In: Spence MTZ, editor. In the handbook, a guide to fluorescent probe and labeling technologies. 10th ed. Eugene, Oregon: Invitrogen, Molecular Probes; 2005. p. 710e1. [9] You Y, Lee S, Kim T, Ohkubo K, Chae WS, Fukuzumi S, et al. Phosphorescent sensor for biological mobile zinc. J Am Chem Soc 2011;133:18328e42. [10] Botchway SW, Charnley M, Haycock JW, Parker AW, Rochester DL, Weinstein JA, et al. Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes. Proc Nat Acad Sci 2008;105: 16071e6. [11] Kuimova MK, Yahioglu G, Levitt JA, Suhling K. Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging. J Am Chem Soc 2008;130:6672e3. [12] Gratton E, Breusegem S, Sutin J, Ruan Q, Barry N. Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods. J Biomed Opt 2003;8:381e90. [13] Svensson FR, Abrahamsson M, Stromberg N, Ewing AG, Lincoln P. Ruthenium(II) complex enantiomers as cellular probes for diastereomeric interactions in confocal and fluorescence lifetime imaging microscopy. J Phys Chem Lett 2011;2:397e401. [14] Tadrous PJ. Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging. J Pathol 2000;191:229e34. [15] Zhang J, Fu Y, Lakowicz JR. Fluorescent metal nanoshells: lifetime-tunable molecular probes in fluorescent cell imaging. J Phys Chem C 2011;115:7255e60. [16] Zhao Q, Huang CH, Li FY. Phosphorescent heavy-metal complexes for bioimaging. Chem Soc Rev 2011;40:2508e24. [17] Lo KKW, Tsang KHK, Sze KS, Chung CK, Lee TKM, Zhang KY, et al. Non-covalent binding of luminescent transition metal polypyridine complexes to avidin, indole-binding proteins and estrogen receptors. Coord Chem Rev 2007;251: 2292e310. [18] Baggaley E, Weinstein JA, Williams JAG. Lighting the way to see inside the live cell with luminescent transition metal complexes. Coord Chem Rev 2012;256: 1762e85. [19] Leung SK, Kwok KY, Zhang KY, Lo KKW. Design of luminescent biotinylation reagents derived from cyclometalated iridium(III) and rhodium(III) bis(pyridylbenzaldehyde) complexes. Inorg Chem 2010;49:4984e95. [20] Lo KKW, Choi AWT, Law WHT. Applications of luminescent inorganic and organometallic transition metal complexes as biomolecular and cellular probes. Dalton Trans 2012;41:6021e47.
M. Chen et al. / Biomaterials 34 (2013) 4284e4295 [21] Lo KKW, Li SPY, Zhang KY. Development of luminescent iridium(III) polypyridine complexes as chemical and biological probes. New J Chem 2011;35: 265e87. [22] Leung SK, Liu HW, Lo KKW. Functionalization of luminescent cyclometalated iridium(III) polypyridine complexes with a fluorous moiety: photophysics, protein-binding, bioconjugation, and cellular uptake properties. Chem Commun 2011;47:10548e50. [23] Kumar R, Ohulchanskyy T, Turowski S, Thompson M, Seshadri M, Prasad P. Combined magnetic resonance and optical imaging of head and neck tumor xenografts using gadolinium-labelled phosphorescent polymeric nanomicelles. Head Neck 2010;2:35. [24] Koo CK, So LKY, Wong KL, Ho YM, Lam YW, Lam MHW, et al. A triphenylphosphonium-functionalised cyclometalated platinum(II) complex as a nucleolus-specific two-photon molecular dye. Chem Eur J 2010;16:3942e50. [25] Yu J, Parker D, Pal R, Poole RA, Cann MJ. A europium complex that selectively stains nucleoli of cells. J Am Chem Soc 2006;128:2294e9. [26] New EJ, Congreve A, Parker D. Definition of the uptake mechanism and subcellular localisation profile of emissive lanthanide complexes as cellular optical probes. Chem Sci 2010;1:111e8. [27] Montgomery CP, Murray BS, New EJ, Pal R, Parker D. Cell-penetrating metal complex optical probes: targeted and responsive systems based on lanthanide luminescence. Acc Chem Res 2009;42:925e37. [28] Crocker J, Nar P. Nucleolar organizer regions in lymphomas. J Path 1987;151: 111e8. [29] De Capoa A, Ferraro M, Lavia P, Pelliccia F, Finazzi-Agrò A. Silver staining of the nucleolus organizer regions (NOR) requires clusters of sulfhydryl groups. J Histochem Cytochem 1982;30:908e11. [30] Heukeshoven J, Dernick R. Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 1985;6:103e12.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
A phosphorescent silver(I)egold (I) cluster complex that specifically lights up the nucleolus of living cells with FLIM imaging Min Chen a, Zhen Lei b, Wei Feng a, Chunyan Li a, Quan-Ming Wang b, *, Fuyou Li a, * a b
Department of Chemistry & Institute of Biomedicine Science, Fudan University, Shanghai 200433, PR China State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemistry, Xiamen University, Xiamen 361005, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2012 Accepted 10 February 2013 Available online 11 March 2013
The phosphorescent silver(I)egold(I) cluster complex [CAu6Ag2(dppy)6](BF4)4 (N1) selectively stains the nucleolus, with a much lower uptake in the nucleus and cytoplasm, and exhibits excellent photostability. This AgeAu cluster, which has a photoluminescent lifetime of microseconds, is particularly attractive as a probe in applications of time-gated microscopy. Investigation of the pathway of cellular entry indicated that N1 permeates the outer membrane and nuclear membrane of living cells through an energydependent and non-endocytic route within 10 min. High concentrations of N1 in the nucleolus have been quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and transmission electron microscopy coupled with an energy dispersive X-ray analysis (TEM-EDXA), which also helped to elucidate the mechanism of the specific staining. Intracellular selective staining may be correlated with the microenvironment of the nucleolus, which is consistent with experiments conducted at different phases of the cell cycle. These results prove that N1 is a very attractive phosphorescent staining reagent for visualizing the nucleolus of living cells. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Silver(I)egold (I) cluster complex Nucleolus Selective staining FLIM imaging
1. Introduction As the most prominent structure in a cell nucleus, the nucleolus plays a central regulatory role in cell growth and proliferation [1,2]. In particular, the nucleolus is the key site in which ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins [3e5]. Therefore, the visualization of nucleolus-related events is a valid demand. In light of the sub-cellular resolution and high sensitivity of fluorescence microscopy, the visualization of nucleolus by fluorescence microscopy has attracted increasing attention. Nowadays, there are several fluorophores used for staining the nucleolus specifically in living cells. For example, Wong et al. developed a 2,7-carbazole-based dicationic salt for two-photon fluorescence imaging of RNA in the nucleolus and cytoplasm [6]. Liu et al. have demonstrated the utility of a bidentate 1,2,3-triazole-based ligand modification of CdS quantum dots (QDs) for targeting the nucleolus of cells [7]. However, their potential toxicity of CdS QDs is still an issue, which limits their utility. To date, Syto RNASelect is the only commercially available dye for live cell RNA imaging [8].
