Two-Photon Imaging of 3D Organization of Bimetallic AuAg

Aug 11, 2017 - We report on two-photon excitation properties of small silver-doped gold nanoclusters (AuAgNCs) and on their three-dimensional arrangem...
1 downloads 9 Views 2MB Size
Subscriber access provided by University of Rochester | River Campus & Miner Libraries

Article

Two-photon imaging of 3D organization of bimetallic AuAg nanoclusters in DNA matrix Katarzyna Brach, Magdalena Waszkielewicz, Joanna Olesiak-Banska, Marek Samoc, and Katarzyna Matczyszyn Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00873 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

TOC 114x88mm (96 x 96 DPI)

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 18

Two-photon imaging of 3D organization of bimetallic AuAg nanoclusters in DNA matrix Katarzyna Brach, Magdalena Waszkielewicz, Joanna Olesiak-Banska*, Marek Samoc, Katarzyna Matczyszyn Advanced Materials Engineering and Modelling Group, Faculty of Chemistry, Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland Corresponding Author: *E-mail: [email protected]

ABSTRACT: We report on two-photon excitation properties of small silver-doped gold nanoclusters (AuAgNCs) and on their three-dimensional arrangement in a hybrid system composed of DNA liquid crystals (LCs) and AuAgNCs. UV-Vis and fluorescence spectroscopy, transmission electron microscopy (TEM) and multiphoton excitation spectroscopy were used to characterize the nanoparticles. We show that AuAgNCs exhibit two-photon excited luminescence (2PL) emission and second-harmonic generation (SHG) and that these properties remain the same in liquid crystalline matrix. The results are described in detail and discussed in the context of possible imaging application of AuAgNC and specific AuAgNCs organization induced by liquid crystalline ordering of DNA molecules.

KEYWORDS: two-photon luminescence, second-harmonic generation, DNA liquid crystals, silver-doped gold nanoclusters, dual nonlinear emission properties. 1 ACS Paragon Plus Environment

Page 3 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Ultra-small nanoclusters are a relatively new class of metallic nanoparticles, which are especially intriguing because of their unique chemical and optical properties, such as strong photoluminescence, large Stokes shift, tunable emission in the visible spectrum, high photostability, good water-solubility, chirality and magnetism.1-8 Due to their size, structure and properties, nanoclusters bridge the gap between bigger nanoparticles and small molecules. Attractive luminescent properties of NCs overcome the limitations of standard fluorescent probes and quantum dots, which may exhibit high toxicity, poor photostability or photobleaching.9-13 Although there are many interesting reports about the application of gold nanoclusters as fluorescent probes in vitro and in vivo,14-22 most of them concern mainly one-photon excitation properties of nanoclusters. On the other hand, two-photon luminescence microscopy (2PLM) has many advantages over traditional one-photon optical microscopy techniques such as possibility of high-resolution deep imaging even in highly scattering environments like living tissues, localized red or infrared excitation in small focal volume and reduced phototoxicity and photobleaching. There are only few reports in which NCs have been successfully applied as fluorescent probes for in vitro two-photon imaging of human mesenchymal stem cells,23 cellular delivery in HeLa cells24-25 or SH-SY5Y human neuroblastoma cells.26 Gold nanoclusters have been shown to exhibit significant nonlinear optical properties with two-photon absorption cross-sections reaching several thousand GM at relevant wavelengths, which make them suitable also for photonic applications such as optical power limiting.5, 7, 27-28 Also second- and third-harmonic generation and multiphoton luminescence were observed,29 which are promising features for application in SHG microscopy and fluorescence microscopy. A majority of the investigations were conducted in solutions, however there were some reports concerning enhancement of optical properties in solid state30-31 or in DNA matrix.32 In addition,

