Biocompatible glutathione capped gold clusters as one- and two-photon excitation fluorescence contrast agents for live cells imaging

Abstract

The one- and two-photon excitation emission properties of water soluble glutathione monolayer protected gold clusters were investigated. Strong two-photon emission was observed from the gold clusters. The two-photon absorption cross section of these gold clusters in water was deduced from the z-scan measurement to be 189 740 GM, which is much higher compared to organic fluorescent dyes and quantum dots. These gold clusters also showed high photo-stability. The MTT assay showed that these gold clusters have low toxicity even at high concentrations. We have successfully demonstrated their applications for both one and two-photon excitation live cell imaging. The exceptional properties of these gold clusters make them a promising alternative for one- and two-photon bio-imaging and other nonlinear optical applications.

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Introduction

Recent developments in optical imaging techniques, in particular multi-photon excitation microscopy that allows studies of biological interactions at a deep cellular level, has motivated intensive research in developing multi-photon absorption fluorophores.^1–10 Biological tissues are optically transparent in the near-infrared (NIR) region.^4,11 Fluorophores that can absorb light in the NIR region by multi-photon absorption are particularly useful in bio-imaging.^4,6 Many organic dyes have been synthesized for multi-photon imaging, but their applications are limited by their small two-photon absorption (TPA) cross sections and rapid photo-bleaching.^2,9,10Quantum dots have been known to be a good alternative for multi-photon imaging due to their large two-photon absorption cross sections.^6,12–15 However, their applications in bio-imaging have been restricted due to their cyto-toxicity and photon blinking behavior.^16–23

Photoluminescence (PL) from noble metals was first observed in 1969 by Mooradian et al.²⁴ In 1986 Boyd et al.²⁵ observed strong photoluminescence from metal films with rough surfaces, in which local field enhancement was believed to be responsible for the observed strong PL. Farrer et al.²⁶ reported highly efficient multi-photon induced luminescence from gold nanoparticles of sizes ranging from 2.5–125 nm. Various metal nanoparticles with different shapes have been proposed as contrast agents for bio-imaging.^1,3,27–32 Among different metal nanoparticles, gold nanorods have been widely used for multi-photon cellular imaging due to their easy preparation of different sizes and aspect ratios.^{3,27,29–31,33–36} However, their relatively large sizes limit their applications for intracellular imaging. In addition, gold nanorods are not suitable for one-photon imaging due to their low quantum yields. Recently a lot of research attention has been paid to fluorescent gold clusters.^28,37–47Gold nanoparticles with sizes close to the Fermi wavelength (<1 nm) of the electron possesses molecule like properties and are denoted as gold clusters.^42,45,48Gold clusters have many attractive features, such as strong fluorescence, excellent biocompatibility, water solubility, long term stability, and low toxicity, which make them promising candidates in many biological applications. So far most of the research has focused on developing various synthetic methods to fine control the number of atoms in a cluster.^{37,46,47,49–51} There are few investigations on their nonlinear optical properties^45,48 and applications in bio-imaging.⁴³ Ramakrishna et al.⁴⁵ recently investigated two-photon absorption properties of quantum sized gold clusters dispersed in toluene, which showed extremely high TPA cross sections at 800 nm. However, those gold clusters are only soluble in organic solvents such as toluene, which makes it difficult to functionalize them with other biomolecules, and limits their application in biological systems. These studies motivated us to investigate the two-photon properties of water soluble gold clusters and their applications in bio-imaging. Among various water soluble gold clusters, glutathione capped gold clusters were known able to be further functionalized for biological applications.^39,42,51 Their linear optical properties have been well characterized.^39,42,51 However, their nonlinear optical properties and applications as fluorescence imaging contrast agents have yet to be explored.