* Corresponding authors. E-mail addresses: [email protected] (Q.-M. Wang), [email protected] (F. Li). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.02.032
When applied in real biological samples, the fluorescent probes often suffer from the interference from autofluorescence of biosamples and scattered excitation light, reducing the signal-to-noise ratio (SNR). To avoid such an interference, one effective solution is to use time-gated luminescence imaging based on long-lifetime emissive materials as probes [9,10]. The advantage of measuring the lifetime of photoluminescence emission is that this parameter is directly dependent upon excited-state reactions but independent of compound concentration, light intensity and photobleaching, so it is an ideal parameter for monitoring the local environment of the materials. Fluorescence lifetime imaging microscopy (FLIM) [11e15] is a technique used to study the nanosecond decay kinetics of the electronic excited state. To date, however, no example of FLIM imaging has been reported for nucleolus staining of living cells. For fluorescence lifetime imaging, a long-lifetime emissive materials for nucleolus staining should be developed. Heavy-metal complexes and lanthanide complexes are the ideal candidates as long-lifetime emissive probes [16e24]. Recently, Lo et al. reported that iridium(III) dipyridoquinoxaline complexes could stain the nucleoli of MDCK cells [19], and Parker et al. reported several europium and terbium complexes selectively stained the nucleoli of cells [25e27]. For these small-molecule stains, cytoplasm staining often accompanies staining of the nucleolus and interferes with the observation of nucleolus staining.
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Scheme 1. Possible pathway of nucleolus staining with the Ag(I)eAu(I) cluster complex [CAu6Ag2(dppy)6](BF4)4 (N1).
It is well known that nucleolus organizing regions (NORs) contains a lot of argyrophilic proteins which have a high affinity of silver (AgNOR proteins) [28,29]. Such NORs can be identified by silver staining, and some silver-staining methods have been developed for immunolocalization of nucleolus proteins [30e33]. To data, however, these reported silver stains are non-emissive. In this present study, we demonstrated a phosphorescent silver(I)egold(I) cluster complex [CAu6Ag2(dppy)6](BF4)4 (N1, Scheme 1), which has a long lifetime of w32 ms, to be an excellent luminescent stain for nucleolus-specific bioimaging. Not only a red emission contrasting with the green fluorescence of commercial SYTO RNASelect, but also well photostability, allow the complex N1 for its continuous tracking studies. Of special note, complex N1, can specifically light up the nucleolus of a living cell by using the fluorescence lifetime imaging method. 2. Experimental section 2.1. Materials and general instruments Phosphate buffered saline (PBS), Fetal bovine serum (FBS), DMSO, 2-deoxy-Dglucose, oligomycin, Cytochalasin D, Filipin, NH4Cl, Lealanine (Ala), Learginine (Arg), Leasparagine (Asn), Leaspartic acid (Asp) Leglutamine (Gln), Leglycine (Gly), Leisoleucine (Ile), Leleucine (Leu), Lelysine (Lys), Lephenylalanine (Phe), Leproline (Pro), Leserine (Ser), Lethreonine (Thr), Letryptophan (Try), Letyrosine (Tyr), Levaline (Val), Leglutamic acid (Glu), Lecysteine (Cys), Lemethionine (Met), Lehistidine (His), bovine serum albumin (BSA), deoxyribonucleotide triphosphate (dNTP) and CT DNA were obtained from Acros. Syto RNASelect, DAPI, DiI, and cell culture reagents were purchased from Invitrogen. Metabolic inhibitors (oligomycin, deoxyglucose) and endocytosis inhibitor (Cytochalasin D, Filipin) were obtained from SigmaeAldrich. UVevisible spectra were recorded on a Shimadzu UV-2550 spectrometer. Steadyestate emission experiments at room temperature were measured on an Edinburgh instrument FL-900 spectrometer with Xe lamp as excitation source. Luminescence lifetime studies were performed with an Edinburgh FL-900 photocounting system with a hydrogenefilled lamp as the excitation source. Luminescence quantum yield (f) of N1 in aerated solution were measured with reference to terpyridine ruthenium Ru (bpy)3 (f ¼ 0.028). The cluster complex N1 was synthesized according to our previous literature [34].