2 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 18

the research about combination of LC and nanoparticles into one hybrid system has shown that liquid crystalline phase may be used as template for nanoparticles ordering, which may enhance and improve properties of both LC and nanoparticles.33-37 Here, we present an investigation concerning two-photon excitation properties of AuAgNCs in DNA LCs. First, we characterized the nanoparticles using common spectroscopic techniques and TEM combined with two-photon excited spectroscopy. Next, we prepared and investigated lyotropic DNA LCs doped with AuAgNCs using polarized light and fluorescence microscopy to analyze the distribution of nanoparticles within liquid crystalline matrix. We also exploited the advantages of 2PLM to further evaluate the properties of the prepared samples and asses the suitability of AuAgNCs as markers for multiphoton imaging as well as that of DNA LC as matrix for organization of nanoparticles. RESULTS AND DISCUSSION AuAgNCs characterization. The size and morphology of AuAgNCs were determined from TEM images (Figure 1A). The synthesis of AuAgNCs led to the formation of well dispersed spherical nanoparticles with the size of 1.78 ± 0.20 nm. Elemental analysis of AuAgNCs showed that Ag:Au mass ratio was equal to 1:75. The absorbance spectrum (Figure 1B) of AuAgNCs was similar to the ones reported before38 and showed strong absorption around 400 nm. Because of their small size induced discrete electronic structure and molecular-like properties, the optical properties of NCs are significantly different from those of plasmonic gold nanocrystals, in particular, the surface resonance plasmon band at 520 nm, characteristic for larger Au nanoparticles, is not observed in the case of NCs UV-Vis spectrum. This suggested also that NCs were the only product of the synthesis and no bigger particles were formed. The NCs were luminescent with a broad emission band at 600 nm (Figure 1C). The excitation spectrum shows 3 ACS Paragon Plus Environment

Page 5 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the main peak at 405 nm. The luminescence quantum yield (QY) was measured by a comparative method using Rhodamine B as a reference and was equal to 13.0 %, markedly higher than the QY in the case of similarly sized gold NCs without silver doping.39 A

B

C

Figure 1. (A) TEM image of AuAgNCs. (B) UV-Vis spectrum of AuAgNCs. (C) Excitation (dashed line, λem = 600 nm) and emission (solid line, λex = 400 nm) spectra of AuAgNCs. Two-photon excitation properties of AuAgNCs. 2PL signals from the solution of AuAgNCs were obtained in the range of excitation wavelengths from 800 nm to 1000 nm with the average power of incident light around 10 mW (Figure 2A). The 2PL intensity of AuAgNCs in the solution was found to decrease with the increase of the excitation wavelength, with the highest 2PL intensity for the excitation at 800 nm. We conducted the same experiments for a thin film of AuAgNCs obtained by depositing nanoparticles solution (CAuAgNC = 1 mg mL-1) onto a cover glass and complete solvent evaporation. Similarly to the solution of AuAgNCs, with the increasing excitation wavelength the decrease of 2PL was observed (Figure 2B). The maximum of the emission for the excitation at 800 nm was around 625 nm and with a decrease of excitation energy it was red shifted, up to 695 nm in the case of 1050 nm excitation. The red shift of luminescence maximum from AuAgNCs from solution and deposited on the glass substrate with the shift of incident laser wavelength suggest a different excitation and emission pathway. A similar behavior was 4 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 18

observed e.g. for Ag nanoclusters.40 Weak SHG signal, increasing with the increase of excitation wavelength, was also recorded in the range of excitations from 850 nm to 1050 nm. We did not observe SHG with the excitation at 800 nm, which is probably due to the SHG generated in this case being strongly reabsorbed through single photon absorption (an inner-filter effect). A

B

Figure 2. 2PL emission spectra of (A) AuAgNCs aqueous solution (CAuAgNC = 1 mg mL-1) and (B) a thin film of AuAgNCs, obtained by depositing nanoparticles solution of CAuAgNC = 1 mg mL-1, at excitation wavelength from 800 nm to 1050 nm. DNA LCs doped with AuAgNCs. In a drying droplet of properly concentrated DNA solution the DNA molecules tend to self-assembly into a liquid crystalline hexagonal columnar phase near the border of the droplet.41 In the beginning of the drying process, DNA chains are homogeneously spread within the volume of the droplet, then, because of the capillary flow induced by contact line pinning and additional undulation caused by radial stress occurring at the end of drying, the DNA strands accumulate at the border of the droplet and liquid crystalline phase with characteristic regular zigzag pattern is formed.41 The orientation of DNA in this type of LC was already investigated in our group by means of 2PLM. The results indicated that DNA

