All Optical Determination of Microscopic and Macroscopic Structure of

Article pubs.acs.org/JPCC

All Optical Determination of Microscopic and Macroscopic Structure of Chiral, Polar Microcrystals from Achiral, Nonpolar Molecules Stijn Van Cleuvenbergen,*,† Gunther Hennrich,§ Pieter Willot,† Guy Koeckelberghs,† Koen Clays,† Thierry Verbiest,† and Monique A. van der Veen*,†,‡ †

Department of Chemistry and ‡Centre for Surface Chemistry and Catalysis, KU Leuven, B-3001 Leuven, Belgium Departamento de Química Orgánica, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

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S Supporting Information *

ABSTRACT: Organic microcrystals obtained from an octupolar molecule are studied by polarized nonlinear optical microscopy. While two-photon fluorescence microscopy is employed to verify the monocrystallinity of the analyzed domains, second-harmonic generation microscopy allowed determination of the point group symmetry of the crystallites. This combined analysis revealed that the achiral, octupolar molecules form chiral and polar conglomerate structures; the former are confirmed by circular dichroism spectroscopy. We additionally present a convenient and straightforward image analysis protocol, based on polarization dependent second-harmonic generation microscopy. This allows assessing the local organization and homogeneity of crystalline structures, which is highly relevant for technological applications, with high resolution and sensitivity.

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frequency ω, interacting with a nonlinear material, combine to create a new photon at the double frequency 2ω. Within the electric dipole approximation, it can be described by the different components of the nonlinear polarization Pi(2ω) at frequency 2ω:

INTRODUCTION While traditional microscopy techniques are based on the linear interaction of light and matter, the interest in nonlinear optical (NLO) microscopy has increased exponentially over the past 15 years.1 Since NLO processes rely on multiphoton excitation in the (near) infrared range of the spectrum, where most biological materials are transparent, as are the chromophores typically studied in NLO, they provide increased penetration depths and reduced photodamage compared with traditional microscopy.1,2 Furthermore, the low probability inherent to simultaneous multiphoton excitation restricts the fraction of photons generated outside the focusing volume, greatly enhancing the imaging contrast and resolution. The majority of publications on NLO microscopy involve biological samples studied by two-photon fluorescence (TPF), which is a thirdorder NLO process and hence not restricted by any symmetry considerations. Lately, however, it has been recognized that second-harmonic generation microscopy, with its inherent sensitivity to symmetry, can reveal additional structural information that is not accessible through (NLO) fluorescence imaging.3 While the focus so far has been mainly on the study of cellular structures,4 SHG microscopy is increasingly used in materials science as well. SHG microscopy has emerged as an excellent tool to probe the organization of abiotic systems such as nanostructures,5 interfaces,6 and crystals.7 For organic crystals the high resolution offered by SHG microscopy allows characterizing the structure of crystallites down to the submicrometer scale, with minimal sample preparation.8,9 Second-harmonic generation (SHG) is a coherent secondorder nonlinear optical process in which two photons at © 2012 American Chemical Society

PiNL(2ω) = χijk(2) Ej(ω) Ek (ω)

where χ(2) ijk is a component of the second-order susceptibility tensor and Ej and Ek are the electric field components of the incoming light. Because the nonlinear susceptibility χ(2) is an odd-rank tensor within the electric dipole approximation, SHG vanishes in centrosymmetric materials.10 In organic materials, this symmetry requirement must be met on the molecular level as well as on the macroscopic level. Efficient organic secondorder NLO materials are traditionally achieved by electric field poling of asymmetrically substituted dipolar conjugated systems doped in polymer films.11 However, for many applications crystalline structures are preferred due to their higher number density, stability, and robustness. In this perspective, octupolar molecules hold great promise. Since these lack a molecular ground state dipole moment, they are more prone to spontaneously form noncentrosymmetric crystals. Progress in crystal engineering and the development of organic crystals for technological applications relies on an accurate assessment of Received: March 20, 2012 Revised: May 10, 2012 Published: May 12, 2012 12219

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Figure 1. Chemical structure of 1,3,5-tris[(4-nitrophenyl)ethynyl]2,4,6-tris(octyloxy)benzene.