24 h at 37 C under 5% CO2. The cluster complex N1 (100 mL/well) at different concentrations (1.6, 3.1, 6.2, 12.5, 25, 50, 100 mM, diluted in RPMI 1640) was added to the wells of the treatment group, and RPMI 1640 to the negative control group. The cells were then incubated for 24 h at 37 C under 5% CO2. Thereafter, MTT (20 mL; 5 mg/mL) was added to each well, and the plate was incubated for an additional 4 h at 37 C under 5% CO2. After changing the culture medium to 100 mL DMSO, the assay plate was allowed to stand at room temperature for 10 min. An enzyme-linked immunosorbent assay (ELISA) reader (infinite M200, Tecan, Austria) was used to measure the OD570 (Absorbance value) of each well with background subtraction at 690 nm. The following formula was used to calculate the viability of cell growth: Cell viability ð%Þ ¼ ðmean of absorbance value of treatment group=mean of A value of controlÞ 100
2.4. Luminescence imaging of live cells incubated with N1 Luminescence imaging including xy-scan, lambda-scan, T-scan, and time-lapseimaging, was performed with an Olympus FluoView FV 1000 confocal fluorescence microscope and a 60 oil-immersion objective lens. Cells incubated with N1 were excited at 405 nm with a semiconductor laser, and the emission was collected at 600 20 nm. Quantization by line plots was accomplished using the software package provided by Olympus instruments. DAPI was excited using a laser at 405 nm, and the emission was collected at 460 20 nm. 2.4.1. Luminescence imaging of live cells after treatment with metabolic and endocytic inhibitors Cells were detached from the culture and were preincubated with 50 mM 2-deoxy-D-glucose and 5 mM oligomycin, with 5 mM Cytochalasin D, or with 5 mM Filipin, or 50 mM NH4Cl in PBS, for 1 h at 37 C. The cells were then washed with PBS and incubated solely with 10 mM in DMSO/PBS （pH 7.4, 1:99, v/v） for 10 min at 37 C. Before imaging, the cells were washed three times with PBS.
2.2. Cell culture (Human epithelial cervical cancer cell line) Hela, (Human nasopharyngeal carcinoma cell line) KB and (Human normal hepatocyte) LO2 cell lines were provided by the Institute of Biochemistry and Cell Biology, (The Chinese Academy of Sciences) CAS. Hela cells were grown in (Dulbecco’s modified Eagle’s medium) DMEM supplemented with 10% v/v FBS and 1% antibiotic/antimycotic solution (penicillin and streptomycin, Invitrogen). KB and LO2 cells were grown in RPMI 1640 supplemented with 10% v/v FBS and with and 1% antibiotic/antimycotic solution. All cells cultures were kept at 37 C in a humidified incubator with 5% CO2. 2.3. Cytotoxicity assay of the cluster N1 The in vitro cytotoxicity was evaluated by performing the methyl thiazolyl tetrazolium (MTT, SigmaeAldrich) assay in Hela cells. Cells growing in log phase were seeded into 96-well cell-culture plate at 5 103/well and then incubated for
Fig. 1. Absorption (black line) and photoluminescence (red line) spectra of the N1 solution in DMSO/PBS (1:99, v/v), lex ¼ 405 nm. Inset: photographs showing the brightfield and luminescence photos of the N1 solution in DMSO/PBS (1:99, v/v) under excitation from UV lamp (lex ¼ 365 nm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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2.4.2. Colocalization imaging of live cells incubated with N1 and Syto RNASelect The living cells were plated on 14 mm glass coverslips and allowed to adhere for 24 h. After washing with PBS, the cells were incubated with 10 mM N1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 37 C, and then further incubated with Syto RNASelect for another 20 min before imaging. 2.4.3. Luminescence imaging of fixed cells incubated with N1 The cells were detached from the culture and were fixed with 4% paraformaldehyde at 4 C for 30 min. After washing with PBS, the fixed cells were incubated with 10 mM N1 in DMSO/PBS （pH 7.4, 1:99, v/v） for 10 min at 37 C. After washing with PBS, the coverslips were separated from the chamber, and the cells were mounted with 10% glycerol and sealed with nail varnish on a glass substrate. 2.5. Fluorescent lifetime imaging microscopy (FLIM) After incubation of N1, the cells were fixed by addition of 4% paraformaldehyde/2.5% glutaraldehyde in PBS (pH 7.4, 1 mL) for 1 h. An inverse
time-resolved laser scanning confocal microscope was employed for FLIM experiments (Nikon, A1R-si). The fixed Hela cells attached onto a slide glass were covered with a thin cover glass, on which an excitation beam was focused. A 488 nm ps pulsed diode laser with 20 MHz repetition rate was used for excitation. The instrumental response function of the system was 30 ps at fwhm. A filter of FITC (515-30) and a single photon avalanche diode were used to collect emissions from the Hela cells. The time-gated emission signals were obtained with a TCSPC technique. Typically, an 80 mm 80 mm sample area consisting of 256 256 pixels was scanned with an acquisition rate of 0.83 ms/pixel. FLIM images and their exponential fits were analyzed using NIS-Elements C software provided by manufacturer. 2.6. Flow cytometry analysis Cellular uptake of N1 under different conditions was analyzed by flow cytometry (Beckman Counter). Cells were filtered through 41 mM nylon mesh in preparation to analyze at a rate of 200e500 cells/s using FACS flow as at the sheath fluid and 10,000 events were recorded for each sample.
