Polymorphism of CMONS Nanocrystals Grown in Silicate Particles

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Article pubs.acs.org/crystal

Polymorphism of CMONS Nanocrystals Grown in Silicate Particles through a Spray-Drying Process Cécile Philippot,† Joséphine Zimmermann,† Fabien Dubois,† Maria Bacia,† Bruno Boury,‡ Patrice L. Baldeck,§ Sophie Brasselet,∥ and Alain Ibanez*,† †

Institut Néel, CNRS (UPR 2940) & Université Joseph Fourier, 25 rue des Martyrs, BP 166, 38042 Grenoble cedex 9, France Institut Charles Gerhardt, CNRS (UMR5253), Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 5, France § Laboratoire Interdisciplinaire de Physique Université Joseph Fourier, CNRS (UMR 5588), BP 87, 38402, St Martin d′Hères Cedex, France ∥ Institut FRESNEL, CNRS (UMR 7249), Université Aix Marseille, Ecole Centrale Marseille, Campus Universitaire de Saint Jérôme, Avenue Escadrille Normandie-Niemen 13397 Marseille cedex, France ‡

ABSTRACT: The preparation of core−shell nanoparticles constituted by organic nanocrystals embedded in amorphous organosilicate shells is based on the controlled drying of micrometer-sized droplets produced from the atomization of sol−gel solutions. This leads to the formation of an organosilicate crust at the first stage of the droplet drying, followed by the confined nucleation and growth of dye nanocrystals in the cores of the resulting silicate particles. We selected CMONS as model dye because it is strongly fluorescent, only in the crystal state. To specify the coupled effects of confined nucleation and growth and that of the nature of silicate matrix on crystallinity and polymorphism of CMONS nanocrystals, we characterized the core−shell nanoparticles by electron microscopies and absorption, fluorescence and second-harmonic generation (SHG) spectroscopies. This polymorphism control of CMONS nanocrystal allows enhancing and tuning their fluorescence and second harmonic generations. This is promising for the development of biological tracers involving fluorescence excited by two-photon absorption. fluorescence observed for a lot of molecule dimers.8 Some recent works present the utilization of a series of organic dyes that exhibit aggregation-enhanced fluorescence to prepare bright nanoparticles for bioimaging applications.9 In this context, we have developed a one-step process to prepare hybrid core−shell NPs constituted by organic nanocrystal cores (dye) surrounded by amorphous organosilicate shells.10 Their preparation is based on the controlled drying process of micrometer-sized droplets (spray-drying) produced from sol−gel solutions leading to the formation of an organosilicate concomitantly with the confined nucleation and growth of dye nanocrystals (NCs) forming the cores of the resulting core−shell NPs (see Figure 1). Thus, at the center of NPs, the organic NCs exhibit diameters of around several tenths of nanometers corresponding to about 105−106 aggregated molecules. These high molecule numbers increase, by several orders of magnitude, the absorption and fluorescence emission cross sections of NCs. Thus, nanocrystals exhibit a significantly higher photoluminescence emission in comparison to single fluorescent molecules. This leads to brilliant NPs, which exhibit generally a good photostability due to the dye crystallization. In this paper,

1. INTRODUCTION Utilization of fluorescent nanoparticles (NPs) is an efficient approach to develop new sensitive protocols for two-photon intravital in vivo imaging.1,2 However, publications in this area remain limited because of the difficulty to prepare nondiffusible and biocompatible contrast agents with high fluorescence quantum yield and sufficient two-photon absorption cross section in physiological environment. Dye-doped silicate NPs exhibit significant advantages over single dye labeling in bioanalysis applications. Indeed, the incorporation of dye molecules inside a silicate matrix protects the dye from its biological environment and increases its photostability.3 Moreover, they are biocompatible and can be easily functionalized by grafting biomolecules through the presence of silanol groups on the NP surface.4 Dye-doped silicate NPs have several advantages for bioanalysis: they are chemically inert and not subjected to microbial attack5 and are easy to obtain by a modified Stöber synthesis6 or by grafting dye molecules onto NPs with covalent bonds (imposition method). Additionally, surfactants can be used to control the NP size distribution between 20 nm and 1 μm by creating microemulsions. These NP syntheses are easy to implement, and grafting processes of dyes on silicate NPs are well-known.7 The major drawback of these NPs is the very low level of fluorescence intensities because dye molecules are dispersed at low concentration in silicate matrices to avoid the typical quenching of molecular © 2013 American Chemical Society

Received: July 3, 2013 Revised: November 8, 2013 Published: November 8, 2013 5241