5 ACS Paragon Plus Environment

Page 7 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

molecules within one columnar domain were parallel to each other and the domains with molecules oriented in different directions were separated by vertical wall defects.42 The polarized light microscopy observation of our sample confirmed the presence of LC with the characteristic zigzag pattern formed at the perimeter of the dried DNA droplet (Figure 3A). We observed multiple columnar domains separated by wall defects. The addition of AuAgNCs did not influence the LC phase formation as well the type of observed phase texture. Atomic force microscopy (AFM) measurements showed that the surface of the dried droplet edge was undulated (Figure 3B) and had a mountain-like shape with numerous peaks and valleys, which is consistent with literature reports.41-42 The maximum height of the undulated region (from a valley to a peak) was around 700 nm. The fluorescence microscopy observation of dried DNA droplets doped with NCs revealed that the nanoclusters were spread within the entire layer of the sample (Figure 3C). However, there were some particular regions of the sample, in which the intensity of the emission was significantly increased and characteristic emission pattern was observed. These regions corresponded to the peaks of the undulated LC phase. This kind of specific accumulation of AuAgNCs was correlated with the presence and ordering of DNA molecules into a lyotropic LC caused by capillary flow. In contrast, preparation of a thin film formed from a drying droplet of NCs solution with CAuAgNC = 1 mg mL-1 resulted in a formation of a “coffee ring” stain without a characteristic zigzag pattern and surface undulation.

6 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

Page 8 of 18

B

C

Figure 3. (A) Polarized light microscopy image of undulated columnar LC phase of DNA doped with AuAgNCs (CAuAgNC = 1 mg mL-1). (B) 2D AFM scan of the dried droplet edge surface. (C) Fluorescence microscopy image of undulated columnar LC phase of DNA doped with AuAgNCs taken in the same region of the sample as in Figure 3A (λexc = 460 – 495 nm, λem > 510 nm). One of the major difficulties in predicting the behavior of nanoparticles and the pattern of their distribution in liquid crystals is the complexity of prepared systems. There is a variety of variables which have to be taken into account, like the properties of mesogens and properties of nanoparticles such as their shape, size and coating. It was shown that even the same type of nanoparticles (size and shape), but with different coating can have different influence on the liquid crystal.43 Concentration of nanoparticles in liquid crystalline phase may be also one of the factors influencing the aggregation behavior of NCs. It was shown that depending on the 7 ACS Paragon Plus Environment

Page 9 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

concentration of nanoparticles different types of liquid crystalline textures and aggregation patterns can be observed, for example increasing the concentration of nanoparticles led to the increased aggregation of nanoparticles in liquid crystal.44-45 Comparing our current results to the ones obtained in the case of DNA LCs doped with gold nanorods (NRs),46 we could observe differential nanoparticles aggregation pattern. NRs were dispersed homogenously, whereas NCs also tended to accumulate in the defects. Taking into consideration the calculations of DNA base pair (bp) mole per nanoparticle mole, we obtained DNA bp:NC ratio ≈ 130:1 and DNA bp:NR ratio ≈ 160·104:1. However, if we look at the results in the category of weight/volume % concentration of nanoparticles doped into LCs, the difference between the nanoparticles concentration is much smaller (CAuAgNC = 1 mg mL-1 and CNR = 0.333 mg mL-1). We prepared DNA LC doped with AuAgNCs at the concentration of CAuAgNC = 0.25 mg mL-1, at which we could still observe defect correlated aggregation of nanoparticles, which was not present in the case of bigger NRs (Supplementary Information, Fig. S1). In order to exclude silver interaction with DNA, which could be responsible for specific defect correlated aggregation of NCs, we performed circular dichroism measurements (Supplementary Information, Fig. S2). There were no significant changes of DNA conformation in the presence of the clusters. Additionally, if silver had affinity to DNA, it would interact with all DNA molecules forming liquid crystal and in this case NCs would be spread homogenously in the volume of the sample. The orientation of DNA molecules and presence of walls separating columnar domains of different molecular orientation should not influence the interaction between silver and DNA, then it should not direct the aggregation of NCs. Furthermore, similar experiments conducted for DNA LCs doped with AuNCs have also shown specific aggregation of NCs (Supplementary Information, Fig. S3). We suggest that differences between NRs and