Fig. 2. Confocal luminescent images of living LO2 cells (a), Hela cells (b), and KB cells (c) incubated with 10 mM N1 in PBS (pH 7.4) for 10 min at 25 C. (d) Corresponding to extracellular region (1 and 4), cytoplasm (2), and nuclear region (3). (e) Quantification of the luminescence intensity profile of N1-treated KB cells shown in panel d. (lex ¼ 405 nm, lem ¼ 600 20 nm). Scale bar: 20 mm.
M. Chen et al. / Biomaterials 34 (2013) 4284e4295 2.7. Cellular uptake and mechanism 2.7.1. Analysis of inhibitor experiment Hela cells preincubated with or without 50 mM 2-deoxy-D-glucose and 5 mM oligomycin in PBS for 1 h at 37 C were incubated with 10 mM N1 in PBS (pH 7.4) for 10 min at 37 or 4 C. After incubation, adherent cells were detached from the surface of culture dishes by treatment with trypsin-EDTA solution (Hangzhou Genom Biomedical & Technology Co. Ltd). The mean fluorescence was measured for 10,000 cells by using flow cytometry. 2.7.2. Cell cycle analysis Hela cells were grown for 24 h in culture (DMEMþ10% FBS) and then incubated DMEM containing colchicine at the final concentrations of 1.0 mg/mL to synchronize cells in the metaphase (M phase). After 7 h, suspended cells which were in M phase were collected by shake off to centrifugate at 1000 g for 10 min. The supernatant solution was removed after centrifugation, and then one-quarter of cells were fixed with cold methanol (70%) at 20 C until analysis. The other three quarters were equally released in three dishes containing fresh culture medium (DMEM) and collected at the indicated time points, then fixed with 20 C cold methanol (70%) for further analysis. To prepare for flow cytometry analysis, all fixed cell suspensions were centrifuged at 1000 rpm for 10 min, and cells were resuspended in 0.25 mL PBS containing 200 mM/mL RNase (SigmaeAldrich) and incubated at 37 C for 30 min. Then, 100 mg/mL of propidium iodide solution was added. After incubating at 37 C for 10 min, cells were already for further analysis by flow cytometry. 2.7.3. Interaction of N1 and biomolecules The interaction of N1 with amino acids, bovine serum albumin (BSA), deoxyribonucleotide triphosphate (dNTP), and CT DNA has been investigated by luminescent emission titration. Herein, L-alanine (Ala), L-arginine(Arg),L-asparagine (Asp), L-glutamine (Gln), L-glycine (Gly), L-isoleucine (Ile),L-leucine (Leu),L-lysine (Lys),L-pheneylalanine (Phe), L-proline (Pro), L-serine (Ser), L-threonine (Thr), L-tryptophan (Try), L-tyrosine (Tyr), L-valine (Val), L-glutamic acid (Glu), L-cysteine (Cys), L-methionine (Met), L-histidine (His) were used as examples of amino acids.
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2.7.4. Determination of gold and silver content After digestion by trypsin-EDTA solution, Hela cells were divided into two parts. When the cells adhered to the culture flask, the culture medium was changed to 10 mL of PBS with 10 mM N1. The cells were incubated with N1 for 10 min at 37 C. Thereafter, they were carefully washed with PBS, and then the nuclei and cytoplasm were extracted using a nucleus extraction kit (Nanjing KeyGen Biotech. Co., Ltd.). The gold and silver concentration in the samples was determined by an inductively coupled plasma atomic emission spectroscopy (ICP-AEC Thermo Elemental Co., Ltd.). 2.7.5. Transmission electron microscopy and energy-dispersive X-ray analysis (TEM-EDXA) After incubation with 10 mM N1 for 10 min, the cells were thoroughly washed with PBS buffer. Cells were then scraped from the culture dish to centrifuge at 2000 g for 5 min, and the supernatant was removed. The cell pellets were fixed by addition 4% paraformaldehyde in PBS (pH 7.4, 1 mL) for 1 h. Then the cells were rinsed with PBS buffer, postfixed using 4% aqueous solution of OsO4 (Caution! Extremely toxic) for 1 h. Subsequently the cells were rinsed with distilled water, and stained with 0.5% uranyl acetate (0.5 mL, in 30% ethanol) for 1 h. Cells were then gradually dehydrated using a series of ethanol solutions (30, 60, 70, 80, and 100%) and embedded in epoxy resin.
3. Results and discussion 3.1. Photophysical properties of complex N1 Complex N1 [CAu6Ag2(dppy)6](BF4)4 was synthesized according to our previous method [34], and can be dissolved in a solution of DMSO/PBS (1:99, v/v). The electronic absorption and emission spectra of N1 in DMSO/PBS (1:99, v/v) solution at room
Fig. 3. Colocalization images of living KB cells incubated with 10 mM N1 in PBS for 10 min at 25 C and then incubated with Syto RNASelect. (a) Green channel for Syto RNASelect (lex ¼ 488 nm, lem ¼ 530 20 nm). (b) Red channel for N1 (lex ¼ 405 nm, lem ¼ 600 20 nm). (c) Bright-field image. (d) Colocalization of green and red luminescence is shown as yellow pixels. Scale bar: 30 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Three-dimensional luminescence images of live HeLa cells loaded with 10 mM N1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 25 C. The nucleus was stained blue with DAPI. Panel a is a xy image obtained at z ¼ 3.35 mm, while panels b and c display the yz and xz cross sections (z ¼ 12.25e18.35 um) taken at the lines shown in panel a, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Real-time monitoring of N1 uptake and intracellular transport in cultured Hela cells over a 700 s period. The views were monitored by confocal luminescence microscope. (a) Luminescence images of living HeLa cells incubated with 10 mM N1 in DMSO/PBS (pH 7.4, 1:99, v/v) at 37 C at selected time points (lex ¼ 405 nm, lem ¼ 600 20 nm; scale bar: 10 mm). T0 indicates a short time (<30 s) for cells entering the focal plane after N1 was added. (b) Luminescence image of Hela cells incubated with N1 for 1038 s. (c) Time course of luminescence intensity in the nucleolus (regions 3 and 4 in b), membrane (region 2 in b) and nucleus (region 1 in b).