this study due to the use of a low numerical aperture (0.32) objective.15 The spatial resolution under these conditions is 1 μm. Depending on the probed process and the corresponding wavelength range of interest, different filter sets are used (400 and 420−650 nm for SHG and TPF, respectively), and a sheet analyzer can be inserted as well. Finally, an EM-CCD (electron multiplying charge coupled device; Hamamatsu) camera collects the transmitted light. In the recorded images, the horizontal and vertical directions correspond to X and Y, respectively. Polarization patterns are recorded by rotating the incident fundamental linear polarization over 360° in steps of 2°, while recording the TPF or SHG images of the horizontally (X) and the vertically (Y) polarized TPF or SHG for each step. For 0° in the polarization patterns, the incident IR light is polarized horizontally, while rotating counterclockwise during the measurement. Additionally, SHG images are recorded for different azimuthal rotation angles of the sample, over 360° in steps of 5°. The sample is then rotated among the Z direction by a rotation stage (Thorlabs, XYR1) which allows for a precise alignment of the center of rotation and the incident beam. Image analysis and processing were performed within the ImageJ software packet. For the analysis of the images obtained by azimuthal rotation of the sample, all images were aligned (stacked) before analysis or processing with stackreg,16 a plugin for ImageJ available online. Circular Dichroism Spectroscopy. CD spectroscopy measurements were performed on a JASCO J810 CD spectrophotometer. The incident beam is always normal to the sample plane, i.e., the glass surface. A sample holder has been designed that allows rotating the sample among its azimuth. It is also possible to turn the sample 180°, such that the front side, directed at the light source, becomes the back side, directed at the detector. To minimize the effects of scattering, the sample is positioned as close to the detector as possible. The sampled area is 2 mm in diameter. Absorption spectra were recorded on a UV−vis spectrophotometer (Perkin-Elmer, Lambda 900).

displayed discotic liquid crystalline mesophases at higher temperatures, have been reported elsewhere.12 The sample was prepared using a standard liquid crystal cell (AWAT Co. Ltd.) composed of two uncoated glass plates separated by spacers of 1.7 μm thickness, which was filled upon heating the compound above its melting point. Subsequently the sample was cooled slowly to room temperature. Nonlinear Optical Microscopy. In the NLO microscope used in this work, the sample is illuminated wide field under normal incidence with femtosecond pulsed infrared (IR) laser light at 800 nm (Spectra Physics, Tsunami).13 The polarization of the incident IR light is varied by a zero-order half-wave plate for 800 nm mounted in a computer-controlled rotation stage (Thorlabs, PRM-Z8). A set of prisms (Newport, SF10) compensates for possible group velocity dispersion caused by the optical elements. The sample is irradiated by a long focal length lens (f = 7.5 cm) so that the incident fundamental light can be considered to a good approximation as a collimated beam and electric field components along the propagation direction (Z) can be neglected. Behind the sample, a highpower objective (Thorlabs, LMU-15X-NUV) combined with the 1.6× objective of the Olympus IX71 microscope frame collimates the transmitted light. While effects due to spherical aberration can be substantial,14 these effects can be neglected in

RESULTS AND DISCUSSION Point Group Determination. The solid state generates a strong SHG signal, up to 3 times greater than that of a 1 mm thick quartz plate. This implies, considering the moderate molecular hyperpolarizability,17 a supramolecular organization of a large number of molecules into highly efficient secondorder NLO structures. A typical image is shown in Figure 2. Needle-like crystals, several hundreds of micrometers in length and grown parallel to the glass surface, penetrate areas where notably smaller structures are formed. The SHG signal of these smaller crystallites, presumably interrupted in their growth by the glass boundaries, is significantly less intense. It has been shown by some of us that for noncentrosymmetric, thus SHG-active, structures the point group symmetry can be determined by polarization dependent SHG microscopy.17 In order to employ this recently developed methodology, it is a prerequisite that the analyzed region is a single uniform structure. In this case this means monocrystallinity. This can be verified by polarization dependent TPF microscopy. For the TPF of monocrystals, the polarization dependence of the excitation and that of the subsequent emission are independent.9 The consequence is that for such a structure the excitation response to the varying polarization will be identical, regardless of the position of the analyzer. The