8 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 18

NCs doping effects are caused mainly by different sizes and shapes of these nanoparticles. The rate at which liquid crystalline phase is formed in a drying droplet is high, thus bigger NRs are too heavy comparing to NCs to follow the capillary flow of DNA molecules from interior to the edge of a drying droplet. Since NCs are smaller it is easier for them to move and in order to minimize the total free energy they tend to aggregate in defects. Two-photon luminescence microscopy of DNA LCs doped with AuAgNCs. In order to evaluate the use of AuAgNCs as possible probes for biological imaging we investigated DNA LCs doped with the nanoclusters using 2PLM. 2PLM was already applied to explore DNA LCs doped with dyes42,47 or gold nanorods.46 The research provided useful information about the orientation of DNA molecules within the liquid crystalline matrix and the possible application of gold nanorods as imaging and theranostic probes. Figure 4A presents XY 2PL intensity scan of the undulated DNA liquid crystalline phase obtained for the phase region shown in Figure 4B. The results are in the agreement with onephoton fluorescence microscopy observations, where specific aggregation of nanoparticles correlated to the position of the peaks formed within the undulated phase. The distance between neighbouring peaks was twice the distance between vertical wall defects, and it varied from 8 to 25 µm. Consequently, the same values were obtained for the distance between linearly organized AuAgNCs. The experimental conditions were optimized in order to obtain good signal to noise ratio and to avoid the photobleaching and photodamage of the sample. The 2PL intensity scans could be performed with the average power of incident light up to 1.5 mW. With the advantage of the intrinsic 3D resolution of 2PLM, we performed several scans in the same region, but with different Z positions of the undulated phase formed at the perimeter of a droplet (Figure 4C). It was found that AuAgNCs not only tended to aggregate on the top of the 9 ACS Paragon Plus Environment

Page 11 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

peaks of the undulated LC phase, but also accumulated within the whole volume of the phase forming a vertical wall defect. It means that in this type of LCs, self-assembly of NCs is templated by phase defects. DNA LCs offer a possibility for 3D self-assembly of nanoparticles on liquid crystalline template. A

B

10 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 18

C

Figure 4. (A) 2PL intensity raster scan of undulated phase (CAuAgNC = 1 mg mL-1) with (B) the corresponding optical image (size of the image 80 x 80 µm), λexc = 800 nm. (C) 2PL intensity raster scans performed at several Z positions of undulated phase at the perimeter of dried droplet of DNA doped with AuAgNCs (CAuAgNC = 1 mg mL-1), λexc = 800 nm. We performed additional 2PLM measurements, which proved the multimodal imaging abilities of AuAgNCs. A slight modification of the experimental setup allowed us to collect the signals originating from SHG emission. Figure 5 presents XY intensity raster scans obtained from the same region of the sample, in which we detected signals from both 2PL and SHG at the same time (Figure 5A) and SHG signals, which were separated from 2PL (Figure 5B). The contribution of 2PL in the two-photon excited emission of AuAgNCs was in this case significantly greater than that of SHG. However, it was still possible to detect SHG signals that were consistent with the aggregation pattern of nanoclusters. The results suggest great potential of AuAgNCs in nonlinear optical imaging of biological samples.