In this work we have investigated linear and nonlinear optical properties of glutathione capped gold clusters. These gold clusters display strong one- and two-photon excitation emissions. We have successfully demonstrated their applications as fluorescence contrast agents for live cell imaging. In addition, they displayed very good photostability and bio-compatibility. These gold clusters were found to have very low toxicity to live cells even at high concentrations. These exceptional properties make them promising materials for bio-imaging and other nonlinear optical applications.

Results and discussion

The glutathione capped gold clusters were prepared by using a modified method from the one reported previously for the synthesis of mercaptosuccinic acid monolayer protected gold clusters by Murray et al.⁵⁰ The preparation procedures are described in the Experimental section. Fig. 1 shows the UV-Vis absorption and emission spectra of the prepared glutathione capped gold clusters as well as their transmission electron microscopic (TEM) images. The TEM images were taken by drop casting a dilute gold-cluster solution onto a copper grid. The particles are quite monodisperse with sizes of 1–1.1 nm, which corresponds to approximately 25 atoms per cluster. The UV-Vis spectrum of glutathione capped gold clusters shows an optical absorption maximum at 665 nm and a strong continuous band on the higher energy side. The band at 665 nm arises from intra-band (sp–sp) transition, which is expected for the 25-atom gold clusters.^45,51 The absorption at the higher energy region arises from inter-band (sp–d) transition. The surface plasmon resonance was not observed in the UV-vis spectrum, suggesting that the sizes of the clusters are less than 2 nm.⁴⁵ The emission spectrum of the gold clusters under one-photon excitation at 650 nm has a dominant contribution in the near IR region with a band maximum at 780 nm. Their absorption and fluorescence spectra are consistent with the previously reported results.⁵⁰

Fig. 1 (a) Absorption and emission spectra, and (b) TEM image of the glutathione capped gold clusters.

The gold clusters also show strong two-photon excitation photoluminescence (TPL). Fig. 2 shows TPL spectra of the gold clusters under excitation at 800 nm by using femtosecond laser pulses of different excitation intensities. A 750 nm short pass filter was used to block the scattering from the excitation source. The obtained TPL spectra of the gold clusters are very broad spanning over from 400 to 650 nm. The near-IR portion was not presented due to strong contamination from the scattering of the excitation beam. The log–log plot excitation power dependent emisison intensities at 500 nm (Fig. 2) give a slope of ∼2, confirming that the emission is due to a two-photon excitation process. When the gold clusters were irradiated with a continuous wave (CW) beam of the same wavelength and same power, the emisison signal disappeared down to the background noise level, which further confirms the emission is due to a two-photon excitation process.

Fig. 2 (a) Excitation intensity dependent TPL spectra of glutathione capped gold clusters under excitation at 800 nm. (b) log–log plot of excitation intensity dependence of their TPL emission at 500 nm.

It has been previously reported that the TPL of gold nanoparticles can be strongly enhanced by their surface plasmon resonance.^30,52 For example, gold nanorods with a longitudinal plasmon band at 800 nm emit strong TPL compared to spherical gold nanoparticles at an excitation wavelength of 800 nm, which was ascribed to plasmon resonance enhancement.^30,53 The TPL intensity of these gold nanorods was reported to be strongly dependent on the excitation wavelength.^30,53 We have measured the TPL of gold clusters over excitation wavelength range from 770 to 830 nm, and little excitation wavelength dependence was observed (Fig. 3a). In addition, the TPL of these glutathione capped gold clusters in water showed very good photostability. The TPL intensity of these gold clusters in aqueous solution remained nearly constant during continuous irradiation with femtosecond laser pulses with power of 230 mW for one hour (Fig. 3b). The good photostability of nanoparticles is a very important factor for their applications as fluorescence contrast agents for bio-imaging.

Fig. 3 (a) TPL intensity of glutathione capped gold clusters at 500 nm under different excitation wavelengths and (b) under 800 nm for different periods of illumination time.