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melting point at 163 °C,13 can also be obtained from the melt, but under lower cooling rates.14 This polymorph can be transformed in form II by mechanical compression.14 These three polymorphs have different absorbance and fluorescence spectra (Table 1). Form I presents a yellow fluorescence with an emission maximum at 557 nm but no SHG due to its centrosymmetrical structure. Form II exhibits a maximum of fluorescence at 596 nm and a weak SHG intensity. Finally, polymorph III has a fluorescence maximum at 539 nm and shows a very strong SHG emission.

we analyzed the crystal quality and polymorphism of organic NC cores inside these new types of fluorescent NPs. We selected α-[(4′-methoxyphenyl)methylene]-4-nitro-benzeneacetonitrile (CMONS) as a model for several reasons, and first because it is strongly fluorescent, only in the crystal state, with good chemical-, thermal-, and photostabilities associated with interesting quadratic nonlinear optical (NLO) properties with efficient second harmonic generation (SHG) emission.11 On the other hand, this compound, easily obtained from a onestep synthesis, presents three transmolecular crystalline forms described in the literature (Table 1). Form I exhibits a centrosymmetric crystal structure with the P21/n space group, which is thermodynamically the most stable polymorph, while forms II and III are noncentrosymmetric with the Cc space group and are thus potentially active for SHG.12−14 The presence of these three identified and very different forms is an effective tool to study the effect of the preparation of core−shell NPs on the polymorphism of the CMONS core (CMONS-NC). Moreover, these polymorphs exhibit different fluorescent properties; therefore, the knowledge and control of the CMONS polymorphism is important to achieve in order to set up the most efficient process for the preparation of highly fluorescent core−shell labels for imaging. The CMONS polymorphism allowed us to study the coupled effects of confined nucleation and growth occurring during the spraydrying process, and those of chemical compositions of the silicate matrix, on the crystal quality and polymorphism of CMONS-NCs grown in the organosilicate shells. We compared our CMONS nanocrystallizations obtained through the confinement in sol−gel microdroplets (1−2 μm in diameter) and under high supersaturations with the previous ones obtained for CMONS-NCs confined in bulk sol−gel matrices,13 and also with typical crystallizations of CMONS microcrystals in free solutions.14

3. EXPERIMENTAL SECTION 3.1. Starting Materials. CMONS was synthesized by condensation of 4-methoxybenzaldehyde and 4-nitrophenylacetonitrile (Sigma) and then recrystallized in toluene and acetic acid, as described in the literature.15 Tetramethoxysilane (TMOS) and 1,2-bis(trimethoxysilyl)ethane (TMSE)16 Si-alkoxides were purchased from ABCR, while 1,16-bis(trimethoxysilyl)-4,7,10,13-tetraoxahexadecane (BTTPOHD) 17 was synthesized according to the literature procedure.17 Tetrahydrofuran (THF, inhibitor free, >99.9%, for HPLC, Aldrich) was directly used as solvent without any other purification. 3.2. Sol Preparations. Previous experiments have shown that specific molar compositions of Si-alkoxides allowed obtaining good dye NC confinements in spherical silicate shells: 0.66 TMOS + 0.33 TMSE (STMSE)18 and 0.89 TMOS + 0.11 BTTPOHD (SBTTPOHD).19 The sols were prepared at room temperature in air atmosphere from these STMSE or SBTTPOHD Si-alkoxide mixtures with THF, because CMONS presents a high solubility in this solvent (0.15 mol/L at 25 °C), and adding a small amount of HCl aqueous solution (pH = 1) to favor the formation of silicate chains through the control of hydrolysis and condensation reactions of alkoxides. THF, water, and CMONS were added according to parameters optimized in a previous work20 using the following molar ratios referred to the alkoxides or alkoxide functions (−OR): s = [solvent]/[alkoxides] = 500, h = [H2O]/[−OR] = 1, and d = [CMONS]/[alkoxides] = 0.1.

2. POLYMORPHISM OF CMONS The different forms of CMONS (Table 1) were previously obtained by crystallizations in solutions, through vapor Table 1. Spectroscopic Data from Ref 13 and Crystallographic Data from Ref 14 of CMONS Polymorphs