the macroscopic organization, which is responsible for the second-order NLO response, and the crystal quality. In this work, we use SHG microscopy to study the symmetry and local organization of organic microcrystals obtained from an octupolar chromophore, with minimal sample preparation. TPF microscopy is employed to verify the monocrystallinity of the sampled areas. The point group symmetry has been determined solely by optical methods through polarization dependent SHG microscopy, in conjunction with circular dichroism (CD) spectroscopy. We found that these molecules adopt a chiral and polar macroscopic structure, although they are achiral and nonpolar in the absence of intermolecular interactions. Such symmetry lowering implies molecular deformation upon crystallization. Finally, we made use of the intrinsic high resolution of SHG microscopy to probe the local organization and crystal quality. A straightforward image analysis allowed us to map the direction of the polar crystal axis and the structural homogeneity of the sample with unprecedented sensitivity.

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EXPERIMENTAL SECTION Sample Preparation. The synthesis and the molecular linear and nonlinear optical properties of the disk-like octupolar (D3h symmetry) compound (Figure 1), which additionally

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Figure 2. SHG microscopy image of the sample (magnification 24×). The detected second-harmonic light at 400 nm is depicted in blue. The area selected for further analysis is highlighted in yellow and indicated by an arrow. The macroscopic X and Y directions are also indicated.

Figure 3. Point group determination via symmetry traces revealed by nonlinear microscopy (arbitrary units are used). (a) TPF polarization pattern for X and Y detection. (b) SHG polarization pattern without analyzer (equivalent to X + Y). (c) SHG polarization pattern for Y detection. (d) SHG pattern for clockwise sample rotation, while the directions of the incident IR and the detected SHG light are parallel along X (0° corresponds to the image shown in Figure 1). The symmetry axes (SA) are shown in red.

intensity of the detected TPF polarization pattern will however depend on the relative position of the crystal and the analyzer. This means that the shape of the TPF polarization pattern of a monocrystal must be identical for X and Y detection, but the amplitude, or intensity, of the pattern may be different. It can be inferred that the polarization patterns of polycrystalline ensembles, with each structure having different scaling factors for X and Y detection, must have different shapes. An analysis of the observed patterns revealed that the large crystals growing parallel to the substrate are monocrystalline, at least over several micrometers, while the intermediate areas are clearly polycrystalline. Care was taken that only monocrystalline regions were selected for symmetry determination with SHG (Figure 3a).17 The distinction between different point groups by SHG microscopy is based on two types of observables or symmetry traces. One is the amount of polarization positions at which the SHG intensity becomes zero during the polarization dependent test. The other is the number of times the function of the SHG intensity behaves as an even function around a minimum or maximum value; these maxima or minima are denominated as symmetry axes. A first series of tests analyzes observables in SHG polarization patterns for the X + Y (equivalent to no analyzer present) and Y polarization directions, labeled SHGX+Y and SHGY, respectively (parts b and c of Figure 3). In another test, labeled SHGXX, the sample is rotated clockwise in steps of 5° while the incoming IR polarization is kept parallel with the detected SHG polarization direction (along X, Figure 3d). While the curves taken at different selected areas will be different, for the majority the observed symmetry traces are the same. A few areas have higher amounts of symmetry traces, which is expected for structures in which part of the natural coordinate system coincides with the sample plane of the microscope (see ref 17). For the SHGX+Y test symmetry traces are generally absent: the SHG intensity never becomes zero and no symmetry axes are present (Figure 3b). The same is the case for the SHGY test (Figure 3c). For the final test (SHGXX), the SHG response curve exhibits clear symmetry traces: it goes to zero twice and