We have also investigated the two-photon absorption (TPA) properties of the glutathione monolayer protected gold clusters by using a z-scan technique. Fig. 4 shows the z-scan measurement results using 800 nm femtosecond laser pulses as the excitation source with a pulse energy of 180 nJ. The data can be well fit using a two-photon absorption model^14,15,54 and the TPA cross section of the gold clusters was calculated to be ∼189 740 GM, which is much higher compared to those of typical organic fluorescent dyes and quantum dots.^9,14,15 The obtained large TPA cross section value for glutathione monolayer protected gold clusters is on the same order of magnitude as the previous results obtained by Ramakrishna et al.,⁴⁵ who reported a TPA cross section of 427 000 GM for the Au₂₅ clusters in toluene. The huge TPA cross section and exceptional photostability makes these gold clusters promising candidates for many nonlinear optical applications.

Fig. 4 z-Scan measurement results on the gold clusters under femtosecond laser excitation at 800 nm. The solid line is the best fit based on a two photon absorption model. The concentration of gold cluster is 1 × 10⁻⁶ M and the pulse energy is 180 nJ.

We have tested the capability of gold clusters for both one- and two-photon excitation cell imaging on SH-SY5Y human neuroblastoma cells (human cancer cells). The cells were incubated with 50 μg ml⁻¹ of gold clusters for 24 h and then subjected to confocal imaging. Fig. 5a shows one-photon excitation (633 nm) fluorescence image of SH-SY5Y human neuroblastoma cells using gold clusters as fluorescence contrast agents. It can be clearly seen that the particles are mainly located at the cytoplasm of the cells. To make sure that the observed fluorescence was indeed from gold clusters, we also performed a control experiment at the same experimental conditions without addition of gold clusters. We did not observe any fluorescence from the cells without incubation with the gold clusters (data shown in the ESI†). The control experiment confirms that the observed fluorescence was due to the gold clusters attached to those cells instead of autofluorescence of the cells.

Fig. 5 One-photon (a) and two-photon (c) excitation fluorescence images of SH-SY5Y neuroblastoma cells that were incubated with the gold clusters. The excitation wavelength is 633 nm. The images on the right (b & d) are the overlaid pictures of the fluorescence images and the corresponding differential interference contrast (DIC) images.

Fig. 5c shows the two-photon excitation fluorescence image of SH-SY5Y human neuroblastoma cells incubated with gold clusters under excitation of femtoseond laser pulses at 800 nm with an excitation power of 140μW. Similar to the one-photon excitation results, the particles inside the cells can be clearly seen and no auto-fluorescence from the cells was observed under the same experimental conditions (See Fig. S1 in the ESI†). The cells were also imaged with a different z-position using a XYZ scanning mode²⁹ under two-photon excitation (Fig. 6). The z-stack sectioning clearly confirms that the glutathione capped gold clusters were internalized inside the cells.

Fig. 6 z-Sectioning of SH-SY5Y neuroblastoma cell images obtained using gold clusters as two-photon fluorescence contrast agents under excitation of 800 nm femtosecond laser pulses.

Many nanomatereials generally have strong cytotoxcity. Biocompatibility is a critical issue for their biological applications such as bio-imaging.^28,55–57 We have examined the toxicity of the glutathione capped gold clusters on the SH-SY5Y human neuroblastoma cells using an MTT test (Fig. 7). The results clearly indicate that the cells are viable even at very high concentrations (400 μg ml⁻¹) of these glutathione capped gold clusters. Glutathione is a known antioxidant⁵⁸ that helps protect cells from reactive oxygen species such as free radicals and peroxides.^58,59 The glutathione capped gold clusters showed quite low toxicity toward the cells.

Fig. 7 The cell viability results obtained using MTT test after overnight incubation with different concentrations of glutathione capped gold clusters.