3.3. Spray-Drying Process. The preparation of core−shell NPs is based on the confined nucleation and growth of CMONS-NCs in sol− gel droplets generated from sol atomizations, which are rapidly dried through heated fluxes in a homemade reactor, Figure 1. The CMONS molar amount d = 0.1 corresponds to the maximum of dye ratio that can be correctly confined in the silicate shell without any phase segregation.20 Just after the sol atomization, each droplet is an homogeneous THF solution containing partially hydrolyzed silicate precursors (black strings in Figure 1 (7)), and dissolved CMONS molecules. These droplets start their drying in the laminar flux of vector gas (nitrogen) at the entrance of the horizontal furnace (T = 150 °C) with the gradual formation of organosilicate crusts at the droplet surfaces, Figure 1 (8). Then when the drying of droplets is highly advanced in the horizontal furnace, nucleation and growth of CMONS-NCs take place, in the confinement of each droplet cores (Figure 1 (9)). Finally, the complete evaporation of THF occurring at the end of this self-organized one-step process leads to core−shell NPs (Figure 1 (10)). These are collected at the exit of the horizontal furnace with an electrostatic filter (10 kV) kept at 140 °C to avoid any vapor condensation of water and solvent. 3.4. Dissolution of Silicate Shell. Bare CMONS-NCs have been obtained by dissolving the silicate shell in 0.1 M NaOH solutions. Thus, NPs were dispersed in NaOH solutions with ultrasound activation for 30 min (150 W). Then the slow dissolution of silicate shells was prolonged at room temperature for one weak in a closed vial followed by the neutralization of solutions with HCl.

condensation or from the melt:13,14 Form I, which melts at 164.5 °C, is predominant from crystallization by vapor condensation with sublimation at 160 °C or by crystallization from the melt, using very slow cooling rates.14 This thermodynamically stable form can also be obtained in solutions under low and high supersaturations with solvents as dichlorometane, dimethylformamide, or alcohols and at low supersaturations with toluene.13 Form II, which melts at 163 °C, crystallizes in toluene at high supersaturations13 and from melt at rapid cooling rates14 (melt quenching). Form III, with a 5242

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Figure 1. Spray-drying reactor: (1) flask containing the initials sols, (2) nitrogen injection in the pneumatic atomizer from TSI, model 3076, (3) annular injection of vector gas, nitrogen, (4) horizontal oven (1.5 m long) at 150 °C, (5) electrostatic filter at 140 °C, and (6) gas outlet. Schematic presentation of the controlled drying of initial sol−gel microdroplets (7), formation of organosilicate crusts at the droplet surfaces (8), confined nucleation and growth of CMONS-NCs in the droplet cores (9), and the resulting core−shell NPs (10). 3.5. Characterizations. To characterize the core−shell NPs, they were dispersed in 10−2 M NaOH solutions with ultrasound activation for 30 min (150 W) in order to slightly dissolve the organosilicate shells and thus favor their disaggregation. The resulting solutions were then neutralized, up to pH = 7−8, by a gradual addition of 10−2 M HCl solution. The as-obtained NP suspensions were colloidally stable during several months. Field emission scanning electron microscopy (FESEM) pictures were recorded using a Carl Zeiss Ultra scanning electron microscope. NPs were previously deposited on doped silicon wafers to favor negative charge evacuation. This FESEM technique offered high resolution imaging at low voltage. Indeed, to reduce the problem of charge accumulation at NP surfaces and their degradation during observations, we worked at 3 kV with a working distance of 3 mm. We determined the size distributions of core−shell NPs and CMONS-NC cores, after shell dissolution, by dynamic light scattering (DLS) using a Zetasizer Nano Zs instrument from Malvern company. These corresponding values were obtained by averaging three measurements performed at 25 °C (laser wavelength 633 nm, acquisition time 5 min). Before the measurements, core−shell NPs and bare CMONS-NCs were dispersed by ultrasound (15 min, 150 W) in aqueous solution (pH = 7). To confirm the presence of crystallized CMONS cores inside NPs and specify the different polymorphs, transmission electron microscopy (TEM) was involved in dark field and diffraction modes by using a Philips CM300 microscope operating at 150 kV. TEM images were acquired with a Gatan 794 CCD camera. Due to the typical high sensitivity of organic NCs to electron beams, TEM observations were carried out at 100 K, using a Gatan liquid nitrogen-cooled holder. Diffraction patterns were recorded with a short exposure time of 0.25 s to avoid any NC amorphization. For phase identification and zone axis determination from observed d-spacings, Calidris softwares,21 ELD and PhIDO, were used. In PhIDO database of possible compounds, three CMONS polymorphs (Table 1 and Sherwood et al.14) and structures of impurities as Si, NaOH, and NaCl were defined. Absorption spectra were registered with a spectrophotometer Perkin-Elmer λ9. Fluorescence emission spectra were measured with a spectrofluorometer Fluoromax@4 Horiba of Jobin-Yvon. Twophoton fluorescence spectra were recorded with a Ti:sapphire femtosecond laser (50 mW) using an acquisition time of 1 s. The fluorescence emission was collected at 90° with an optical fiber of 600 μm diameter coupled with a CVI spectrometer. In all cases, the core− shell NPs were colloidally dispersed in aqueous solutions. The two-photon emission properties (SHG and fluorescence) of single NPs were performed by depositing and drying on microscope