11 ACS Paragon Plus Environment

Page 13 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

A

B

Figure 5. Comparison between (A) 2PL combined with SHG intensity raster scan and (B) SHG intensity raster scan (λexc = 920 nm) of nanocluster doped undulated DNA phase (CAuAgNC = 1 mg mL-1). CONCLUSION In summary, we synthesized monodispersed thiol-stabilized silver-doped gold nanoclusters and showed their two-photon excitation properties. Then we introduced the NCs into DNA matrix, where the nanoparticles emitted strong 2PL and weak SHG signal. In the presence of liquid crystalline DNA the optical properties of nanoclusters remained unchanged, which makes them attractive probes for imaging of biological samples. Additionally, we observed that undulated liquid crystalline phase of DNA formed at the perimeter of a drying droplet induced the 3D organization of nanoclusters in the wall defects. It is an easy method for nanoclusters ordering, which may find the possible application in photonic devices construction. Further investigation concerning the properties and distribution of NCs within different types of liquid crystalline phases is required to fully understand the phenomena of liquid crystalline induced NCs aggregation and these studies are currently performed in our laboratory. METHODS 12 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 18

Chemicals. Deoxyribonucleic acid sodium salt from salmon testes, HAuCl4•3H2O (99.9%), glutathione (≥98.0%) and AgNO3 (99+%) were purchased from Sigma Aldrich. All chemicals were used as received, except when mentioned specifically. Milli-Q water of resistivity 18.2 MΩ•cm was used to prepare all solutions. AuAgNCs synthesis. AuAgNCs were prepared according to a slightly modified protocol of Le Guevel.38 Aqueous gold salt solution was mixed with silver nitrate at an Ag:Au ratio equal to 1:47. Glutathione was used as a reduction agent at a molar ratio of 1:1 to gold. The sample was incubated for 48 h at 65 °C. The purification steps consisted of centrifugation at 3000 rpm for 5 min and double precipitation with acetone. DNA liquid crystal preparation. The DNA liquid crystal preparation consisted of deposition of 10 µL of AuAgNCs doped DNA solution (CDNA = 10 mg mL-1 and CAuAgNC = 1 mg mL-1) on the top of a thin cover slip and drying under 55 °C until total solvent evaporation. The samples were stored at room temperature. Instrumentation. The morphology of AuAgNCs was examined using a FEI Tecnai G2 20 XTWIN transmission electron microscope. The UV-Vis absorption spectra were recorded in 10 mm quartz cuvettes at room temperature using a JASCO V-670 spectrophotometer. The fluorescence

spectra

were

measured

at

room

temperature

using

a

FluoroMax-4

spectrofluorometer. The ICP analysis was performed with ARL 3410 ICP (Fisons Instruments). The samples were observed under polarized light and using a conventional widefield epifluorescence Olympus BX60 optical microscope. The surface of dried droplets was investigated by AFM in tapping mode (Dimensional V scanning probe microscope, Veeco). Two-photon excited luminescence measurements were performed with a custom-built two-photon microscope in which luminescence was excited in the range of 800 nm to 1050 nm with a mode-locked

13 ACS Paragon Plus Environment

Page 15 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Ti:sapphire laser (Chameleon, Coherent Inc.). The laser beam was focused tightly by a high numerical aperture Nikon Plan Apo oil immersion objective (100×/1.4 NA) or by a Nikon LU Plan Fluor objective (100×/0.9 NA). The sample was mounted on an XYZ piezoelectric scanning stage (TRITOR 102) and two-photon excited emission was collected in epi-fluorescence mode through the same microscope objective. The emitted signal was separated from the incident light on a dichroic mirror and detected by avalanche photodiodes operating in the photon counting regime. SHG intensity raster scan was recorded in the same experimental setup with additional Semrock 460/14 nm BrightLine® single-band bandpass filter placed before the avalanche photodiodes. The two-photon excited emission spectra were recorded using a Shamrock 303i Spectrograph from Andor. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ACKNOWLEDGMENT This

work

was

supported

by

the

National

Science

Centre

under

grant

DEC-

2013/09/B/ST5/03417, DEC-2013/10/A/ST4/00114 and by a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wroclaw University of Technology. SUPPORTING INFORMATION AVAILABLE Polarized light and fluorescence microscopy images (Figures S1 and S3), circular dichroism measurements (Figure S2) This information is available free of charge via the Internet at http://pubs.acs.org/. 14 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 18