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Fig. 6. Effect of incubation temperature and endocytic inhibitors on cellular uptake of N1 in Hela cells. (a) The cells were pretreated with 50 mM 2-deoxy-D-glucose and 5 mM oligomycin in PBS for 1 h at 37 C, and then incubated with 10 mM N1 at 37 C for 10 min (bec) The cells were incubated with 10 mM N1 for 10 min at 4 C, 37 C, respectively. (def) The cells were pretreated with endocytic inhibitors Cytochalasin D (5 mM), Filipin (5 mg/mL) and NH4Cl (50 mM), respectively, and then incubated with 10 mM N1 at 37 C for 10 min (lex ¼ 405 nm, lem ¼ 600 20 nm).
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temperature are shown in Fig. 1. N1 has intense absorption bands in the ultraviolet region of 280e480 nm. Moreover, upon excitation at 405 nm, N1 exhibits visible emission at 650 nm. With reference to terpyridine ruthenium Ru(bpy)3 as a standard (f ¼ 0.028) in deionized water, the luminescence quantum yield of N1 in DMSO/ PBS solution was measured to be 1.11% (f) at room temperature and 1.44% in a degassed solution. Compared with the luminescence quantum yield of 30% in chloroform solution, the photoluminescence emission of N1 shows a significant dependence on solvent polarity. In addition, the luminescent lifetime of N1 in DMSO/PBS solution was measured to be w32 ms (Fig. S1 in the Supporting Information). Such a long lifetime on the microsecond scale suggests the emission has a phosphorescent nature. 3.2. Selective nucleolus staining Three kinds of cells, including two cancer cells (Hela and KB) and one normal cell (LO2), were employed for bioimaging testing. Typically, a cell sample was incubated with 10 mM N1 for 10 min and then cell images were recorded by confocal fluorescence microscopy (Fig. 2). The emission spectrum of living cells incubated with N1 is shown in Fig. S2. Interestingly, an intense luminescence signal was detected in the nucleolus region, while the luminescence in the remainder of the cells was very weak (Fig. 2c). Quantification of the luminescence intensity profile of N1-treated KB cells revealed an extremely high signal intensity ratio for the nucleolus (region
3, z4000) compared to both the nucleus and cytoplasm (regions 1, 2 and 4, z0). Such high nucleolus/nucleus and nucleolus/cytoplasm luminescence intensity ratios suggest an exclusive staining of the cell nucleolus with N1 (Fig. 2d,e). Similarly, upon incubation with N1, the normal cell line LO2, also exhibited intense luminescence in the nucleolus region (Fig. 2a). These facts indicate that nucleolus-specific staining by N1 occurs regardless of the type of cell line. To confirm the luminescence spots observed in the nucleolus (Fig. 2), colocalization experiments were carried out with a confocal fluorescence microscope (Fig. 3). Living cells were loaded with N1 and the commercial nucleolus stain Syto RNASelect. The luminescence (red) of N1 (excitation at 405 nm, emission at 600 20 nm) was completely colocalized with that (green) obtained at the wavelength for Syto RNASelect (excitation at 488 nm, emission at 530 20 nm). The colocalization was evident with the observation of bright yellow spots in the region of the nucleolus (Fig. 3d), suggesting that the targeted organelles of N1 are nucleoli. To further verify that the localization of N1 is internalized within the nucleolus, three-dimensional (3D) imaging of live cells was carried out. With the advantage of the confocal technique, which allows filtering by depth of field, the 3D image is built up through separately recorded images of optical slices (Z-scans) of the sample. In this experiment, Hela cells were coloaded with N1 and the commercial nucleus stain DAPI, and were then imaged by serially scanning at increasing depths along the z-axis. As shown in Fig. 4a,
Fig. 7. Flow cytometric histogram profile (a) and the ICP-MS analysis (b) of cellular uptake of N1 in Hela cells. Hela cells were incubated with 10 mM N1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 37 C, 4 C, and 37 C after cells had been preincubated with 50 mM 2-deoxy-D-gluscous and 5 mM oligomycin in PBS for 1 h, respectively.
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the nucleus was stained blue with DAPI, while the nucleolus regions were clearly lighted up by N1. The nucleoli of Hela cells were perfectly visualized in the yz and xz cross-sectional images (Fig. 4b,c) through 3D reconstruction of serial xy sections, which indicates that N1 stains the nucleolus rather than other locations in the cell. In addition, the presence in the nucleolus of the Ag-Au cluster N1 was further confirmed by transmission electron microscopy (TEM) of the cells incubated with N1 (Fig. S3).