plates, of a droplet of NP colloidal suspension. The NP concentration was chosen to allow a sufficiently high interdistance between particles (more than 1 μm). Two-photon microscopy imaging was performed using an excitation source from a Ti:Sa laser (pulse width 100 fs, repetition rate 80 MHz) set at a wavelength of 800 nm, for a twophoton excitation wavelength (400 nm) located at the vicinity of the linear excitation maximum, and far enough from the fluorescence emission in order to avoid fluorescence leakage in the SHG detection spectral range. The light was focused on the sample using a water immersion high numerical aperture objective (NA 1.15, ×40) after reflection on a dichroic mirror. The incident average power at the sample plane was typically 1 mW, which is significantly below that for the photodamage of NPs. The sample was scanned using a galvanometric scanner moving the beam focus within a lateral area of typically 20 × 20 μm2, with a spatial sampling of 150 × 150 pixels and a pixel integration time of 100 μs. The typical pixel size was therefore about 130 nm, which was sufficient to resolve a diffraction limit spot since the lateral resolution of the microscope is about 300 nm. SHG and TPEF emissions, collected by the same objective in the backward direction, were detected by large area photomultiplier tubes working in the photon counting mode. The SHG and TPEF (TwoPhoton Excited Fluorescence) signals were filtered by a 400 nm (width 20 nm) interference filter and by a 540 nm (width 50 nm) filter, respectively. Polarization dependence responses were recorded by rotating an achromatic half wave plate in front of the microscope, and recording the emission signals for 32 different incident linear polarization angles varying from 0° to 180° with respect to the horizontal axis in the sample plane (denoted X). For SHG and TPEF polarization responses detection, the emission was projected along two polarization analysis directions (denoted X and Y) using a polarization beam splitter, allowing the simultaneous recording of two polarization dependence responses IX(α) and IY(α). To depict polarization responses, the data from a single particle were averaged over 4 × 4 pixels around the particle center. In the images, the SHG and TPEF efficiencies are given as sums over all polarization angles. Statistics on the global SHG efficiency of nanoparticles depending on their preparation conditions were performed for 40 particles.

4. RESULTS AND DISCUSSION The good control of sol−gel chemistry, coupled to optimized spray-drying parameters, allows the perfect confinement of nucleation and growth of organic cores in silicate shells.20 For that, we must carefully select Si-alkoxides to avoid significant chemical interactions between CMONS molecules and the 5243

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are highly delicate for molecular NCs with diameters below 150 nm due to crystal sublimation under the electron beam. Similar dissolution behaviors and CMONS crystal shapes were observed for the two types of silicate matrices STMSE and SBTTPOHD, Figure 3. For the largest NP diameters, of around 1

silicate chains, resulting from the hydrolysis and condensation reactions of alkoxides. Indeed, these interactions such as hydrogen bonds, between silanol groups of silicate chains and nitro moieties of CMONS, can lead to the grafting of dye molecules on the sol−gel network and can affect the confined nucleation and growth of CMONS-NCs. In order to screen these dye−matrix interactions, TMOS was always mixed with another type of silicon alkoxide bearing specific moieties. To obtain NPs with hydrophilic shells required for in vivo tracers, the choice of these additional silicon alkoxides has been focused on TMSE and BTTPOHD.19 These Si-alkoxides exhibit organic spacers between two silyl functions (ethylene group for TMSE and polyethyleneglycol group for BTTPOHD), which reduce the dye−silicate interactions while maintaining the hydrophilic character of NP surfaces. In order to specify the influence of silicate sol−gel nature on crystallinity and polymorphism of CMONS-NCs, we have characterized the core−shell NPs by FESEM, TEM, and absorption, fluorescence, and SHG spectroscopies. Thus, CMONS-NCs were grown from confined nucleations in two types of silicate shells prepared from STMSE or SBTTPOHD sols, using drying (furnace, Figure 1 (4)) and collecting (electrostatic filter, Figure 1 (5)) temperatures of 150 and 140 °C, respectively, which are the minima required to prepare well-dried and spherical core−shell NPs with the geometry of our spray-drying reactor, Figure 1. 4.1. First Observations of Core−Shell NPs and CMONS Cores. For the two compositions of the silicate matrices, STMSE and SBTTPOHD, we observed by FESEM imaging well-defined spherical and individual NPs. The absence of organic crusts or CMONS needles, on the particle surfaces, indicates a good confinement of the dye during the preparation of core−shell NPs, as was previously described in ref 20 (Figure 2). In

Figure 3. FESEM images of crystalline organic cores after the total dissolution of their silicate shells: (a) CMONS microcrystal coming from particles with initial diameters of around 1 μm; (b) CMONS-NC coming from NP with initial diameter of around 150−200 nm.