behaves as an even function over its respective minima and maxima, with four symmetry axes in total (Figure 3d). The outcome of these tests corresponds with a set of three point groups, i.e., C4, C6, and C∞. These both polar and chiral groups which only differ in the occurrence of either a 4-fold, a 6-fold, or an ∞-fold rotation axis are indistinguishable by the applied methodology, since they have the same χ(2) ijk tensor components governing the SHG response.17 The point group symmetries C4, C6, and C∞ have the following nonzero secondorder tensor components χijk(2): χzzz; χzxx = χzyy; χxzx = χyzy = χxxz = χyyz; χxyz = χxzy = −χyzx = − χyxz; χzxy = −χzyx. The z-index corresponds to the polar axis. The tensor components containing three different indices correspond to the chiral symmetry of the system, which is a three-dimensional property. In all other components, as can be seen, the x- and y-indices can be replaced by each other, which is a reflection of the presence of either a 4-fold, a 6-fold, or an ∞-fold rotation axis corresponding to the z-axis. The symmetry found by second-harmonic generation reflects (2) the occurrence of the different tensor components χijk associated with this symmetry. As the structure is in resonance at the wavelength of the second-harmonic light, 400 nm (see the absorption spectrum in Figure 4a), all symmetry-allowed tensor components are expected to be significant.18 This means that the symmetry in the SHG polarization patterns manifests as the true supramolecular symmetry of the extended conjugated system. The methodology used is developed to discriminate between all noncentrosymmetric point group symmetries. None of the identified polarization dependent tests can be used to discriminate among all chiral groups on one hand and all achiral groups on the other hand. The set of observables corresponding to the set of tests is specific for each 12221

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Figure 4. (a) CD and absorption spectra. (b) CD spectra for different azimuthal orientations. (c) CD spectra for both the front and back sides of the sample. (d) Ellipticity at 445 nm plotted for different sample positions. A spot corresponding to an area with a diameter of 2 mm has been measured in each CD spectrum; this corresponds roughly to the occurrence of 1000 needle crystals.

the probability of generating a chiral superstructure is even more pronounced for molecules with a 3-fold rotation axis since they are able to adopt two propeller-like enantiomeric configurations,20 and indeed several C3 symmetric octupolar molecules have been reported to do so.22 The propeller-like structure has also been reported for a very closely related molecule upon aggregation,23 meaning that the occurrence of this chiral structure at the molecular level is likely in the crystals reported here. We also found several examples in the literature of octupolar molecules crystallizing in polar space groups, even though the number of reports on octupolar crystals is so far still limited.24 However, to the best of our knowledge this is the first case of an achiral octupolar compound adopting a chiral and polar symmetry. To obtain the macroscopic polarity of a system built up from a single component, in this case a molecule, it is necessary that this molecular unit has a dipole moment. Via rotation of the aliphatic chains of the molecule, a conformation with a dipole moment can easily be accomplished; however, it is unlikely that this small dipole moment with low polarizability can manifest itself that strongly in second-harmonic generation measurements. Therefore, we suggest that these compounds adopt an umbrella-like shape by bending the three conjugated arms substituting the central benzene ring. Indeed, aggregation can induce molecular deformation that breaks the planarity of the disk-like compounds.23 Circular Dichroism Spectroscopy. To confirm the chiral nature of the bulk, circular dichroism (CD) spectroscopy has been employed. Figure 4a displays a typical CD spectrum. Strong CD bands with maxima around 445 nm are observed, at the edge of the sample’s absorption bands. Due to the large extinction coefficient of the chromophores, practically all light

point group symmetry. We would like to note that the previously reported SHG-based methods to distinguish between chiral and achiral systems, such as SHG-CD, can only be applied to samples of a known, specific orientation with respect to the incident laser light and are even then only correct for certain point group symmetries.19 The methodology used here is not limited by these conditions. Of the three possibilities, C4, C6, and C∞, the C6 point group is most consistent with the 3-fold molecular symmetry and the occurrence of hexagonal columnar liquid crystalline mesophases for structurally closely related compounds.12 A rectangular ordering of the columns generally has a 2-fold rotation axis associated with it, but no 4-fold rotation axis as in C4, meaning that rectangular ordering can be ruled out. The occurrence of C∞ is still a possibility and corresponds to a disordered packing of the columns, or another phase with disorder in two dimensions as an ∞-fold rotation axis is associated with C∞. This leads to an interesting observation: while the molecular building blocks are clearly achiral and nonpolar (octupolar), they adopt a crystal structure that is chiral and polar (C4, C6, and C∞). Although counterintuitive, it has been shown that about 8% of achiral organic compounds crystallize in chiral space groups.20,21 Such a chiral organization is promoted by a molecular deformation, e.g., bond rotation, or a supramolecular helical arrangement of the molecules, and can be translated through the lattice by the stacking of the discotic octupoles into columnar superstructures. Structurally very closely related C̅ 3 symmetric, π-expanded alkynyl benzenes typically form a columnar structure by stacking of the planar π-conjugated cores. A helical arrangement of preferred handedness suffices to obtain macroscopic chirality. Finally, it has been suggested that 12222