3.3. Kinetic processing of cellular uptake of N1 To monitor the dynamic process of nucleolus staining by N1 in real time, continuous imaging of living Hela cells was performed (Fig. 5 and movie in Supporting Information). In the cells prior to incubation with N1 (<500 s), luminescence was observed near the plasma membranes. After incubation with the N1 solution for 500 s, the luminescence signal of N1 appeared in the nucleolus. At 700 s, the red luminescence became strong in the nucleolus, indicating more and more N1 was transported to and accumulated in the nucleolus.
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Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2013.02.032. Quantification of the photoluminescence intensity in the microscopic image of the Hela cells revealed that the addition of N1 triggered a continuous increase in luminescence intensity at the nucleolus, while the signal from the cytoplasm remained negligible (Fig. 5b, c). In addition, the fluorescence signals of the membrane shown in Fig. 5b are much stronger than those in Fig. 2. It is probable that the cells in Fig. 5b were continuously incubated with N1, whereas the medium containing the AgeAu cluster which were not taken up by the cells in Fig. 2 was washed by the PBS buffer. Those results indicate that a 700 s incubation of N1 at 37 C is sufficient to detect a significant intensity increase in the nucleoli and that specific nucleolus staining can be effectively performed by N1 even in the absence of a molecular transporter. 3.4. The pathway of cellular uptake of N1 The internalization of extracellular material can occur through energy-independent (facilitated diffusion, passive diffusion) and
Fig. 8. The histogram of the cell cycle obtained using flow cytometry and the confocal images in specific cell cycle phase cells treated 10 mM N1 for 10 min. (a) Histogram of the cell cycle obtained from Hela cells cultured under different collected time after synchronization. (b) Bright-field images of Hela cells in cell cycle images. (c) Luminescence images. (d) Overlay of luminescence image and their corresponding brightfield images.
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energy-dependent (endocytosis, active transportation) pathways [35]. Herein, a detailed investigation of the cellular internalization mechanism and uptake pathway of N1 was carried out, at different temperatures with various inhibitors of endocytic pathways (Fig. 6). The effect of temperature on the cellular uptake of N1 was investigated by confocal luminescence microscopy. As shown in Fig. 6b, luminescence was hardly observed for N1-incubated cells at 4 C, in contrast to the case at 37 C (Fig. 6c). Cell metabolism and physiological activity would be suppressed under low-temperature conditions. Hence, N1 is likely to enter the cells by an energydependent pathway. To confirm this deduction, we investigated the effect of metabolic inhibitors 2-deoxy-D-glucose and oligomycin on cellular uptake of N1. As shown in Fig. 6a, when the cells were preincubated with metabolic inhibitors, the intracellular luminescence was significantly suppressed. To quantitatively analyze the cellular uptake effect for the AgeAu cluster, flow cytometry and ICP data were used to study the intracellular fluorescence and metal contents (Fig. 7). The mean fluorescence of Hela cells incubated at 37 C was higher than that of cells incubated at 4 C or preincubated with metabolic inhibitors. The ICP-MS data confirmed this result. These results imply that energy plays a very important role during the process by which N1 cross the plasma membrane and eventually reach the nucleolus. Next, we investigated the possibility of cellular entry by N1 through an endocytosis or positive pathway that is an energy-
dependent route. As shown in Fig. 6def, when cells were preincubated with an endocytic inhibitor such as cytochalasin D, filipin or NH4Cl, which block different endocytic pathways, no significant reduction in luminescence signal in the nucleolus was observed. That is, there was no measurable decrease in the cellular uptake of N1 under endocytosis-inhibiting conditions. Therefore, the nucleolus-specific staining of living cells by N1 is related to an energy-dependent non-endocytic pathway across the cell membrane and nuclear membrane. The cell membrane controls the movement of substances in and out of cells. When the cells were fixed, the plasma membrane and the nuclear membrane became permeable. Interestingly, when the fixed cells were incubated with N1, an intense luminescence signal from N1 (lex ¼ 405 nm, lem ¼ 600 20 nm) was detected in the whole fixed cells, including the nucleus and cytoplasm (Fig. S4). The generalized, diffuse whole-cell staining pattern for the fixed cells was significantly different from the specific nucleolus staining seen in living cells. Therefore, it can be safely deduced that N1 is transported by an active mechanism rather than passive diffusion. 3.5. The mechanism of selective nucleolus staining of N1 Considering that N1 is red emissive in specific staining of N1 in the nucleoli may possible causes: either N1 is specifically nucleolus or N1 is distributed throughout
solution (Fig. 1), the be attributed to two concentrated in the the whole cell, and