μm, we observed well-faceted microcrystals with typical shapes of perfect single crystals, Figure 3a. For smaller particles, with diameters ranging between several hundreds of nanometers and 100 nm, we still saw some crystal faces, Figure 3b. Finally, for NPs with diameter less than 100 nm, the corresponding CMONS-NC cores exhibit a spherical shape, typical of molecular NCs of these sizes, as previously observed for organic NCs grown in sol−gel thin film22 or molecular NCs directly obtained in aqueous solution by “jetlike precipitation”.23 Then, we roughly estimated from these FESEM observations the average diameters of initial core−shell NPs compared to those of CMONS-NC obtained after shell dissolutions in sodium hydroxide solutions. This allowed estimating the diameter ratio between organic cores and core−shell NPs, DC‑NP, ranging between 0.6 and 0.75. As these indirect measurements are qualitative, we then specified by DLS the size distribution of core−shell NPs and CMONS-NCs and the corresponding diameter ratio, DC‑NP. Figure 4 shows the typical size distributions (NP diameters) calculated from DLS measurements performed for core−shell

Figure 2. Typical FESEM image of hybrids NPs constituted by crystalline organic cores (CMONS) confined in amorphous silicate shells (STMSE).

agreement with previous results, we obtained polydisperse particles, due to the pneumatic atomization of sols that allows high yields of NP production; with more than 65% of NP diameters lower than 100 nm and without any influence of the sol−gel matrix nature. Then, to directly observe the CMONS-NCs by FESEM, we dissolved the silicate shells in sodium hydroxide solutions. Indeed, this process is known to gradually dissolve the silicate matrices without any damage of the embedded organic NCs.22 The resulting colloidal suspensions of CMONS-NCs were then observed by FESEM. It is noteworthy that imaging conditions

Figure 4. DLS size distributions of core−shell NPs (solid line) and corresponding CMONS-NC cores obtained after shell dissolution in sodium hydroxide solutions (dotted line).

NPs and CMONS-NCs dispersed in aqueous solutions. We registered similar curves for the two types of silicate shells, STMSE and SBTTPOHD. Moreover, these DLS results led to the same size distributions than those estimated by FESEM images with a maximum for diameters of about 80 nm and a significant majority of NPs having diameters less than 100 nm. After the 5244

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shell dissolution, CMONS-NCs exhibit logically smaller sizes, with a maximum for diameters of about 60 nm and a diameter ratio between organic cores and core−shell NPs, DC‑NP, of around 0.75 in good agreement with the first FESEM observations. 4.2. Polymorphism of CMONS-NCs Characterized by Electron Diffraction. To reduce the strong electron scattering resulting of amorphous silicate shells, TEM observations were performed on CMONS-NCs obtained after the total dissolution of shells in sodium hydroxide aqueous solutions. When the latter were initially embedded in STMSE silicate shells, using the smallest available selected area aperture (70 nm in specimen plane), it has been possible to record a lot of diffraction patterns typical of single crystals, as one of the most representative is shown in Figure 5a. Using the Calidris

Figure 6. (a) Typical electron diffraction pattern registered for CMONS-NCs cores grown in SBTTPOHD shells, crystallized in form III in zone axis near [1̅32]; (b) corresponding TEM image recorded in the dark field mode of CMONS-NCs; (c, d) other dark field images illustrating the generality of the presence of alternating domains. (Notice A and B in the diffraction pattern.)