REFERENCES 1. Jin, R. C., Quantum sized, thiolate-protected gold nanoclusters. Nanoscale 2010, 2, 343362. 2. Lu, Y. Z.; Chen, W., Sub-nanometre sized metal clusters: from synthetic challenges to the unique property discoveries. Chem. Soc. Rev. 2012, 41, 3594-3623. 3. Knoppe, S.; Burgi, T., Chirality in thiolate-protected gold glusters. Accounts Chem. Res. 2014, 47, 1318-1326. 4. Kumar, S.; Jin, R. C., Water-soluble Au-25(Capt)(18) nanoclusters: synthesis, thermal stability, and optical properties. Nanoscale 2012, 4, 4222-4227. 5. Russier-Antoine, I.; Bertorelle, F.; Vojkovic, M.; Rayane, D.; Salmon, E.; Jonin, C.; Dugourd, P.; Antoine, R.; Brevet, P. F., Non-linear optical properties of gold quantum clusters. The smaller the better. Nanoscale 2014, 6, 13572-13578. 6. Noguez, C.; Garzon, I. L., Optically active metal nanoparticles. Chem. Soc. Rev. 2009, 38, 757-771. 7. Philip, R.; Chantharasupawong, P.; Qian, H. F.; Jin, R. C.; Thomas, J., Evolution of Nonlinear Optical Properties: From gold atomic clusters to plasmonic nanocrystals. Nano Lett. 2012, 12, 4661-4667. 8. Luo, Z. T.; Zheng, K. Y.; Xie, J. P., Engineering ultrasmall water-soluble gold and silver nanoclusters for biomedical applications. Chem. Commun. 2014, 50, 5143-5155. 9. Parak, W. J.; Pellegrino, T.; Plank, C., Labelling of cells with quantum dots. Nanotechnology 2005, 16, R9-R25. 10. Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Javier, A. M.; Gaub, H. E.; Stolzle, S.; Fertig, N.; Parak, W. J., Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles. Nano Lett. 2005, 5, 331-338. 11. Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N., Probing the cytotoxicity of semiconductor quantum dots. Nano Lett. 2004, 4, 11-18. 12. Sukhanova, A., et al., Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells. Anal. Biochem. 2004, 324, 60-67. 13. Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T., Quantum dots versus organic dyes as fluorescent labels. Nat. Methods 2008, 5, 763-775. 14. Zhang, Z. Y.; Xu, L. J.; Li, H. X.; Kong, J. L., Wavelength-tunable luminescent gold nanoparticles generated by cooperation ligand exchange and their potential application in cellular imaging. RSC Adv. 2013, 3, 59-63. 15. Bian, P. P.; Zhou, J.; Liu, Y. Y.; Ma, Z. F., One-step fabrication of intense red fluorescent gold nanoclusters and their application in cancer cell imaging. Nanoscale 2013, 5, 6161-6166. 16. Palmal, S.; Basiruddin, S. K.; Maity, A. R.; Ray, S. C.; Jana, N. R., Thiol-directed synthesis of highly fluorescent gold clusters and their conversion into stable imaging nanoprobes. Chem. Eur. J. 2013, 19, 943-949. 17. Zhang, W. J.; Ye, J.; Zhang, Y. Y.; Li, Q. W.; Dong, X. W.; Jiang, H.; Wang, X. M., One-step facile synthesis of fluorescent gold nanoclusters for rapid bio-imaging of cancer cells and small animals. RSC Adv. 2015, 5, 63821-63826. 18. Xu, S. H.; Liu, P. P.; Song, Q. W.; Wang, L.; Luo, X. L., One-pot synthesis of biofunctional and near-infrared fluorescent gold nanodots and their application in Pb2+ sensing and tumor cell imaging. RSC Adv. 2015, 5, 3152-3156.