Fig. 9. Comparison of photobleaching of N1 and Syto RNASelect in confocal luminescence microscopy imaging. The living Hela cells were incubated with DAPI (blue) and N1 (red), respectively. (a) Confocal luminescence microscopy images of 10 mM N1-pretreated Hela cells incubated with Syto RNASelect for another 20 min. Green channel for Syto RNASelect (lex ¼ 488 nm, lem ¼ 530 20 nm), Red channel for N1 (lex ¼ 405 nm, lem ¼ 600 20 nm). (b) Quantitative analysis of the changes in fluorescence intensities of N1 (red line) and Syto RNASelect (black line) in panel a. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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enhanced luminescence is generated in the nucleolus. To verify the real reason, the amounts of N1 in the nucleolus and the remaining parts of the cell should be measured. Herein, a quantitative method utilizing transmission electron microscopy coupled with an energy dispersive X-ray analysis (TEM-EDXA) was used to determine the concentrations of N1 in the nucleolus and cytoplasm. As shown in Fig. S5, gold was detected in the nucleolus at a concentration of 3.85% dry weight, which is 30 times higher than the concentration in the cytoplasm, which was approximately 0.10% dry weight. Furthermore, the amounts of Au in the cytoplasm and nuclei of N1 stained cells were quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AEC). The results of quantitative distribution revealed similar proportions in the nuclei (0.38 pg Au/ cell) and cytoplasm (0.32 pg Au/cell), indicating that more than 54.29% of Au accumulated in the nuclei. Considering the very small volume of the nucleolus compared with the cytoplasm and nuclei, the concentration of N1 in the nucleolus is much higher than that in the cytoplasm. These results proved that N1 is concentrated in the nucleolus instead of being distributed over the whole cell. It was unclear whether the interactions between N1 and the biomolecules affect the luminescence characteristics of N1 or not. To address this question, we investigated the luminescence change of N1 upon addition of numerous substances (including CT DNA, BSA, RNA, various amino acids and triphosphates) by luminescence analysis techniques. As shown in Fig. S6, the emission intensity of N1 in BSA increased nearly three times compared with that of N1 in PBS, and two-fold enhancement was observed in DNA. Fig. S7 shows that the quantum yield of N1 is strongly influenced by the solvent polarity. So, it can be deduced that N1 in nonpolar (or hydrophobic) microenvironment will display intense luminescence emission, which is relative to the increase in luminescence quantum yield of N1 in BSA and DNA. But there was nearly no change in the RNA solution. This is quite different from the case of Syto
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RNASelect, which gives rise to an enhanced fluorescence on binding to RNA [8]. The N1 in solutions of various amino acids and triphosphates behaves similarly to that in TriseHCl/NaCl. These results indicate that high concentration in nucleolus may be very important for specific staining. As we know, the nucleolus is a dynamic structure that is disassembled and reassembled at different phases of the cell cycle. Therefore, the distribution of N1 during the different phases of the cell cycle was examined by flow cytometry and confocal microscopy imaging (Fig. 8). For convenience, the cycle has been classified into four phases (G1/S/G2/M). Between the mitosis (M) phase and the S phase is the G1 phase, which has a normal diploid (2N) DNA content, whereas the G2 (between the S phase and M phase) cells have tetraploid (4N) DNA content, the same as the M phase. S phase cells contain intermediate amounts of 2N and 4N [36e39]. In practice, Hela cells were collected at the indicated time point in accordance with the lengths of the individual phases of the cell cycle after the cells were synchronized by colchicine. The collection of cells was carried out by monitoring the content of DNA intercalated with propidium iodide (PI) dye to identify the different phases. As shown in Fig. 8a, the percentages of cells existing within the various phases of the cell cycle were calculated by gating on G0, G1, S, and [G2 þ M] cell populations (it is difficult to differentiate between cells in the G2 and M phases, as both have double DNA), and the prominent peak represents the specific cell cycle state in the histogram. Fluorescence microscopy provides a sensitive means of acquiring information about the organization and dynamics of complex cellular structures. Confocal images of stained cells clearly illustrate the distribution of N1 compared with cycling cells (Fig. 8bed). The M phase cells have no nucleoli, whereas cells in other phases all have nucleoli. Interestingly, N1 stains whole cells in the M phase but only lighted up the nucleoli in the other three phases. The special environment of the nucleolus enables N1 to
Fig. 10. Fluorescence lifetime microscope images of Hela cells treated with 10 mM N1 for 10 min (aed) Images of N1 luminescence which was measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. (e) False color fluorescence lifetime images of N1 in Hela cells under TCSPC FLIM equipment, excited at 488 nm.
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distinguish the nucleolus from the other parts of the cell. These data distinctly verify that specific staining by N1 is closely related to the environment of the nucleolus. On the basis of the above discussion, we propose a mechanism for nucleolus staining by N1 as follows: (a) the specific staining is mainly associated with N1 concentration in the nucleoli; (b) the microenvironment of the nucleolus may account for N1 of the specific staining in the nucleolus. 3.6. Cytoxicity and photostability of N1 A key consideration is whether the cells studied remain healthy after incubation with N1. By using the MTT assay, cell viabilities were estimated to be greater than 80% in the presence of 10 mM N1. This indicates that N1 has low toxicity under luminescence nucleolus imaging conditions (Fig. S8). Moreover, the photostability of N1 was compared with that of the known fluorescent dye, DAPI. Hela cells were incubated with N1 (10 mM, 10 min), and thoroughly rinsed with fresh medium to remove floating cells. The cells were then treated with DAPI (50 mM, 15 min), and the extracellular solution was removed completely before imaging. Continuous illumination with a 405 nm laser with a power of 1.4 mW in the focal plane induced significant photobleaching of DAPI, whereas N1 exhibited a better photostability (Fig. S9). In addition, N1 is more photostable than Syto RNASelect in fixed cells (Fig. 9). The results indicate that N1 displays a better photostability than DAPI and Syto RNASelect do. 3.7. Fluorescent