spots, but it was not feasible to distinguish interplanar spacings closer to 0.02 Å. The second difficulty consisted of the impossibility to record diffraction patterns through another zone axis for the same crystal CMONS-NC due to its rapid amorphization under the electron beam (in a few seconds). On the other hand, this phenomenon of fringes has already been viewed at the macroscopic scale by Sherwood et al.14 for bulk crystals of CMONS grown from the melt by the Bridgman method. They observed alternating fringes by photomicrographs on cleaved Bridgman boules, leading to (010) and (100) fractures faces. They specified the origin of these fringes by the alternating presence of polymorphs III and II (characterized by X-ray diffraction) due to a partial structural transition of form III crystals in the form II as a consequence of thermomechanical strains induced in the Bridgman boule during the growth. By this remarkable analogy between these photomicrographs of cleaved centimeter-sized crystals and dark field TEM images of CMONS-NCs, Figure 6, an obvious parallel can be drawn between the bulk crystal growth of CMONS Bridgman boules and CMONS-NCs in SBTTPOHD silicate shells. This SBTTPOHD matrix is based on the mixing of two silicon alkoxides: TMOS that is an efficient cross-linker and BTTPOHD, which exhibit a flexible polyethylene-glycol spacer. During the drying of droplets, the cross-linking of the inorganic Si−O−Si network occurs, leading to the organosilicate shell, a chemical process that generates high mechanical stresses on the CMONS-NCs cores through typical capillary forces. Thus, taking into account the previous observations of Sherwood et al.14 and the spectacular similarity of our observations at the nanoscale, we can assume that the formation of these alternating fringes in CMONS-NCs is due to a structural transition between form III and form II, favored by thermomechanical stresses, during the drying of SBTTPOHD shells. This phenomenon was not observed for the STMSE shell, which favors only the crystallization of polymorph I. 4.3. Spectroscopy Analysis. The one-step nucleation and growth process of organic nanocrystals in sol−gel droplets is highly confined in small volumes (initial droplet diameter of around 1 μm). Thus, after the nucleation in each droplet and during the growth, all the CMONS molecules are strongly attracted to the thermodynamically stable crystal phase. Consequently, in the resulting core−shell nanoparticles, the concentration of dispersed CMONS molecules in the silicate shell is null or negligible. Previous studies have shown that the various polymorphs of CMONS present differences in absorption, fluorescence, and/or SHG spectra.13,14 The

Figure 5. (a) Electron diffraction patterns of CMONS-NCs grown in STMSE silica shells and crystallized in form I observed here near the [11̅1] zone axis; (b) CMONS-NCs grown in SBTTPOHD silica shells crystallized in form II near of zone axis [011̅ ].

software, we specified from these diffraction patterns the monoclinic primitive cell parameters a = 3.8 Ǻ , b = 12.3 Ǻ , c = 28.1 Ǻ , β angle = 90.71°, two inter-reticular distances: 11.3 Ǻ that does not exist in forms II and III of CMONS, and 3.8 Ǻ which corresponds to a crystal orientation through the [11̅1] zone axis of form I of the CMONS crystal structure with the monoclinic P21/n space group (Table 1). Electron diffraction patterns of this form I were sometimes observed for CMONS-NCs grown in the second type of matrix SBTTPOHD. This result confirms that this form I, which is thermodynamically the most stable, is the predominant one for the CMONS polymorphism.14 But with this SBTTPOHD silicate shell, we generally observed diffraction patterns such as that shown in Figure 5b, with the principal inter-reticular distances, 4.5 and 4.0 Å, and an angle of 61.1° between them. These crystallographic data clearly correspond to CMONS-NCs of polymorph II, observed here along the [01̅1] zone axis. Moreover, a lot of diffraction patterns of CMONS-NCs grown in SBTTPOHD silicate shells looked like that presented in Figure 6a. These diffraction patterns, which can be approximately indexed with the structures of form II and III, are closely related and typical to crystals containing planar defects. This result was then completed by TEM observations in the dark field mode, using the (023)̅ diffraction spot, which confirmed for many CMONS-NC grown in SBTTPOHD silica shells the presence of parallel fringes inside of CMONS-NCs, Figure 6b−d. Furthermore, when identifying diffraction pattern in Figure 6a, we found that it can correspond to a twin near to the [13̅2̅] zone axis of the polymorph III of CMONS (Table 1). In Figure 6a, we present one of the possible indexation of diffraction 5245

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Figure 7. (a) Absorption spectra (dotted lines) and one-photon fluorescence spectra (λex = 400 nm) (solid line); (b) two-photon fluorescence spectra (λex = 800 nm) of core−shell NPs. Black curves correspond to CMONS-NCs grown in STMSE shells, while the gray ones correspond to CMONS-NCs grown in SBTTPOHD sol−gel matrix.