15 ACS Paragon Plus Environment

Page 17 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

19. Zhao, C. Q., et al., Biosynthesized gold nanoclusters and iron complexes as scaffolds for multimodal cancer bioimaging. Small 2016, 12, 6255-6265. 20. Chen, D.; Li, B. W.; Cai, S. H.; Wang, P.; Peng, S. W.; Sheng, Y. Z.; He, Y. Y.; Gu, Y. Q.; Chen, H. Y., Dual targeting luminescent gold nanoclusters for tumor imaging and deep tissue therapy. Biomaterials 2016, 100, 1-16. 21. Chen, L. F.; Zhang, Y. Y.; Jiang, H.; Wang, X. M.; Liu, C. Y., Cytidine mediated AuAg nanoclusters as bright fluorescent probe for tumor imaging in vivo. Chinese J. Chem. 2016, 34, 589-593. 22. Zhang, Y. Y.; Li, J. C.; Jiang, H.; Zhao, C. Q.; Wang, X. M., Rapid tumor bioimaging and photothermal treatment based on GSH-capped red fluorescent gold nanoclusters. RSC Adv. 2016, 6, 63331-63337. 23. Liu, C. L., et al., Thiol-functionalized gold nanodots: Two-photon absorption property and imaging in vitro. J. Phys. Chem. C 2009, 113, 21082-21089. 24. Oh, E., et al., PEGylated luminescent gold nanoclusters: Synthesis, characterization, bioconjugation, and application to one- and two-photon cellular imaging. Part. Part. Syst. Char. 2013, 30, 453-466. 25. Shang, L.; Dorlich, R. M.; Brandholt, S.; Schneider, R.; Trouillet, V.; Bruns, M.; Gerthsen, D.; Nienhaus, G. U., Facile preparation of water-soluble fluorescent gold nanoclusters for cellular imaging applications. Nanoscale 2011, 3, 2009-2014. 26. Polavarapu, L.; Manna, M.; Xu, Q. H., Biocompatible glutathione capped gold clusters as one- and two-photon excitation fluorescence contrast agents for live cells imaging. Nanoscale 2011, 3, 429-434. 27. Ramakrishna, G.; Varnavski, O.; Kim, J.; Lee, D.; Goodson, T., Quantum-sized gold clusters as efficient two-photon absorbers. J. Am. Chem. Soc. 2008, 130, 5032-5033. 28. Olesiak-Banska, J.; Waszkielewicz, M.; Matczyszyn, K.; Samoc, M., A closer look at two-photon absorption, absorption saturation and nonlinear refraction in gold nanoclusters. RSC Adv. 2016, 6, 98748-98752. 29. Knoppe, S.; Vanbel, M.; van Cleuvenbergen, S.; Vanpraet, L.; Burgi, T.; Verbiest, T., Nonlinear Optical Properties of Thiolate-Protected Gold Clusters. J. Phys. Chem. C 2015, 119, 6221-6226. 30. Ho-Wu, R.; Yau, S. H.; Goodson, T., Linear and nonlinear optical properties of monolayer-protected gold nanocluster films. ACS Nano 2016, 10, 562-572. 31. Kumar, S.; Shibu, E. S.; Pradeep, T.; Sood, A. K., Ultrafast photoinduced enhancement of nonlinear optical response in 15-atom gold clusters on indium tin oxide conducting film. Opt. Express 2013, 21, 8483-8492. 32. Yau, S. H., et al., Bright two-photon emission and ultra-fast relaxation dynamics in a DNA-templated nanocluster investigated by ultra-fast spectroscopy. Nanoscale 2012, 4, 42474254. 33. Shuai, M., et al., Spontaneous liquid crystal and ferromagnetic ordering of colloidal magnetic nanoplates. Nat Commun 2016, 7. 34. Gharbi, M. A.; Manet, S.; Lhermitte, J.; Brown, S.; Milette, J.; Toader, V.; Sutton, M.; Reven, L., Reversible nanoparticle cubic lattices in blue phase liquid crystals. ACS Nano 2016, 10, 3410-3415. 35. Liu, Q. K.; Cui, Y. X.; Gardner, D.; Li, X.; He, S. L.; Smalyukh, I. I., Self-alignment of plasmonic gold nanorods in reconfigurable anisotropic fluids for tunable bulk metamaterial applications. Nano Lett. 2010, 10, 1347-1353. 16 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 18