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Figure 5. Image analysis. The analysis is limited to the interval 0−180°, since this is equivalent to the interval 180−360°. Intermediate areas between the large crystallites are masked, except the area highlighted in green. (a) The direction of maximal SHG for test SHGY is depicted for each pixel. (b) The direction of the crystallographic z-axis, derived from test SHGXX, is depicted for each pixel. This series of images was aligned (stacked) before analysis by the appropriate software.16 Please note that, as the sample is rotated, parts move in and out of the image. Only the parts that are visible on all recorded pictures are depicted; the others appear black.

signature.26 This allows for mapping the local symmetry pixel per pixel, thus taking advantage of the full image and high resolution offered by SHG microscopy. A first image analysis protocol analyzes for which polarization direction of the incident IR light the SHG response curve becomes maximal in the presence of an analyzer. In Figure 5a the value and color for each pixel corresponds to the polarization direction of maximal response, between 0 and 180°, for Y detection (test SHGY). Hence, within a crystal, monocrystalline areas will have the same color and defects or impurities will have a different one, enabling a visual assessment of the crystal quality with high contrast and resolution. For clarity the areas between the large crystals have been masked. This is easily achieved by introducing an intensity threshold since these domains display a far weaker response. Only in one area (highlighted in green) this mask has not been applied, to illustrate how these intermediate segments are organized. As expected, the response from this region is very heterogeneous, since it is made up of many small and to a good approximation randomly distributed crystals. Although the large structures display a more homogeneous response, a substantial variation is observed in certain regions within seemingly uniform crystals, demonstrating the formation of differently structured microdomains within seemingly single crystals. In Figure 5b the images corresponding to test SHGXX (Figure 3d) are analyzed. The nonzero susceptibility components χ(2) ijk of the crystals all contain at least one zindex (vide supra). This z-index corresponds to the crystallographic z-axis of the crystals, which is the polar axis. For the SHGXX test, in which the planes of polarization of the incident light and of the detected light are kept parallel, this means that when the plane of polarization is oriented perpendicular to the crystallographic z-axis, no SHG can be generated. In other words, the z-axis is oriented 90° from the direction for which the SHG intensity becomes zero. The orientation of the z-axis projected in the sample plane, corresponding to angle φ in Figure 6, is determined for each pixel and depicted in Figure 5b. In the intermediate regions (highlighted in green in Figure 5), the small crystallites are randomly distributed, adopting all possible orientations. The larger crystallites on the other hand show a profoundly homogeneous response. Indeed, the variation over large regions within these structures is as small as 5°. It can be seen that the direction of the polar z-axis lies perpendicular to the long axis of the crystallites. Accordingly,

is absorbed below 425 nm and the CD signal vanishes, even for thin films. In both absorption and CD spectra light scattering (circular differential scattering for the latter) is observed. It is well-known that sample anisotropies, characteristic of the solid state, can give rise to false CD signatures. However, these effects can be accounted for.25 First, the effect of azimuthal sample rotation on the CD spectra is investigated, as shown in Figure 4b. A true CD effect is invariant under this operation. As the CD changes upon this rotation, it is clear that macroscopic anisotropies contribute to the observed CD spectrum, but this contribution is small in comparison with the observed CD band. It is therefore apparent that this artifact cannot explain the observed CD effect. A second test consists of turning the sample 180°, so the front side becomes the back side. Likewise, a true CD effect is invariant under this operation, while artifacts due to sample anisotropies such as linear dichroism and linear birefringence will change sign, giving rise to the opposite spectrum. Again the variation of the response is negligible in comparison to the appearing CD band. These observations unambiguously demonstrate that the response predominantly stems from a true CD effect, confirming the chiral character of the bulk. Since there is no chirality at the molecular level, there should be no preference toward one enantiomeric form, and a racemic mixture is expected. Different positions of the sample indeed give rise to positive and negative CD bands (compare, for instance, parts a and b of Figure 4). Since the observed CD effect is averaged over the sampled area (2 mm in diameter), this implies the occurrence of spontaneous deracemization over large domains. This is in agreement with the formation of large, conglomerate structures, such as the crystallites observed through (NLO) microscopy. In Figure 4d the ellipticity at 445 nm is plotted for different sample positions. An equal probability for both enantiomeric forms is found, confirming the racemic nature of the crystallization. Image Analysis. The local organization in the crystallites can be visualized by processing the recorded polarized SHG images with appropriate software. An analysis of the SHG images recorded in the polarization dependent tests discussed above (Figure 3) allows determining the polarization dependent SHG response for each pixel composing the image. For monocrystalline domains this response will be identical for the entire monocrystalline area. Since SHG is extremely sensitive to symmetry, even small deviations will give rise to a different 12223