These gold clusters have unique advantages compared to the previously reported bio-imaging nanomaterials. Previously Wu et al.³¹ have shown that gold nanorods are very good materials for two-photon imaging. However, gold nanorods are not proper for one-photon imaging due to it low quantum yields. Quantum dots are known as good imaging agents for both one-photon and two-photon excitation imaging. However, their cytotoxicity have limited their applications in bio-imaging. In addition, quantum dots typically show photo-blinking behavior.^20,23 It has been reported that Au clusters do not have this problem.²⁶ We have also done some preliminary tests on the photo-blinking properties of our gold clusters and they showed significantly reduced photo-blinking behavior compared to the quantum dots (see the ESI†). Most importantly, compared to gold nanorods and quantum dots, these gold clusters have much smaller sizes, which is particularly important for bio-imaging. Their small sizes make them easy to be introduced into cells and able to target the inside portions of the cells using these gold clusters, which may not be possible using larger size nanoparticles. These gold clusters are capable of acting as both one-photon and two-photon excitation imaging agents, which give similar imaging effects for conventional situations. However, their extra two-photon imaging capability allows them to penetrate deeper into the biological tissues and enable in vivo imaging with larger depths. In addition to their low toxicity and good biocompatability, the glutathione protected gold clusters can be further functionalized with biomolecules using the COOH group in glutathione for further applications.

Conclusions

In summary, one- and two-photon emission properties of glutathione capped gold clusters were investigated. The two-photon absorption properties of these monolayer protected quantum size gold clusters were studied by using two-photon excitation photoluminescence measurements and femtosecond z-scan technique. The TPA cross section of these glutathione capped gold clusters was measured to be ∼189 740 GM in water, which is much larger compared to those of organic fluorescent dyes and quantum dots. In addition, these gold clusters show exceptional photo-stability. The gold clusters also displayed very low toxicity to cells even at high concentrations of gold clusters. We have successfully demonstrated their applications in both one- and two-photon excitation live cell fluorescence imaging. The exceptional properties of these gold clusters make them a good alternative for bio-imaging and other nonlinear optical applications.

Experimental section

Synthesis and characterizations of glutathione capped gold clusters

The glutathione capped gold clusters were prepared by using a method modified from the one reported previously for the synthesis of mercaptosuccinic acid monolayer protected gold clusters by Murray et al.⁵⁰ Briefly, 80 ml of 1.25 × 10⁻² M HAuCl₄ methanol solution and 40 ml of 0.075 M glutathione aqueous solution were mixed under stirring. 10 ml of 1.0 M NaBH₄ aqueous solution was quickly added into the mixture. The solution immediately turned a brown colour and the stirring was continued for one hour. The solvents was evaporated at 43 °C to concentrate the solution and excess methanol was added to precipitate the clusters. The precipitate was then filtered, dissolved in ∼2 ml of distilled water and precipitated with methanol again to remove any impurities. The obtained gold clusters precipitate was finally dispersed in 100 ml of water for further characterizations. UV–Vis and emission spectra of the gold clusters were measured by using a Shimadzu UV 2450 spectrometer and a Perkin Elmer L55 fluorimeter, respectively. The TEM images were taken by using a JEOL2010 electron microscope.

Two-photon photoluminescence (TPL) measurement

The excitation source for two-photon photoluminescence (TPL) measurement was a Spectra Physics femtosecond Ti:sapphire oscillator (Tsunami), which gives output laser pulses with a tunable central wavelength from 770 to 830 nm and a repetition rate of 80 MHz. The samples were excited by directing a tightly focused laser beam onto the sample. The emission from the sample was collected at a 90° angle, perpendicular to the incoming excitation beam. The fluorescence signal was directed into a monochromator (Acton, Spectra Pro 2300i) coupled CCD (Princeton Instruments, Pixis 100B) with an optical fiber. A short pass filter with a cut-off wavelength at 750 nm was placed before the spectrometer to minimize the light scattering from the excitation beam.