CMONS-NCs are constituted by two bands, centered at around 390 and 450 nm. This is due to the CMONS crystal cell composed of four CMONS molecules similar to two by two for the three polymorph structures. Moreover, the significant enlargement observed for CMONS-NCs grown in SBTTPOHD shells is well consistent with the presence of the three polymorphs I−III as evidenced by electron diffraction. For CMONS-NCs grown in STMSE shells, a blue shift of the spectroscopic data was observed compared to the characteristic value obtained for the CMONS polymorph I (Table 1), which can be attributed to different origins such as a small amount of polymorph III, interactions between the surface of the CMONS

corresponding spectroscopies of CMONS-NCs were performed on colloidal suspensions with core−shell NP diameters less than 100 nm. The as-recorded spectra, for absorption and one- or two-photon fluorescence, are gathered in Figure 7, while the corresponding spectroscopic data are summarized in Table 2. The absorption spectra (Figure 7a, dotted curves) of Table 2. Spectroscopic Data of CMONS-NCs Grown in STMSE and SBTTPOHD Shells STMSE SBTTPOHD

cutoff wavelength (nm)

emission max (nm)

SHG

494 525

544 556

very weak strong

Figure 8. (a) SHG image and (b) TPEF image of NPs based on CMONS-NCs grown in SBTTPOHD. The intensity scale is the total intensity (in photon counts) summed over all 32 incident polarization angles. (c−e) SHG and TPEF polarization-dependent responses analyzed in the X horizontal (red) and Y vertical (green) directions, for some of the NPs obtained from CMONS-NCs in SBTTPOHD. The responses to a varying incident polarization from 0° to 180° in 32 steps in the sample plane are represented as polar graphs. Left: SHG theoretical adjustment. Middle: SHG experimental (markers) superimposed with a continuous line obtained from a Fourier series decomposition of the data. Right: TPEF experimental (markers). The complete 0−360° response is reconstructed by central symmetry. The left graphs are obtained based on the following parameters: (c) form III unit cell, Euler set of angles (θ = 0°; ϕ = 30°; ψ = 0°); (d) form III unit cell, Euler set of angles (θ = 30°; ϕ = −15°; ψ = 0°); (e) incoherent addition of a form III unit cell (θ = 70°; ϕ = −50°; ψ = 0°) and a form II unit cell (θ = 40°; ϕ = 40°; ψ = 0°). 5246

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nanocrystals and the sol−gel matrix, and finally to particle shape or surface defects. Moreover, these different suggested origins could be coupled. In conclusion, it is rather delicate to clearly specify this slight fluorescence shift, but the very weak SHG signal for CMONS-NCs in STMSE (Figure 7b) seems to be in agreement with the existence of the preferential centrosymmetric polymorph I associated to a weak amount of polymorph III. On the other hand, we can notice a red-shift for CMONSNCs grown SBTTPOHD referred to those obtained in STMSE sol− gel droplets, Table 2 and Figure 7, that can be explained by the contribution of form II (Table 1). We can also observe a significant SHG signal for CMONS-NCs grown SBTTPOHD due to the presence of an important contribution of polymorph III. In conclusion, for CMONS-NCs in SBTTPOHD, the fluorescence red-shift is due to the presence of form II, while the strong SHG is arising from form III. This confirms that CMONS-NCs in SBTTPOHD are constituted of a mixture of forms II and III as previously shown by dark field TEM image, Figure 6b and c. To complete these qualitative results, we then performed SHG and TPEF polarization resolved imaging on single CMONS-NCs grown in STMSE and SBTTPOHD matrices. Typical SHG and TPEF images performed on a same sample region of NPs made of CMONS-NCs are shown in Figure 8a and b. NPs can be recognized as bright spots with a size close to the diffraction limit, which correspond to objects below 100 nm in diameter. All particles are efficient for both SHG and TPEF, to different extents: indeed optical efficiencies can vary from one NP to another due to their variations in size and orientation. To study quantitatively the SHG efficiency of single NCs and their capacity to form noncentrosymmetric nano-objects, it is important to discard aggregates of NPs that can be produced during the particle deposition on the substrate. Since these agglomerates can be smaller than the diffraction limit, they are not necessarily distinguishable by their spot size on the SHG image. To perform this distinction, we used the information given by polarization resolved TPEF signals. It has been demonstrated that pure monocrystalline nano-objects can be recognized by their identical polarization responses for TPEF, whatever the analyzer direction in the detection path.11,24 Examples of such particles are depicted in Figure 8c−e, for which TPEF responses show visibly identical polarization response shapes (with different magnitudes) along the X and Y analysis directions. In the following, only such data are considered, since they are representative of monocrystalline NPs. Using such a diagnostics, which was demonstrated to be efficient for detecting CMONS single nanocrystals,11 we extracted and analyzed data over about 40 single crystals for CMONS-NCs grown in both STMSE and SBTTPOHD. The SHG efficiency of each NP was first calculated as its X and Y summed signal averaged over the 32 incident polarization directions, allowing a rough average over their orientational variations. This total signal was then averaged over 40 NPs per preparation condition, to average over possible size dispersion effects. In the case of NPs grown in STMSE, which are expected to be purely centrosymmetric, about half of the TPEF-active NPs exhibit measurable SHG efficiency. This non-negligible SHG, which is in agreement with two-photon spectroscopy observations (Figure 7b), can be assigned to surface effects or particle shape defects, which are the dominant source of SHG in centrosymmetric bulk nano-objects or by the presence of a small amount of the noncentrosymmetric polymorph III. The polarization dependence of the SHG signals from such NPs