lifetime imaging microscopy (FLIM) As phosphorescent gold-silver cluster exhibit long lifetime, the N1 stained cells can be monitored with time-gated microscopy to minimize contributions from autofluorescence or coloaded dyes (Fig. 10). The photoluminescence lifetime images were acquired for an 80 mm 80 mm sample area consisting of 256 256 pixels at an acquisition rate 0.84 mm/pixel. A 488 nm ps diode laser was employed for excitation. Fig. 10a reflects the apparent excited-state lifetime of N1, fitted with one lifetime in each pixel. As seen from the colors in the FLIM image (scale range from 0 (blue) to 26.21 (red)), N1 has a longer apparent lifetime inside the cell than DiI dye. N1 displays the strongest emission intensity inside of the nucleolus (red), whereas the lifetimes of DiI staining in the membranes of nuclei and cells (green and blue, respectively) are much shorter than that of the nucleolus. The AgeAu cluster N1 possesses a long emission lifetime, which avoids interference from autofluorescence by biomolecules when using FLIM. The development of time-gated microscopy renders the AgeAu cluster N1 a good alternative to the commercial nucleolus stain [40,41]. 4. Conclusions In summary, we have presented the intensely luminescent silver (I)-gold (I) cluster as a new nucleolus stain that is specifically concentrated in the nucleolus of living cells. The complex is not only compatible with living cells, but also more photostable than DAPI and Syto RNASelect. Of special note, FLIM can be applied to discriminate the long-lived excited states of N1 from autofluorescence and conventional dyes, and this gives new insight into the interaction between N1 and the cellular microenvironment and into nucleolus accessibility. This makes this complex a new candidate as a selective nucleolus fluorescent probe. Moreover, we carried out a systematic investigation of the cellular internalization mechanism for N1 and concluded that N1 was highly concentrated in the nucleoli compared to the membrane and cytoplasm through an energy-dependent, non-endocytic pathway. Interestingly,
specific staining by this complex may be related to the microenvironment of the nucleolus. The experiments involving the cell cycles are consistent with this conclusion. N1 will be of particular interest for the development of new agents for living cell-related studies. Acknowledgment This work was financially supported by China National Funds for Distinguished Young Scientists from the NSFC (20825101 and 21231004), SSTC (11XD1400200 and 10431903100), the State Key Basic Research Program of China (2012CB932403), and SLADP (B108). Thanks to Prof. C. Yan and Dr. J. Zhou for help in FLIM experiment. Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2013.02.032. References [1] Boisvert FM, van Koningsbruggen S, Navascues J, Lamond AI. The multifunctional nucleolus. Nat Rev Mol Cell Biol 2007;8:574e85. [2] Lam YW, Trinkle ML, Lamond AI. The nucleolus. J Cell Sci 2005;118:1335e7. [3] Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JHD, et al. Crystal structure of the ribosome at 5.5 Å resolution. Science 2001;292:883e96. [4] Carmo FM, Mendes SL, Campos I. To be or not to be in the nucleolus. Nat Cell Biol 2000;2:E107e12. [5] Balakin AG, Smith L, Fournier MJ. The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions. Cell 1996;86:823e34. [6] Liu X, Sun Y, Zhang Y, Miao F, Wang G, Zhao H, et al. A 2,7-carbazole-based dicationic salt for fluorescence detection of nucleic acids and two-photon fluorescence imaging of RNA in nucleoli and cytoplasm. Org Biomol Chem 2011;9:3615e8. [7] Shen R, Shen X, Zhang Z, Li Y, Liu S, Liu H. Multifunctional conjugates to prepare nucleolar-targeting CdS quantum dots. J Am Chem Soc 2010;132: 8627e34. [8] Haulgland RP. Assays for cell viability, proliferation and function. In: Spence MTZ, editor. In the handbook, a guide to fluorescent probe and labeling technologies. 10th ed. Eugene, Oregon: Invitrogen, Molecular Probes; 2005. p. 710e1. [9] You Y, Lee S, Kim T, Ohkubo K, Chae WS, Fukuzumi S, et al. Phosphorescent sensor for biological mobile zinc. J Am Chem Soc 2011;133:18328e42. [10] Botchway SW, Charnley M, Haycock JW, Parker AW, Rochester DL, Weinstein JA, et al. Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes. Proc Nat Acad Sci 2008;105: 16071e6. [11] Kuimova MK, Yahioglu G, Levitt JA, Suhling K. Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging. J Am Chem Soc 2008;130:6672e3. [12] Gratton E, Breusegem S, Sutin J, Ruan Q, Barry N. Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods. J Biomed Opt 2003;8:381e90. [13] Svensson FR, Abrahamsson M, Stromberg N, Ewing AG, Lincoln P. Ruthenium(II) complex enantiomers as cellular probes for diastereomeric interactions in confocal and fluorescence lifetime imaging microscopy. J Phys Chem Lett 2011;2:397e401. [14] Tadrous PJ. Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging. J Pathol 2000;191:229e34. [15] Zhang J, Fu Y, Lakowicz JR. Fluorescent metal nanoshells: lifetime-tunable molecular probes in fluorescent cell imaging. J Phys Chem C 2011;115:7255e60. [16] Zhao Q, Huang CH, Li FY. Phosphorescent heavy-metal complexes for bioimaging. Chem Soc Rev 2011;40:2508e24. [17] Lo KKW, Tsang KHK, Sze KS, Chung CK, Lee TKM, Zhang KY, et al. Non-covalent binding of luminescent transition metal polypyridine complexes to avidin, indole-binding proteins and estrogen receptors. Coord Chem Rev 2007;251: 2292e310. [18] Baggaley E, Weinstein JA, Williams JAG. Lighting the way to see inside the live cell with luminescent transition metal complexes. Coord Chem Rev 2012;256: 1762e85. [19] Leung SK, Kwok KY, Zhang KY, Lo KKW. Design of luminescent biotinylation reagents derived from cyclometalated iridium(III) and rhodium(III) bis(pyridylbenzaldehyde) complexes. Inorg Chem 2010;49:4984e95. [20] Lo KKW, Choi AWT, Law WHT. Applications of luminescent inorganic and organometallic transition metal complexes as biomolecular and cellular probes. Dalton Trans 2012;41:6021e47.
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