Two-photon absorption (TPA) cross section measurement using z-scan method

The femtosecond z-scan measurements were performed by using a mode-locked Ti:sapphire oscillator seeded regenerative amplifier, which gives output laser pulses with a central wavelength at 800 nm and a repetition rate of 1 kHz. The laser beam was focused onto the gold cluster solution (1 × 10⁻⁶ M) samples contained in a 1 mm thick quartz cuvette, with a beam waist of ∼20 μm. The pulse duration of the laser pulse at the sample is ∼200 fs. The sample was moved along the z axis towards and away from the focus point while the transmitted signal was monitored with a lock-in amplifier. The experiment was done with an open aperture configuration. The TPA coefficient (β) was obtained by fitting the Z-scan data with a two-photon absorption model and then the TPA cross section (σ) was calculated by using the following equation.^9,28
where N_A is the Avogadro constant, h is the plank constant, d is the concentration of the gold cluster solution and ν is the frequency of the incident laser.

Cell culture and incubation for imaging

SH-SY5Y neuroblastoma (ATCC, USA) cells were grown at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM/F12 (1 : 1); Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% antibiotic (penicillin streptomycin, Gibco, USA). The cells were placed in a 8-well chamber with a 0.17 mm coverslip bottom (Nunc, Denmark) one day prior to the addition of the gold clusters. The cells were washed with HBSS/HEPES buffer and incubated with 50 μg ml⁻¹gold clusters for 24 h. Cells were then washed three times again with the same buffer solution and suspended in indicator free DMEM for imaging.

One- and two-photon fluorescence imaging

One-photon imaging. The imaging was performed on an Olympus FV300 inverted confocal microscope. A 633 nm He–Ne laser (Melles Griot, Singapore) with an excitation power of 100 μW was directed by a long pass dichroic mirror and scanning mirrors to a water immersion objective (60×, NA1.2, Olympus, Singapore), which focuses the light onto the samples. The fluorescence signal was then collected by the same objective and detected by the photomultiplier tube (PMT) after passing through a 660 nm long pass filter.

Two-photon imaging. The two-photon imaging was performed on a Olympus IX70 conofcal microscope using a femtosecond Ti:sapphire laser (Mira 900, Coherent, Santa Clara, CA) as the excitation source. The output laser pulse has a repetition rate of 76 MHz and the pulse duration of 200 fs. The laser beam was directed into a confocal microscope, with the excitation power of 140 μW on the cells. The 60× water-immersion objective was used to focus the laser beam onto the gold cluster incubated live cells. The TPL was collected through the same objective, separated from the excitation laser by a dichroic mirror. The signal was directed to a photomultiplier tube (PMT) placed in the back port of the microscope. A 565 nm longpass filter was placed before the PMT to collect the TPL of gold clusters. All the imaging experiments were performed at 37 °C using a temperature controller.

Toxicity assay

In order to test the toxicity of the glutathione capped gold clusters on SH-SY5Y neuroblastoma (ATCC, USA) cells, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay kit (Invitrogen, Singapore) was used. The MTT stock solution was prepared according to the protocol given in the company manual.⁶⁰ The cells were seeded in a 24 well plate in 1 ml of culturing medium 24 h prior to nanoparticle incubation. The cells were subsequently incubated with different concentrations of glutathione capped gold clusters (50, 100, 200 and 400 μg ml⁻¹) for 24 h. Cells were then washed three times with HBSS/HEPES buffer followed by addition of 10 μL MTT solution to each well and incubated for 2 h at 37 °C. Then 100 μL of SDS-HCl solution was added to each well and further incubated for 4 h at 37 °C. The entire content of each well was then mixed thoroughly and finally the absorbance of the resulting solution was measured at 570 nm using a UV 2450 spectrometer.

Acknowledgements

This work is funded by the Faculty of Science, National University of Singapore (R-143-000-341-112) and DSTA (R-143-000-432-422). We thank Prof. Throsten Wohland for allowing us to use the confocal microscope facility in his laboratory.

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Footnote

† Electronic supplementary information (ESI) available: Control experiments; blinking studies of gold clusters. See DOI: 10.1039/c0nr00458h

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