resembles one-dimensional anisotropic responses, which can be interpreted as pure surface responses.25 In the case of SBTTPOHD based particles, a great majority of the TPEF-active NPs is SHG active. This is a signature of the higher probability to find noncentrosymmetric structures (form II or III) in CMONS-NCs grown in SBTTPOHD. The SHG signals from NPs in SBTTPOHD are also about twice more efficient than in STMSE. Additional structural information was provided using an analysis of the SHG polarization dependencies of CMONS-NCs (Figure 8c−e). Using a model previously developed,11 which considers the nonlinear tensor of unit-cells of either form II or III, a fit of such responses was performed introducing as a variable parameter the 3D orientation of the NPs given by its Euler set of angles (θ, ϕ, ψ). The examples given in Figure 8c,d show that CMONS-NCs grown in SBTTPOHD can be well compared to theoretical calculations obtained from a type III CMONS symmetry, adapting the crystal orientation to fit at best the experimental data (note that the found (θ, ϕ, ψ) solution might be nonunique). In SBTTPOHD, part of the CMONS-NCs could not be fitted by a single orientation of noncentrosymmetric form. However, a fit by the incoherent addition of two forms III and II of different orientations was able to reproduce the observed responses with a reasonable agreement (Figure 8e). These cases are visibly constituted by coexisting polymorphs II and III, as observed by TEM in the dark field images (Figure 6).

5. CONCLUSION Through the confined nucleation and growth of CMONS used as model dye in these preliminary works, we demonstrated the feasibility of the self-organization of core−shell NPs prepared by a one-step spray-drying process by involving two different types of sol−gel matrices. These NPs are constituted by single crystal cores of dye well embedded in organosilicate shells. We synthesized polydisperse particles, due to the pneumatic atomization of sols that allows high production yields of NPs, with more than 65% of NP diameters lower than 100 nm, and with a diameter ratio between organic cores and core−shell NPs of around 0.75. Moreover, we did not observed any influence of the sol−gel matrix nature on the size and spherical shape of NPs. On the contrary, we clearly noted a significant effect of the nature of organosilicate matrix on CMONS polymorphism. For CMONS-NCs grown in the highly crosslinked STMSE silicate network, the CMONS crystal structure corresponds to form I, which is the more thermodynamically stable one. On the other hand, in SBTTPOHD silicate shells, involving PEGylated flexible chains, CMONS-NCs exhibit parallel fringes, observed by TEM in the dark field mode, which can be explained by the alternating presence of polymorphs III and II. Indeed a remarkable analogy can be drawn between previous photomicrographs of cleaved centimeter-sized crystals grown from the melt by the Bridgman method and the dark field TEM images of CMONS-NCs prepared from their confined nucleation from the drying of sol−gel droplets. This obvious parallel allowed us to conclude that the formation of alternating fringes in CMONS-NCs is due to the structural transition between form III and form II, which is induced by thermomechanical stresses, during the drying of SBTTPOHD shells through typical capillary forces. All the results of the coupled methods used in this study, electron microscopies and absorption, fluorescence, and SHG spectroscopies, are very consistent between themselves. These first results on the polymorphism control of molecular nanocrystals open new 5247

dx.doi.org/10.1021/cg401000t | Cryst. Growth Des. 2013, 13, 5241−5248

Crystal Growth & Design

Article

(24) Gasecka, A.; Tauc, P.; Lewit-Bentley, A.; Brasselet, S. Phys. Rev. Lett. 2012, 108, 263901. (25) Heinz, T. F.; Chen, C. K.; Ricard, D.; Shen, Y. R. Phys. Rev. Lett. 1982, 48, 478−481.

opportunities to tune and enhance the spectroscopic properties such as fluorescence properties and second harmonic generations of dye nanocrystals. This is particularly promising for the development of biological tracers by involving fluorescence excited by two-photon absorption.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (33) 4 76 88 78 05. Fax: (33) 4 76 88 10 38. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was performed with the support of the French National Research Agency (ANR) and the Rhône-Alpes region. REFERENCES

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dx.doi.org/10.1021/cg401000t | Cryst. Growth Des. 2013, 13, 5241−5248