36. Qi, H.; Hegmann, T., Formation of periodic stripe patterns in nematic liquid crystals doped with functionalized gold nanoparticles. J. Mater. Chem. 2006, 16, 4197-4205. 37. Mitov, M.; Bourgerette, C.; de Guerville, F., Fingerprint patterning of solid nanoparticles embedded in a cholesteric liquid crystal. J. Phys. Condens. Matter 2004, 16, S1981-S1988. 38. Le Guevel, X.; Trouillet, V.; Spies, C.; Li, K.; Laaksonen, T.; Auerbach, D.; Jung, G.; Schneider, M., High photostability and enhanced fluorescence of gold nanoclusters by silver doping. Nanoscale 2012, 4, 7624-7631. 39. Roy, S.; Baral, A.; Bhattacharjee, R.; Jana, B.; Datta, A.; Ghosh, S.; Banerjee, A., Preparation of multi-coloured different sized fluorescent gold clusters from blue to NIR, structural analysis of the blue emitting Au-7 cluster, and cell-imaging by the NIR gold cluster. Nanoscale 2015, 7, 1912-1920. 40. Russier-Antoine, I.; Bertorelle, F.; Hamouda, R.; Rayane, D.; Dugourd, P.; Sanader, Z.; Bonacic-Koutecky, V.; Brevet, P. F.; Antoine, R., Tuning Ag-29 nanocluster light emission from red to blue with one and two-photon excitation. Nanoscale 2016, 8, 2892-2898. 41. Smalyukh, I. I.; Zribi, O. V.; Butler, J. C.; Lavrentovich, O. D.; Wong, G. C. L., Structure and dynamics of liquid crystalline pattern formation in drying droplets of DNA. Phys. Rev. Lett. 2006, 96, 177801. 42. Olesiak-Banska, J.; Mojzisova, H.; Chauvat, D.; Zielinski, M.; Matczyszyn, K.; Tauc, P.; Zyss, J., Liquid crystal phases of DNA: Evaluation of DNA organization by two-photon fluorescence microscopy and polarization analysis. Biopolymers 2011, 95, 365-375. 43. Cordoyiannis, G.; Kurihara, L. K.; Martinez-Miranda, L. J.; Glorieux, C.;Thoen, J., Effects of magnetic nanoparticles with different surface coating on the phase transitions of octylcyanobiphenyl liquid crystal., Phys. Rev. E 2009, 79, 011702. 44. Al-Zangana, S.; Turner, M.; Dierking, I., A comparison between size dependent paraelectric and ferroelectric BaTiO3 nanoparticle doped nematic and ferroelectric liquid crystals. J. Appl. Phys. 2017, 121, 085105. 45. Liu, B.; Ma, Y.; Zhao, D.; Xu, L.; Liu, F.; Zho, W.; Guo, L., Effects of morphology and concentration of CuS nanoparticles on alignment and electro-optic properties of nematic liquid crystal. Nano Res. 2017, 10, 618-625. 46. Olesiak-Banska, J.; Gordel, M.; Matczyszyn, K.; Shynkar, V.; Zyss, J.; Samoc, M., Gold nanorods as multifunctional probes in a liquid crystalline DNA matrix. Nanoscale 2013, 5, 10975-10981. 47. Olesiak, J.; Matczyszyn, K.; Mojzisova, H.; Zielinski, M.; Chauvat, D.; Zyss, J., Liquid crystalline phases in DNA and dye-doped DNA solutions analysed by polarized linear and nonlinear microscopy and differential scanning calorimetry. Mater. Sci.-Poland 2009, 27, 813823.

17 ACS Paragon Plus Environment