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indistinguishable C4 and C∞ groups), which is both chiral and polar. CD spectroscopy additionally confirmed the chiral nature of these structures and demonstrates the formation of large conglomerate structures. Finally, the local organization and quality of the crystallites, of fundamental importance for technological applications, was assessed with high contrast and resolution by straightforward image analysis protocols. Very subtle deviations from single crystal morphology, which are likely to go undetected with traditional microscopic methods, were imaged with large sensitivity. Evidently, the analysis presented here allows for a highly sensitive quality control of a variety of noncentrosymmetric crystalline structures.

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ASSOCIATED CONTENT

S Supporting Information *

Polarized optical microscopy image of the sample. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 6. Schematic representation of the system frame (xyz) and the laboratory frame (XYZ) with the corresponding Euler angles for the C6 point group (θ, φ).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (M.A.vdV.); [email protected] (S.V.C.). Fax: +32 16 321998 (M.A.vdV.); +32 16 327982 (S.V.C.). Phone: +32 16 32 7159 (M.A.vdV.); +32 16 327508 (S.V.C.).

the maximal TPF intensity is obtained in this direction, which suggests that the two-photon excitation primarily occurs along the polar axis. For the C6 point group, the projection of the relevant SHG tensor components of the crystal frame on the macroscopic frame depends on two angles, as depicted in Figure 6. The first corresponds to the angle the crystallographic z-axis makes with the macroscopic XZ plane (φ), determined by test SHGXX and depicted in Figure 5b. The second corresponds to the out-ofplane angle (θ) the z-axis makes with the substrate, or the XY plane. Since the former angle, φ, is as good as constant within the large crystallites, the variation in SHG response in Figure 5a can be explained, for the same crystalline structure, by a different out-of-plane angle θ, or also by polycrystallinity, crystal defects, the occurrence of areas of different handedness, or a combination of these. Finally, remark that the elongated morphology of the crystals would be interpreted by optical microscopy as single crystals. Also, due to the uniform in-plane orientation of the z-axis, i.e., the optical axis, this is the likely conclusion to be drawn from polarized light microscopy (polarized microscopy image of the sample illustrating this is provided in the Supporting Information).27 As can be seen, SHG microscopy provides a much more detailed and sensitive picture of the organization of microdomains than polarized microscopy. This is due to the fact that polarized light microscopy is sensitive to differences in refractive index, while SHG microscopy is directly sensitive to the organization and structure of a system. Like phase contrast microscopy and differential interference microscopy, traditionally used imaging techniques to evaluate macroscopic crystallinity are based on differences in refractive index, SHG microscopy should surpass also these techniques in terms of sensitivity to crystal defects.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the Onderzoeksfonds KU Leuven/ Research Fund KU Leuven. This work was supported by the University of Leuven (GOA/2011/03). S.V.C. and P.W. are grateful for the financial support from the Agency for Innovation by Science and Technology (IWT) Flanders for their bursaries. M.A.vdV. thanks the FWO Flanders for a postdoctoral fellowship. G.H. thanks the Spanish government for financial support (Project No. CTQ2010-18813).

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CONCLUSION In conclusion, we have studied the symmetry properties of microcrystals obtained from an octupolar chromophore by using all optical methods. These crystals give rise to a significant SHG signal. A combination of polarization dependent TPF and SHG microscopy revealed that these achiral, nonpolar molecules crystallize in the C6 point group (or in the 12224

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dx.doi.org/10.1021/jp302665t | J. Phys. Chem. C 2012, 116, 12219−12225